MOLECULAR TAXONOMY OF CERTAIN PLASMID HARBOURING … · 2018-01-04 · molecular taxonomy of...
Transcript of MOLECULAR TAXONOMY OF CERTAIN PLASMID HARBOURING … · 2018-01-04 · molecular taxonomy of...
MOLECULAR TAXONOMY OF CERTAIN PLASMID HARBOURING ENTEROBACTERIA
DISSERTATION SUBMITTED.IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF
Muittv of ^dtlo^oplip IN
Microbiology (Ag.)
BY
ABDUb MAblK
A G R I C U L T U R E C E N T R E / D E P A R T M E N T O F B I O C H E M I S T R Y
F A C U L T Y O F L I F E SCIENCES A L I G A R H M U S L I M U N I V E R S I T Y
A L I G A R H ( INDIA)
1991
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t\cc Nei
DS1719
DEPARTMENT OF BIOCHEMISTRY Ai i nARH MUSLIM UNlvrr?'-.! r v AUIGARH-202002 u, n. rwDiA
TfiLHIX ! 3642.10 c r A IN I'll rJK o r | - . - n 7 l l
CERTIFICATE
This i s to cer t i fy that the r e s e a r c h work
embodied in th i s d i s se r t a t ion ent i t led "MOLECULAR
TAXONOMY OF CERTAIN PLASMID HARBOURING
ENTEROBACTERIA" i s an or ig inal work, unless o the rwi se
s t a t ed , c a r r i ed out by Mr. ABDUL MALIK under my
superv is ion and i s su i t ab l e for submission for the award
of M.Ph i l , degree in Microbiology ( A g . ) .
Dated 7 ' ' ^ Jti-rv^-, n 1 ' ( MASOOD AHMAD )
DEDICATED
TO
MY
PARENTS
ACKNOWLEDGEMENT S
I f i n d words i n a d e q u a t e t o e x p r e s s my d e e p s e n s e of g r a t i t u d e and huinble r e g a r d s t o my e s t e e m e d t e a c h e r and s u p e r v i s o r . D r . Masood Ahmad, R e a d e r , Depa r tmen t of B i o c h e m i s t r y , \ander whose g u i d a n c e , keen i n t e r e s t , c o n s t a n t s u p e r v i s i o n and u n c e a s i n g e n c o u r a g e m e n t , I h a v e b e e n a b l e t o c a r r 7 o u t t h i s s t u d y . My a p p r e c i a t i o n f o r h i s s y m p a t h e t i c g u i d a n c e and c o n s t r u c t i v e c r i t i c i s m canno t ' ^e^q i ressed i n few w o r d s . He h a s b e e n c o n s t a n t s o u r c e of i n s p i r a t i o n t o me. Whatever m e r i t s t h i s d i s s e r t a t i o n p o s s e s s l i e w i t h h i m .
I am h i g h l y i n d e b t e d t o P r o f . S. I s r a r H u s s a i n , C o o r d i n a t o r A g r i c u l t u r e C e n t r e and P r o f . K h a l i d Mahmood f o r t h e i r v a l u a b l e s u g g e s t i o n s and c o o p e r a t i o n .
I am g r a t e f u l t o P r o f . M. S a l e e m u d d i n , Cha i rman , D e p a r t m e n t of B i o c h e m i s t r y , F a c u l t y of L i f e ) S c i e n c e s f o r h e l p f u l s u g g e s t i o n s and p r o v i d i n g n e c e s s a r y f a c i l i t i e s t o c a r r y o u t t h i s w o r k .
I am h i g h l y t h a n k f u l t o P r o f .AfM. S i d d i q i f o r f r u i t f u l a d v i c e and encou ragemen t t h r o u g h o u t t h i s s t u d y .
My s i n c e r e t h a n k s a r e due t o P r o f . S«M- H a d i , D r ( s ) A.N.K. Y u s u f i , M r s . B . Bano, M r s . N. Bano , F a s i h Ahmad, R iaz Mahmood, Qaiyyvim H u s s a i n and M i s s I . Naseem f o r t h e i r v a l u a b l e s u g g e s t i o n s .
A t o k e n of deep a p p r e c i a t i o n t o D r . J a v e d M u s a r r a t f o r h i s c o n s t r u c t i v e s u g g e s t i o n s and k i n d h e l p t h r o u g h o u t t h i s s t u d y .
I am d e l i g h t e d t o r e c o r d my deep a p p r e c i a t i o n and s i n c e r e t h a n k s t o Mis s Zehra Rehana f o r h e r a m i c a b l e h e l p and c o o p e r a t i o n d u r i n g t h i s w o r k .
I am a l s o t h a n k f u l t o M i s s S. I s l a m and Mr . S.A, Q a d r i f o r t h e i r h e l p f u l c o o p e r a t i o n .
My s i n c e r e t h a n k s a r e a l s o due t o my f r i e n d s and l a b c o l l e a g u e s , S h a h i d , R a s h i d , A s h g a r , S a i d , A d i l , Fahiro, Qasim, J a l a l , I b r a h i m and G o p a l , Mis s ( s ) R. Z a i d i , M.Mir2ay., R .D. G u p t a , T . Z e h r a , A i s h a , Veena , M r s ( s ) Afshan Naheed , A. Z a i d i , Pabeha and F a r a h .
M i n i s t r y of Env i ronmen t and F o r e s t s , Government of I n d i a , New D e l h i a r e g r a t e f u l l y acknowledged f o r f i n a n c i a l a s s i s t a n c e .
UL MALIK)
OONTENTS
C e r t i f i c a t e
Ac)aiowledgeiT»ent
L i s t o f a b b r e v i a t i o n s
L i s t of t a b l e s
L i s t of i l l u s t r a t i o n s
P r e f a c e ,
I n t r o d u c t i o n
M a t e r i a l and Methods
R e s u l t s
D i s c u s s i o n
B i b l i o g r a p h y
P a g e
V-
1.
36 .
5 3-
8 8 -
10 4-
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LIST OF ABBREVIATIONS
A d iT ip ic l l l l n
Ak a m i k a c i n
B b a c i t r a c i n
C c h l o r a n p h e n i c o l
CPU c o l o n y fofming u n i t s
Ct c h l o r o t e t r a c y c l i n e
Cx c l o x a c i l l i n
E e r y t h r o m y c i n
F r f u r a z o l i d o n e
FS f e c a l s t r e p t o c o c c i
G g e n t a m y c i n
h h o u r
K kanamyc in
MIC minimum i n h i b i t o r y c o n c e n t r a t i o n s
min m i n u t e s
ug micrograms
jul microxiters
MPN most probable number
Na nalidixic acid
N neomycin
P penicillin
Pb polymyxin B
R rifanpicin
S streptomycin
Sec seconds
T tetracycline
TBC total bacterial counts
TC total conforms
TEC total enterobacterial counts
ii
LIST OF TABLES
Table Page
1 Properties determined by bacterial plasmids. 1^-17
2 Taxonomic Relationship of the Entero- 29 bacteriaceae
3 Location of different sampling sites ^^
4 Seasonal variation in bacteriological para- 5 4-55 meters in the sewage water.
5 Seasonal variation in bacteriological para- 5 6-57 meters in the soil.
6 Locational variation and average bacterio- 59 logical parameters in the domestic sewage water.
Locational variation and average bacteriological parameters in the industrial sewage water.
60
8 L o c a t i o n a l v a r i a t i o n and average b a c t e r i o l o - - ^2 g i c a l pa r ame te r s of s o i l i n t h e v i c i n i t y of domest ic sewage w a t e r .
9 L o c a t i o n a l v a r i a t i o n and average b a c t e r i o l o - ^4 g i c a l pa rame te r s of s o i l i n t h e v i c i n i t y of i n d u s t r i a l a r e a .
10 A n t i b i o t i c r e s i s t a n c e p a t t e r n of E . o o l i ^5 s t r a i n s i s o l a t e d from sewage w a t e r .
11 A n t i b i o t i c r e s i s t a n c e p a t t e r n of E . c o l i 57 s t r a i n s i s o l a t e d from s o i l . ""
12 P e r c e n t r e s i s t a n c e a g a i n s t i n d i v i d u a l a n t i - 68 b a c t e r i a l agent moni tored i n t h e sewage w a t e r .
13 P e r c e n t r e s i s t a n c e a g a i n s t i n d i v i d u a l a n t i - 69 b a c t e r i a l agent moni tored in t h e s o i l . ,
14 I n c i d e n c e of metal r e s i s t a n c e and t h e i r MIC 7 i v a l u e in E . c o l i i s o l a t e d from sewage w a t e r .
15 . I n c i d e n c e of metal r e s i s t a n c e and t h e i r MIC 7 2 v a l u e in E . c o l i i s o l a t e d from s o i l .
C o n t d , , ,
iii
16. Incidence of metal resistance and their KIC 73 value in Enterobacter aeroqenes isolated from sewage water,
17. Antibiotic resistance pattern of 18 Enterobacter 75 aeroqenes strains isolated from sewage water.
18. Transforming-ability of AB1157 with the six test 79 plasmids.
19. Conjugative potential of multiply resistant 8 2 E.coli isolates with recipient AB1157 strain,
E.coli strains used in this study 44
i v
LIST OF ILLUSTRATIONS
F i g u r e P a g e
1 Aga rose g e l e l e c t r o p h o r e s i s p a t t e r n s of _ , p l a s m i d DNA i s o l a t e d from 5 E . c o l l Si and Sd i s o l a t e s .
2 Agarose g e l e l e c : ± r o p h o r e s i s p a t t e r n s o f 77 p l a s m i d DNA i s o l a t e d from 8 E . c o l i sewage w a t e r i s o l a t e s .
3 P l a s m i d c u r i n g and s u r v i v a l o f p l a s m i d 80 h a r b o u r i n g E . c o l i S i^ and Wd, s t r a i n s by t r e a t m e n t w i t h e t n i d i u m b r o m i d e .
Agarose g e l e l e c t r o p h o r e s i s of t h e p l a s m i d s p e c i e s i s o l a t e d from E . c o l i i s o l a t e s from w a t e r .
83
M o l e c u l a r s i z e Vs m o b i l i t y p l o t of t h e 85 t e s t s p e c i e s and t h e s t a n d a r d DNA.
Agarose g e l e l e c t r o p h o r e s i s of m u l t i p l e 8 6 p l a s m i d s p e c i e s i s o l a t e d from E . c o l i (Wig, Wd^, Wd^) s t r a i n s .
M o l e c u l a r s i z e Vs m o b i l i t y p l o t of t h e 97 t e s t ( m u l t i p l e s p e c i e s o f t h e R - p l a s m i d s i s o l a t e d from Wic# Wd^ and Wd s t r a i n s ) and s t a n d a r d DNA. " ^
P R E F A C E
The gram negative, non sporeforming bacilli make up a
large group of bacteria that include intestinal commensals/
such as Escherichia and Proteus, enteric pathogens, such as
Salmonella and Shigella and some strains of Escherichia and
Klebsiella as respiratory and urinary tract pathogens
Yersinia including the plague bacillus, and a variety of
saprophytes and plant pathogens (Freeman, 1985),
Application of sewage sludge on agricultural lands for
enhancing productivity has been a common practice in our
country for many years. Nowadays a serious attention is
being paid to the heavy metal contents of sewage sludge
before its land application, because of the tendency of up
take of toxic metals and metalloids like Hg, Cd, Cu, Co, Cr,
Ag, Mo, Ni, Pb, Se, Te, and Zn by food crops and plants
(Webber, 1972; Wood, 1975; Lagerwerf e_t al_., 1976; Bhowal
et al,., 1987) .
Metal resistance in bacteria is frequently coded by
genes located on plasmids and transposons and is often
transferable intergenerically, interspecially and also from
in situ microflora to indigenous microflora (Datta and
Hedges, 1972; Smith, 1978; Bale et al_., 1988). Moreover the
metal resistance is often associated with resistance to
single or multiple drugs also (Kondo et a_l., 1974; Allen
et al., 1977; Foster, 1983) .
vi
Aiigarh city is famous for lock manufacturing facto
ries. Hundreds of small and large scale factories are
supposed to spill tremendous amount of heavy metals into
the sewage and in the form of industrial effluents (Ajmal
fit al., 1980) .
The concentration of metal pollutants in the environ
ment is usually low excepting in specific areas which are
polluted by various hospital and industrial wastes. However
unpolluted environment may also harbour metal resistant
organisms or organism that readily adapt to high concentra
tion of metals. The incidence of plasmid bearing strain is
more in polluted site than in the unpolluted zone (Hada and
Sizemore, 1981) .
It is important to know the existence of plasmid
encoded metal resistances, their physiological and bioche
mical characteristics, the mechanism of resistance and the
limiting factors in natural ecosystem so as to evolve a
"super bug" through recombinant DNA technology. It would
also be a fascinating task to search for new microbes that
would withstand natural milieu containing the metal pollu
tants. Such microbes could be readily used to clean up
waste waters and soils containing toxic metals.
The constant exposure of the body flora of man and
animals to antibiotics has led to the selection and wide
dissemination in human population of antibiotic resi-stant
v i i
s t r a i n s of bac te r i a , R-factors which car ry a n t i b i o t i c and
metal r e s i s t ance can also be t ransmiss ible have become common
in the non-pathogenic E ,co l i of the alimentary t r a c t of man
and domestic animals. Many R"*" organisms e s p e c i a l l y those of
human and animal or ig in are expected to en te r sewage and
from t h i s , obviously to s o i l s (Smith, 1970; Bhowal e^ a l . ,
1987), E ,co l i with t ransmiss ib le r e s i s t ance to some broad
spectrum a n t i b i o t i c i s considered to be p o t e n t i a l l y very
dangerous because i t s r e s i s t a n c e might be t ransmit ted to
other pathogenic bac t e r i a l s t r a i n s , thus rendering the t r e a t
ment of in fec t ious diseases very d i f f i c u l t . Hence th i s sewage
water i s not onxy a source of infect ion but can also promote
the spread of aBbibiot ic r e s i s t a n c e .
In view of the c l i n i c a l and ecological s ignif icance
of the work as well as the urgency of the task and magnitude
of the problem coupled with the meagre work car r ied out in
t h i s d i r e c t i o n , attertpts have been made to study the i n c i
dence of R-piasmids harbouring s t r a i n of en terobac ter ia in
sewage water and soi l of the Aligarh c i t y . Another dimension
of the problem which i s equa l ly ra ther sometimes more iitpor-
tan t happens to be the taxonomy of the plasmid harbouring
organisms. Our study would also provide an appropriate index
for t he i r c l a s s i f i c a t i o n .
INTRODUCTION
General Properties of Enterobacteriaceae
The aerobic and facultatively anaerobic bacterial
flora of the large intestines of man and animals are corrpo-
sed of non-spore forming, nonacid fast, gram negative baci
lli. They exhibit general morphological and biochemical
similarities and are grouped together in the large and
coirplex family Enterobacteriaceae (Ananthanarayan and
Paniker, 1986) . This family includes several genera like
Escherichia and Proteus, inhabiting in the intestines as
commensals. Salmonella and Shigella as enteric pathogens.
Yersinia as plague bacillus and a variety of other sapro
phytes and plant pathogens (freeman, i985;?&'''»""*» ?£ oJ*/* " .
Since the enteric bacilli are closely related in
morphology, structure, biochemistry and antigenic make up,
some of their common properties are as follows :
Morphology i
Enteric bacteria are aerobic, facultatively anaerobic,
gram negative, nonsporulating bacilli. Capsules are formed
by a few, like Klebsiella and some strains of Enterobacter
and Serratia. Loose slime is frequently produced by Erwinia.
Most of the enteric bacilli are motile and when motility
occurs it is by means of peritrichous flagella.Shigella,
Klebsiella and Yersinia are typically nonmotile. Indivi
dual cells vary some what in size, but typically they are
in range of 1.0 to 1.5 x 2.0 to 6.0 um. They occur singly
or in pairs (Braude, 1983) . The variation in size and
morphology is encountered, even within a species. In
some group, e.g. Proteus, there is evidence of filaments,
Spheroplast and other aberrant forms. Yersinia tend to be
some what coccoid or oval in shape and are smaller than
other members. Fimbriae are produced by many enterobacteria,
being found principally in Escherichia, Klebsiella, Proteus,
Salmonella and Shigella (Edwards and Ewing, 197 2) .
In colonial morphology, the enteric bacteria are all
very much alike, and it is not possible to make valid distin
ction by these characters. On ordinary bacteriological media,
they tend to be smooth, convex with entire margins, moist
and shiny and are usually gray in colour. Rough colonial
forms are frequently seen. These are often dry, friable
and not easily emulsifiable in saline. The presence of cap
sules or loose siime is usually reflected in dome shaped
colonies of mucoid and viscous consistency. Some strains
particularly of Escherichia and Proteus, are haemolytic on
blood agar. Although pigment production is rarely seen in
most genera, Serratia produce a red pigment under certain
growth conditions, while many Erwlnia colonies are cream
to yellow in colour (Martin and Washington, 1980) ,
Antigenic structure ;
Among the Enterobacterlaceae the designation of genus,
and in many cases species is based primarily on biochemical
parameters. Further differentiation to specific rank in
some instances and to serogroups/ serotypes, and varieties
is dependent upon serological reaction that detect surface
antigens. These antigens fall into three major classes i
somatic 0 antigen, flagellar H antigens, and capsular or
envelope K antigens.
0 antigens j The somatic 0 antigens are the heat stable
lipopolysaccharide common to all smooth, gram negative bac
teria. They are present in outer membrane and are responsible
for the endotoxic activity allied with gram negative bacteria.
The antigenic specificity of these 0 antigens resides in the
0 specific side chains of the lipopolysaccharide and is
dependent upon the Kinds of sugar residues and their arran
gement in the side chain (Simmons, 1971; OrsKov, 1977) ,
H antigens : The flagella of motile bacteria contain serolo
gically specific proteins (flagellins) which unlike the 0
antigens, are heat labile and are destroyed by ethanol.
When these motile bacteria are reacted with specific anti-
flagellar antibodies, microscopic observation reveals a
loose association of many cells with tangling and inter
twining of their flagella.
K antigens » A third class of antigens, the K antigens,
appear in Enterobacteriaceae as capsule or envelope anti
gens which overlie the surface 0 antigens. Because of this.
the K antigens block agglutination by 0 specific antibody.
Antigens of this class are generally polysaccharides and
their reactivity is usually altered by heating, so that
treatment in this way yields strains agglutinable with 0
antiserum. The K antigens terminology varies some what in
different genera. In few Salrtonella that posses K antigens*
it is designated the Vi antigen. The K antigens of Bscheri-.
chia are generally polysaccharide, but in few cases, they
are coirposed of protein and occur as fimbriae on the cell
surface (Orskov, 1977) ,
Enterobacterial common antigens t In 1962, Kunin and his
colleagues described a haptenic substance of wide distri
bution among enterobacteria that is now called the entero
bacterial common antigen (EGA) or Kunin antigen. The hapten
occurs in two aggregative forms in the outer membrane. In
a few bacteria, the hapten is linked to the lipopolysaccha-
ride (LPS) and is immunogenic in the majority of enteric
bacteria, however it is in a free form and is not capable
of eliciting antibody response. The LPS-linked EGA is
relatively rare but is found in some R-mutants of Escherichia
coli and Shigella (Makela and Mayer, 1976; Mannel and
Mayer, 1978) ,
Flmbrial antigens : Most members of the Enterobacteriaceae
produce fimbriae or pili. The common pili are numerous on
the bacterial surface whereas the F or Sex pili are limited
in number 1-4 per cell and differ morphologically from
common pili (Rinno et al,, 1980),
Pyotein antigens t In common with other gram negative
bacteria, the Enterobacteriaceae contain several major
proteins in the outer membrane. These are antigenic and
contribute to the antigenic mosaic of enterobacteria.
There is considerable serological cross reactivity between
major outer membrane proteins from related strains e.g.
Escherichia coli/ and at least one of these may be common
to all Enterobacteriaceae (Hofstra and Dankert, 1979).
Besides major outer membrane proteins, Enterobac
teriaceae also appear to possess a common lipoprotein
antigen (Griffiths et al,,, 1977),
Toxins I
In addit ion to the endotoxic a c t i v i t y associated
with the l ipopolysaccharide of the outer membrane, and
common to a l l wild s t r a i n s of en te robac te r i a , several
other toxins may be produced.
Those en te robac te r ia t h a t exh ib i t haeitolytic a c t i
v i t y such as enteropathic s t r a i n s of E ,co l i and some Proteus ,
usua l ly produce e x t r a c e l l u l a r haemolytic toxins t ha t may
have other cytotoxic p rope r t i e s as we l l . The diarrheagenic
enterotoxin produced by some s t r a i n s of E .co l i are of
considerable s ignif icance since these are responsible for
the secret ion of f l u id s and e l e c t r o l y t e s in to the lumen of
the snai l i n t e s t i n e . Several Shigellae especia l ly Shigella
dysentr iae type 1, produce substances with cytotoxic and
entero toxic a c t i v i t i e s . Enterotoxins are also occasional ly
encountered in ce r ta in Salmonella (Griesman, 1969; Richards
and Doughlas, 1978; Bergan and Olsvik, 1980; Brown, 1982).
Sal ient Features of Various Species In t h e Family Entero-
bac te r iaceae
Escherichia c o l i and i t s c h a r a c t e r i s t i c fea tures :
Escherichia co l i for t he f i r s t t ime was described
by Buchner (1885) and t h e genus was named a f t e r Theoder
Escherich (1888) who made a de ta i l ed study of t h i s micro
organism. E.col i i s the predominant f acu l t a t i ve anaerobic
species in the la rge i n t e s t i n e and thus the species name
represents t h i s c h a r a c t e r i s t i c f ea tu re . According to the
Sergey 's manual/ the genus Escherichia has been grouped
under the family of Enterobacteriaceae (Bergey's manual,
1984), I t i s a gram negat ive s t r a igh t rod, measuring ( l -3 )x
(0.4-0.7) micron arranged singly or in p a i r s (Alcamo, 1987).
Cul tural c h a r a c t e r i s t i c s and colonial mozTpholoqy ; When
grown in l i qu id media, E .co l i produces a well dispersed
t u r b i d i t y in the temperature range of 10-46°C, but 37^C
i s t h e optimum temperature . Most E.col i s t r a i n s have f l a -
ge l l a and are mo t i l e . I t forms a var ie ty of colonies on
solid media e.g. large, thick, moist, smooth, greyish,
white or colourless, opaque or partially transluscent.
Smooth (S) type strains form shining convex colonies but
when repeatedly subcuitured they become rough (R) type and
form lustureless, granular colonies. The S —> R variation
is associated with loss of surface antigen and, usually
of virulence. Encapsulated variants produce mucoid colonies,
particularly when incubated at low teinperature and when
grown in media containing limited amount of nitrogen and
phosphorus and a high concentration of carbohydrate. Typi
cal E.coli colonies are easily recognised by their character
istic appearance on certain differential media. This bacter
ium is largely a lactose fermenter and forms bright pink
colonies on MacConkey's medium. The colonies on the eosin-
methylene blue and the endo agar display a metallic sheen
characteristic.^-haemolysin is also produced on blood agar
in certain cases (Davis et al,, 1989),
Biochemical reactions t E.coli usually ferments carbohydrates
with the production of acid and gas, A few strains are how
ever, anaerogenic producing acid without gas. Other subs
tances with which different biotypes of E.coli react differe
ntly include raffinose, rhamnose, sucrose, xylose, arginine,
glutamic acid and ornithine. Gram's staining and IMViC
reactions are commonly used for the identification of
enteric bacilli, and their identification is of prime
8
inportance in cont ro l l ing i n t e s t i n a l in fec t ions by preventing
contaminations of food and water suppl ies , E .col i d isplays
indol p o s i t i v e , methyl red p o s i t i v e , Voges-prauskaur nega
t i v e and c i t r a t e u t i l i z a t i o n negative reac t ion i . e . IMViC++ ,
E .co l i i s also d is t inguished from other coliforms by i t s
a b i l i t y to form gas from lac tose in t e s t system incubated
a t 44 C (Cappucino and Sherman, 1987).
Host p a r a s i t e r e l a t i o n s h i p t E .col i i s a commensal t h a t
th r ives bes t in the human i n t e s t i n e . However, some micro
organisms have the a b i l i t y to change g e n e t i c a l l y and become
v i r u l e n t . E .col i which was long considered an av i ru lent
commensal to humans, some times becomes oppor tunis t ic and
a t t a i n s pathogenic charac ter because ce r t a in toxin-producing
s t r a i n s have been i so l a t ed in the outbreaks of human d ia
rrhoea and ur inary t r a c t infect ions (Middiebrook and
Dorland, 1984) .
Salmonella t
The Salmonella are gram negative b a c i l l i whose
c e l l u l a r morphology c lose ly resembles and i s i nd i s t ingu i sh
able from other en te robac te r i a . All members except S ,
e n t e r i t i d i s serotypes pullorum and gal l inarum are a c t i v e l y
motile by pe r i t r i chous f l a g e l l a . No capsules are d i sce rn ib le
by l i g h t microscopy, although a polysaccharide surface
antigen designated as Vi, occurs in most s . typhi and a few
other Salmonella (Nolan, 1981) ,
The bac te r i a of "this group have simple n u t r i t i o n a l
requirements, growing readi ly on the usual nu t r i en t media.
Adequate growth obta ins in syn the t ic media containing
ammonium s a l t s as a ni t rogen source and simple carbon
sources such as glucose, pyruvate or l a c t a t e . A great
majority of s t r a ins do not requi re vitamins or amino ac ids ,
however some s t r a i n s of S.typhi require added t ryptophan.
They are f acu l t a t i ve anaerobes, growing equally well under
e i t h e r aerobic or anaerobic condi t ions . On the bas i s of
biochemical reac t ions , t h ree species of Salmonella are
d i s t ingu i shed . They are Salmonella cho le ra - su i s , S.typhi
and S . e n t e r i t i d i s (Harvey and P r i ce , 1979; Martin and
Washington, 1980) .
Salmonellosis in animals : Salmonellae infect a wide v a r i e t y
of animals and t h e i r importance as animal r e se rvo i r s of
human infec t ion i s well known. Food producing animals, i nc lu
ding swine, c a t t l e and fowl, may be na tu ra l ly infec ted . Man
cont rac t s Salmonellosis by consumption of infected f lesh
of such animals (Blaser, 1980) .
The Salmonellae responsible for animal in fec t ions
include S.cholera-suis and several serotypes of S . e n t e r i t i d i s .
Salmonella typhi in contras t t o most o ther Salmonella, i s
almost completely nonpathogenic for lower animals, but a
d isease closely resembling typhoid fever i s produced- in
Chimpanzees by ora l inoculat ion (Gaines, 1968) .
10
Salmonella enterltldls is generally responsible for
infections in rats and mice, and these animals become heal
thy carriers of the bacilli (Kochan et al_., 1978).
Birds are quite commonly infected with Salmonellae,
Epidemics due to typhimurium serotype cause great destruc
tion among canaries and other songbirds. Two barnyard
diseases of great importance are due to specific types,
the bacillary white diarrhoea of chicks is caused by sero
type pullorum and fowl typhoid is caused by gallinarum
(Pelczar e^ al_., 1986). It is well known that cold blooded
animals may be infected with Salmonella. Ordinarily such
reservoirs of infection do not relate to human disease,
but a number of human infection have been acquired from
turtles and other aquarium pets (Chiodini and Sundberg,
1981), As a part of this problem fresh water aquarium
snails may also harbour these microorganisms (Bartlett and
Trust, 1976) .
Salmonella infections of man : Salmonellosis in humans is
almost always acquired by the ingestion of the microorganisms,
usually in contaminated food, milk or water. There are two
kinds of salmonellosis, one an acute gastroenteritis cha
racterised by vomiting and diarrhoea in which a small propo
rtions of patients usually the young children, become
septicaemic. The other is typhoid also called enteric fever.
11
a disease in which bacteremia and tissue invasion is noted
in the initial stages and synptoms are more general in
nature (Cohen and Gangarosa, 1978; Blaser and Newman, 198 2)
Enterobacter and Serratla :
The Enterobacter are found as commensals in the
intestine and are frequently isolated from soil and water.
They are only rarely encountered as infectious agents in
the community but may be responsible for a variety of
hospital associated or nosocomial infections (John et al.,
1982) . The predominant species responsible are E.cloacae
and E.aerogenes.
Serratia are, for the most part, free living sapro
phytes but they are indeed capable of causing serious
infections under some circumstances and are regarded as
opportunistic pathogens. They have been iirplicated in infe
ctions of the respiratory tract, UTI, meninges, and endo
cardium (Yu# 1979). The most familiar of the Serratia are
the red pigmented varieties of S»marcescens«
Proteus, Providentia and Morqanella ;
The tribe Proteae is made up of three genera :
Proteus, Providentia and Morqanella. These were until
recently classified as Proteus and are thus related to
one another as well as to other enteric bacilli. Except
12
for some of the Providentla, these microorganisms are found
with some frequency in the intestinal tract of normal humans.
They are common inhabitants in soil and water that contain
decaying organic matter of animal origin and are usually
found in sewage. The phenomenon of swarming is typical of
Proteus and is consequence of the production of elongated
cells with excessive numbers of flagella (Williams and
Schwarzhoff, 1978). These microorganisms are regarded as
opportunistic pathogens and most infections are hospital
associated (Chow/ 1979). Among them urinary tract infect
ions are the predominant type and these are most often due
to Proteus mirabilis (Turck and Stamm, 1981),
Klebsiella »
The genus Klebsiella consists of nonmotile, capsu-
lated rods that grow well on ordinary media, forming large
dome shaped, mucoid colonies of varying degrees of sticki
ness, Klebsiellae are widely distributed in nature, occu
rring both as commensals in intestines and as saprophytes
in soil and water. They have been classified into three
species based on biochemical reactions and into over 80
serotypes based on their capsular (K) antigens, K.pneumoniae
is the second most populous member of the aerobic bacterial
flora of the human intestines. It has become very impor
tant cause of nosocomial infections, even replacing E,coli
in some centres (Martin and Washington, 1980; Baurnefied
et al_,, 1981) ,
13
Shigella ;
Shigella are gram negative, nonspore forming rods
related to other enteric bacteria. Some of them resemble
anaerogenic Escherichia and the typhoid bacillus in that
they ferment carbohydrates with the production of acid
but without gas. None of the dysentery bacilli are motile,
hence they do not contain flagellar antigens (Simmons, 1971)
Shigella are aerobic and facultatively anaerobic,
and their optimum temperature for growth is 37°C, Their
nutritive requirements are not conplex, since they can
grow upon ordinary nutrients media containing peptone and
beef extract. Colonies on MacConkey agar are colourless
due to the absence of lactose fermentation (Martin and
Washington, 1980) ,
Erwinia i
Erwinia is the irrportant phytopathogenic genus
belonging to the family Enterobacteriaceae and characteri
sed by motile rods which normally do not require organic
N conpounds for growth. They produce acid with or without
visible gas from a variety of sugars. They invade tissues
of living plants and produce dry necroses, galls, wilt
and soft rots. In the latter cases the enzyme protopecti-
nase destroys the middle lamellar substances of the cells
(Singh, 1986) .
14
Soft rot of vegetables including potatoes caused
by different species of soft rot coliform bacteria is a
common storage and transit disease. In potatoes, these
bacteria not only cause soft rot of tubers but also black
leg and wilt of plants in the field. Several species of
Erwinia which cause plant diseases are E.carotovora«
E.atroseptica, E.aeroideae and E.phytophthora. Black leg
and soft rots of potatoes are caused by E.phytophthora
(Stapp, 1961).
Plasmids and their Characteristics Features
Plasmids are covalently closed circular extra-
chromosomal DNAs capable of autonomous replication. These
are ubiquitous among the bacterial genera and are also
found in some eucaryotic organisms (Davis and Rownd, 1972;
Meynell, 1973/ Lewin, 1977/ Broda, 1979).
Plasmids were discovered soon after the discovery
of bacterial conjugation in early 19501s by J, Lederberg
(Lederberg, 195 2) in family Enterobacteriaceae when it
became clear that there were two genetically determined
'mating* types and that genetic information was physically
transferred from a 'male' or donor type to a 'female' or
recipient type presumably involving the transfer of F
(Fertility) factor, Lederberg demonstrated that this
character for maleness was present in an extrachromosomal
15
genetic elements and in 195 2 he coined the term plasmid
to refer to all such extrachromosomal genetic system,
Plasmid encode a number of specialised function
that are generally nonessential for their bacterial host.
However, these plasmid encoded functions provide their
host cells with versatility and adaptability for growth
and survival under a variety of conditions, Plasmids and
transposable elements that they often contain may also
have played an integral role in the evolution of bacterial
species by promoting the distribution and exchange of
genetic information (Arber et al_, / 1985),
Plasmid mediated functions include genetic tran
sfer, bacteriocin production and their resistance, anti
biotic resistance and their production, resistance to
heavy metals and DNA damaging agents, metabolism of carbo
hydrates and hydrocarbons, toxin and hemolysin production,
virulence and colonisation factors, tumorigenicity and
nitrogen fixation in plants. Recently, plasmids have be
come the indispensable tools of modern molecular bio
logical research (Womble and Rownd, 1988) , A detailed
list of properties determined by bacterial plasmids is
given in table I.
Classification of Plasmids
Identification and classification of plasmids are
16
Table - I i Properties determined by bacterial plasmids
1. Resistance properties ;
(a) Resistance to antibiotics - Aminoglycosides ( e . g . streptomycin, gentamycin,
amikacin - as a resu±t of enzymic modification by N-acetyla t ion. 0-nucxeotidylat lon or 0-phosphory-la t ion) ,
- Chloranphenicol (as a r e s u l t of enzymic modificat ion by 0 - a c e t y l a t i o n ) .
- Fusidic acid - Furans - p-Lactam antibiotics (e.g. benzyl penicillin, arrpi-
clllin, carbenicillin (as a result of enzymic cleavage of the lactam ring).
- Sulphenamides, trimethoprim (as a result of alternative drug-resistant target enzymes) .
- Tetracyclines (as a result of altered transport system)
- Erythromycin (as a result of altered ribosome binding site,
(b) Resistance to heavy-metal cations - Mercury and organomercurials (as a result of reduc
tases and hydrolases) - Nickel, cobalt, lead, cadmiumum, bismuth, antimony,
zinc, silver, thallium
(c) Resistance to anions - Arsenate, arsenite, tellurite, borate, chromate
(d) Other resistances - Radiation (e.g. u.v.. X-rays) - Phage and bacteriocin resistance (e.g. Inc P2 plas
mids) - Plasmid - specified restriction, modification sys
tems (e.g. Inc N plasmids)
2. Metabolic properties
- Ant ib io t ic and bac te r ioc in production ( e .g . by Streptomvces, Escher ichia , Pseudomonas, Baci l lus , Streptococcus)
- Metabolism of s inp le carbohydrates ( e . g . l ac tose , sucrose, raffinose)
- Metabolism of coirplex carbon compounds (e .g . octane and other n-alkanes, p or m-xylenes, p or m-toluenes, cartphor, nicoline)
- Metabolism of p r o t e i n s (e .g . case in , gelat in) - Metabolism of opines (by Ti^ Aqrobacterium) - Nitrogen f ixation (by Rhizobivim)
17
- S"-Endotoxin by sporulating B. thurlnqlensls - Other properties (e.g. citrate utilization, H2S production, leucine biosynthesis)
3. Properties contributing to pathogenicity or sygribiosis
- Antibiotic resistance and bacteriocin production - Toxin production (e.g. enterotoxins of Escherichia coli and Staphylococcus aureus, exfoliative toxin by S.aureus, haemolysins by Escherichia, Staphylococcus and Streptococcus)
- Colonization antigens of E.coli (e.g. K88, K89, CFAI, CFAII) "•
- Tumorigenicity (by Ti Aqrobacterium) - Host specificity (of Aqrobacterium and Rhizobium) - Nodulation (by Rhizobium)
4. Conluqal properties
- Sex pili and associated sensitivity to pilus-specific phages
- Surface exclusion - Response to and inhibition of pheromones (in Streptococcus)
- Fertility inhibition
5, Replication-maintenance properties
- Sensitivity to curing agents -, Inconpatibility - Host range - Copy number - Tenperature-sensitive replication
6, Other properties
- Gas vacuole formation in Halobacterium - Interference in sporulation of Bacillus pumilis - Sensitivity to bacteriocins (in Aqrobacterium and Streptococcus)
- Translucent-opaque colony change in Mycobacterium - Regulation of melanin production in Streptomyces
Adopted from "Methods in Microbiology" (1984), Bennejt, P.M. and Grinsted, J. (eds.). Academic Press.
18
especially inportant in medicine because genes for clini
cal traits such as drug resistance and virulence factors
are frequently present on plasmids (Couturier £i 5 .«
1988), Three major classes of plasmids have been charac
terised most extensively, which include conjugative (F)
plasmids, bacteriocinogenic plasmids and drug resistance
(R) plasmids. The F plasmid is the classic fertility
factor that is capable of its own transfer, the transfer
of other plasmids and the transfer of the host bacterial
chromosome during bacterial conjugation, Col-plasmids and
R-plasmids in contrast mediate the production of colicins
and confer drug-resistance to their bacterial host cell
respectively (Womble and Rownd, 1988).
Some authors have classified R-plasmids on the
basis of (a) their transfer by conjugation (b) inconpati-
bility, and (c) curing or elimination (Novick, 1969;
Couturier e_t aJ,., 1988) .
R-Pla?mid3 and their Salient Features
The best known plasmids from the stand point of
the human medicine are those that specify antibiotic resis
tance (Eiwell and Shiply, 1980) , R-plasmids in bacteria
have resistance markers, either for one antibiotic (Thom
son and Biigeri, 1982; Hirsh et al ,, 1989) or for more
than one antibiotics (Hardy and Haifeli, 1982; Amyes, 1989
19
Hirsh et al,., 1989). R-plastnid mediated antibiotics
resistance in enteric bacteria also referred to as mul
tiple antibiotic resistance or infectious antibiotic
resistance is known ever since its discovery in Japan
in 1959 (Akiba, 1959; Feary et aJ,,, 1972; Womble and
Rownd, 1988). The R-plasmids are most common in bacteria
from clinical and veterinary sources where exposure to
antibiotics is likely to be high (Smith, 1977; 0 Brien
et al,, 1986), Many R-organisms especially from human
origin, would be expected ultimately to enter the sewage
(Smith, 1970). Many authors have reported their occurre
nce at low frequency In bacteria from water and other
sources (Sizemore and Colwell, 1977; Talboot et al.,19e0).
Besides antibiotic resistance, R-plasmids can often
confer certain additional properties on their host cell
and can be accordingly characterised. Some of the proper
ties include resistance to heavy metals and their salts
like arsenate, arsenite, nickel and cobalt ions (Smith,
1967; Dabbes and Sole, 1988), Plasmids also confer resis
tance to UV radiations in Streptococcus, Pseudomonas and
Enterobacteriaceae (Jacob et al ,, 1977; Clewell, 1981) ,
In addition to their resistance properties some other
properties of R-plasmids Include : fertility inhibition
to a male bacterium, propagation of specific phages,
phage restriction, colicin production, colicin resistance.
20
plasniid transfer, plasmid inconpatibility, plasmid insta
bility and plasmid curing (Siccardi, 1966; Meynell et al.*
1968; Bannister and Qlover, 1968j Novakova, et al.., 1969|
Khatoon et a_l.. # 1972; Stanisich, 1984; couturier et al.,
1988; Womble and Rownd, 1988; Hirsh et al.., 1989; Jewell
and Collins-Thoitpson, 1989) .
Plasmid encoded resistance »
(i) Metal resistance t Heavy metal resistance in bacteria
is frequently coded by genes located on plasmids and trans-
posons and is often transferable integenerically, inter-
specially and also from iri situ microflora to indigenous
microflora (Datta and Hedges, 197 2; Smith, 1978; Bale
et £l., 1988). The metal resistance is often associated
with resistance to single or multiple drugs (Hall, 1970;
Kondo et al ., 1974; Fostor, 198 3), phenolic compounds
(Pickup et al,., 198 3) and pesticides (Don and Parriberton,
1981) .
The concentration of metal pollutants in the envi
ronment is usually low excepting in specific areas which
are polluted by various hospital and industrial wastes.
However, unpolluted environments may also harbour metal
resistant organism or organisms that readily adapt to high
concentration of metals. The incidence of plasmid bearing
strains is more in polluted site than in unpolluted region
(Hada and Sizemore, 1981) .
21
Resistance to toxic metals such as mercury, arsenic
and cadmium is frequently plasmld encoded in both gram
positive and gram negative bacteria (Summers and Silver,
1978; Tynecka et al,, 198l7 Mobiey and Rosen, 198 2).
Trevors et al.. (1985) have reported four basic mechanisms
by which plasmids encode resistance against metals and
these are -
i) inactivation of the metal
ii) alteration of the site of inhibition
iii) impermeability of the metal
iv) metal by pass mechanism
Plasmld determined mercury and orqano-mercury resistance ;
Mercury resistance determinants are common, among the
naturally occurring and clinically isolated bacterial
populations and are frequently found on plasmids (Trevor's
et al., 1985; Summers, 1986; Foster, 1987; Silver and
Misra, 1988) .
The earliest observation of the presence of bacteria
with strong resistance towards mercury and organomercurials
came from Japan where resistance bacteria were isolated
from organomercurlal polluted soil (Vaczi e^ al.., 1962;
Tonomura and Kanzaki, 1969) and from the united kingdom
among the penicillinase producing clinical isolates of
S. aureus (Richmond and John, 1964; Dyke et al,., 1970) and
subsequently, it was described in Escherichia coli (Smith,
1967) .
22
Many of the antibiotic resistance plasmids in both
gram negative and gram positive bacteria have the genes
2+ to determine resistance to Hg (Novick and Roth, 1968;
Schottel et al_., 1974; Summers and Jacoby, 1978) . In the
Hammersmith hospital London, in a collection of over 800
plasmids transferred into a common host, E.coli K12 from
a wide range of host species including, Proteus, Provi-
dentia. Salmonella, Serratia, Shigella and Klebsiella,
the frequency of genetic determinants of mercury resis
tance was about 25% (Schottel et ad., 1974) .
Plasmid determined cadmium resistance : Genes resljstant
to cadmium are located on both plasmids and chromosomes
(NovicK and Roth, 1968; TynecKa et al,., 1981; Surowitz
1984), In S.aureus, cadmium resistance is attributed to
reduced uptake of Cd via Mn system (Chopra, 1971;
Chopra, 1975). Recently Silver and Kisra (i988) have
suggested six or more cadmium resistance systems in bac
teria. The first two systems involved are Cad A and Cad B
of S.aureus plasmids that also confer resistance to seve
ral other heavy metals (Perry and Silver, 198 2) .
Plasmid determined arsenic resistance j Role of plasmids
in resistance to arsenate has been studied by Silver
et al. (1981), though it has been known for years in S_,
aureus (Novick and Roth, 1968) and Escherichia coli (Hed
ges and Baumberg, 1973). The plasmids involved are pl524
23
and R773. Arsenite resistance is associated with anti
biotic (Smith, 1978; Summers et ad,, 1978) and mercury
resistance (Nakahara et al.., 1977), Silver et al_. (i98l)
have reported that piasmid mediated arsenate, arsenite
and antimony (III) resistances are coded for by an indu
cible operon like system in both , aureus and E.coli.
Mobley et al . (i983) have demonstrated that in IncF
plasmid R773 of E.coli, arsenate, arsenite and antimonate
resistance are carried by a29Kb EcoRI fragment, Beliiveau
et al, (1987) suggested that three plasmids encoded pro
teins Ars A,B and C are involved in the resistance of
E.CO1i to arsenate. Arsenite is not oxidised to the less
toxic arsenate by the plasmids of E.coli or S,aureus
(Bellieveau e_t al., 1987) ,
A 69Kb conjugative plasmid pDG 10l was found in
Corynebacterium flaccumfaciens sub spp, oortii which codes
for resistance to arsenate, arsenite and antimony (Hende-
rick et al,, 1984) ,
Other plasmid determined metal resistance t Tellurite and
tellurate resistances have been detected from plasmid
mediated determinants (Summers and Jacoby, 1911 i Taylor
and Summers, 1979). Jobling and Ritchie (1987, 1988) have
recently cloned the tellurium determinants from a large
Alcaliqenes plasmid into E.coli, where it was expressed
from 3,55 Kb DNA fragment.
24
Resistance to cobalt and nickel has been reported
in 12 clinical isolates of Enterobacter spp, harbouring
R-plasmids (Smith, 1967) .
Some Staphylococcus aureus plasmids determine resis
tance to inorganic lead conpound (Novick and Roth, 1968),
Copper resistance is associated with an antibiotic
resistance plasmid in E.coli (Ishihara et al_,, 19 78).
(ii) Drug resistance : The increasing use of antimicrobial
agents has provided a strong selective force favouring the
survival of those bacterial strains that have acquired
resistance to such agents either by mutation or by inheri
ting R-plasmids from unknown sources (Davis and Smith,1978),
Resistance plasmids are found in wide variety of
bacterial genera including both gram positive and gram
negative organism, Plasmids may be endowed with the capa
city of self transmission (Conjugative) or non transmi
ssible (non conjugative) .
Various biochemical mechanisms have been proposed
for resistance to different antibiotics (Davis and Maas,
195 2; Davis and Smith, 1978) . These resistance mechanisms
include (a) alteration of the target site in the bacterial
cell, (b) interference with drug transport, (c) enzymatic
detoxification of antibiotics, and (d) by-pass mechanism.
In addition to these mechanism there are some unknown drug
resistance mechanisms also (Davis and Smith, 1978).
25
Some Inpor tan t Charac te r i s t i c s for Studies on the
R-plasmld Harbouring Cells
The a b i l i t y of a proper ty to be t ransfer red from
one bacterium to another through conjugation and/or t r a n s
formation provides a good presunptlve evidence of plasmid
involvement, p a r t i c u l a r l y i f the frequency of t ransfer i s
high. Transfer experiments t ha t are conducted by sirrply
mixing cu l tu res of the t e s t bacterium with a su i t ab le r e c i
p i en t s t r a i n can give r i s e to progenyby conjugation and
transformation.
(1) Con luqation »
Conjugation i s the highly spec i f i c process whereby
DNA is t ransfer red from donor to r e c i p i e n t bac ter ia by
a mechanism involving c e l l to c e l l contac t (Lederbeirg, 195 2) .
Conjugation has been detected in many members of the Entero-
bac ter laceae (Jacob e_t al. , , 1977) and in other gram negative
and gram var iable b a c t e r i a .
Almost a l l types of R-plasmlds of more than 5x10
daltons are conjugative in nature . R-plasmids are composed
of two g e n e t i c a l l y and phys ica l ly d i s t ingu i shab le compo
nents J (1) a r e s i s t ance t ransfer factor (RTF) tha t har
bours the genes for se l f t r a n s m i s s l b i l i t y ( tra) and auto
nomous r e p l i c a t i o n ( rep) , and (11) a r e s i s t ance determinant
(R-determinant) , The R-determinants harbours the majority
of r e s i s t ance genes (Clowes, 1972; Davis and Rownd, 1972) ,
26
The plasmid transfer by conjugation is possible
both within and outside the boundaries of the genus (Marniur
et al,,, 1963; Datta and Hedges, 1972; Datta, 1975). E.coli
is also reported to transfer its R-plasmids to various
pathogenic as well as non pathogenic bacteria (Baron and
Faikow, 1961; Oleson and Gonzalez, 1974; Jeanclaude et al.,
1988) .
(ii) Transformation j
This is the process by which DNA from one cell (the
donor) is taken up by another (the recipient) directly
from the surrounding medium (Cohen e_t al.., 197 2) , There
are many genera of bacteria in which transformation has
been successfully demonstrated e.g. all genera in Entero-
bacteriaceae. Bacillus. Haemophilus (Smith e_t £1./ 1981) ,
Staphylococcus (Lacey, 1975) and Streptococcus (Clewell,
1981) , Some genera of bacteria are able to undergo physio
logical transformation, but E.coli is not conpetent to
physiological transformation (Saunders et al_,, 1984).
However the E.coli can be made susceptible to take up for
eign DNA under artificial conditions like treatment with
transition metals (Ca"*"*", Rb"*"*") , with intermittent heat
shock etc. (Lederberg and Cohen, 1974; Kushner, 1978).
(ili) Plasmid instability and curing ;
One of the ways to ascertain whether or not a
particular character is associated with plasmid, the
27
el iminat ion (curing) of plasmids provides an appropriate
experimental b a s i s . The c r i t e r i o n of curing can be used
both in conjugative and non-conjugative plasmids{Khatoon
and Ali-Mohammad, 1986). I t i s expected, therefore , t h a t
in any growing population of plasmid harbouring bac t e r i a ,
p lasmid- less segregants w i l l occasional ly be produced as
the e r ro r in the process of plasmid r e p l i c a t i o n or pajfti-
t ioning to daughter b a c t e r i a . Thus t h i s loss of plasmid,
or curing can occur e i t h e r spontaneously (Novick, 1969)
or under the influence of the physical ( s t ad l e r and Adel-
berg, 197 2) and chemical agents (Novick, 1969; Stanis ich
1984) . Some of these curing agents can mutate DNA (Will-
e t t e s , 1967) , These agents are ind iv idua l ly e f fec t ive
only aga ins t some plasmids, and t h e i r e f f ec t on newly
discovered plasmid cannot be predicted (Mitsuhashi e t a l . ,
1961; S tan is ich , 1984).
The genera l ly used curing agents are acr idine
dyes (Hirota, 1956; HirOta and l i j lman, 1957) , ethldium
bromide (Bounchaud e t al, . , 1969; Jones and Sneath, 1970;
Hardman e t al,, , 1986; Khatoon and Ali-Mohammad, 1986;
El-Syed e^ SJL»» 1988), many mutagens (Wi l l e t t e s , 1967;
Chakrabarty, 1972), some a n t i b i o t i c s (Ikeda e t al^., 1967;
Yoshikawa and Sevag, 1967; Hahn and Ciak, 1971; Lacey,
1975; McHugh and Swartz, 1977; Molnar e^ a_l,, 1978;
Selan e_t al_,, 1988), chemicals l ike sodium dodecyl s u l -
28
phate (Tomoeda et ed., 1968; Inuzaka et a^., 1969;
Tomoeda et ad., 1970), metal ions like Ni, Co (Hirota,
1956) and also some physical agents like heat (May e_t al.,
1964; Stadler and Adelberg, 1972; Jayaratne et al,, 1987) .
Classification of Bacteria and their Taxonomic Studies
The classification of organisms or their taxonomy
has two purposes : one is to group the organisms of same
kind, the descriptive classification and the second pur
pose is to provide a natural phylogenetic classification.
In the descriptive classification, organisms have been
grouped on the basis of information collected about a given
kind, that can be pooled and conpared. This type of class
ification is based on the characters of the organisms
which a taxonomist can enploy (Mandel, 1969) ,
Bacteria have been classified by microscopic exa
mination on the basis of shape, size and staining charac
teristics. Bacteria also possess certain characteristic
structures such as the capsules, flagella, spores, etc.
whose presence or absence is used as a tool in identifi
cation. Morphological, cultural and colony characteristics
provide useful information about the different organisms
but these can not be used as the sole criteria for class
ification. Further, biochemical, serological and specific
tests are often required to be performed before bacteria
are cleariy identified (Pelczar et al.., 1986).
29
Tabic 2 J 'lnx<tn<)inic Rela t ionships of (he F.u(cr<»l)ac(criacc'ac
Tribe
llsi.hcrichic.ic
ildvk,ini!>icllc.ic
S.ilnioncllc.ic
KlehMcllciic
I'rotccac
Ycn>m)C3c
[lr^^lnlC.lC
Genus
l.schfnchia
.Shij^'fllii
I'llwtirdsielta . .
r Salmonella
Arizona
r
Ciirobacter
Klebsiella ..
Enierobacter.
Hafnia.
Serrano
Proteus
Provideniia.
[_ Morganella.
Yfrsinia
Erwiniu
I'ectinohacienitm
Species
/. col,
S dvseniernie S flexnen S hovdii S ionnei
E tarda
S. cholerasut^ S. typhi S. enteniidi-s
A. /iias/iuvMi
C frcundn C divcrsus C anialonaiiciis
i' •>i\c,iii\i)n\ae h ozacnae K rhinoscleromaiis K oxyioca
E aerogenes E cloacae E. agfitomeraiis E. saKdsakii
[_ E. gergoviae
H alvei
S. marcescens S liquifaciem S. rubtdaca
P. mirabdis P. vulgaris
P. reltgeri P. sluartii P. alcaliquifaciens
M. morganii
Y. pesiis Y pieudoiubcrculoiis Y. enterocolitica y. ifiwrmedia Y frcdenksenii Y. ruckeri
Adapted from ihc projiDs.ils in Brenner,_c/ d/_. , ( 1 9 7 7 )
30
A c l a s s i f i c a t i o n system was given by Kaufman and
his co l labora tors in 1940s. In th i s system E,col i was
subdivided on the bas is of antigen l i k e 0, K and H (Vahlne,
1945; Kaufman, 1975) , In 1976, an analys is of OjH se ro
types and biotypes was used for c lass i fy ing E.col i causing
diarrhoeal diseases (OrsXov e t al_., 1976) , Numerous taxo-
nomists continue to use serotyping and biotyping for
c lass i fy ing bac te r ia , not s o l e l y because of t r a d i t i o n ,
but these r e s u l t s also allow ready pigeon holing of E ,co l i
i s o l a t e s and often c o r r e l a t e well with disease s p e c i f i c i t y .
A successful taxonomic analys is of E .co l i population
s t ruc tu re s would include quan t i t a t i ve est imates of the
re la tedness between b a c t e r i a l groups. This might yield
valuable ins ights into microbial ecology and pathogenic i ty
(Achtman and Pluschke, 1986),
Many c l a s s i f i c a t i o n methods have been used in E .co l i
taxonomic s t u d i e s . Among these , s tarch gel e l ec t rophore t i c
analysis of cytoplasmic isoenzymes, a c l a s s i c a l technique
in the taxonomy of eucaryot ic organisms has been ex tens ive
ly used. Other methods based on r e s i s t ance to co l i c ins
and other toxic chemical, DNA-DNA hybr id iza t ion or seque
nce analys is of rRNA or p ro te ins have a lso been employed
in taxonomic s tudies (Achtman and Pluschke, 1986) ,
Moreover, SDS-PAGE CMP (Outer Membrane Protein)
p a t t e r n s and OiK serotypes have also been used to c l a s s i f y
31
E.coll from epldendologlcal sources into various groups
(Paahkanen e^ al,., 1979; Jaan and Jaan, 1980).
Isoenzyme analysis (Ochman and Salander, 1984) ,
OMP analysis and serotyping collectively defined E.coli
into 15 different clones (Achtman and Pluschke, 1986).
Whereas, it was typed into 160 0 groups, by Orskov and
Orskov, (1984). The 0 serotyping is based on the reacti
vity of the somatic 0 sugar chains of LPS (lipopolysaccha-
ride) with hyperimmune rabbit antigens.
Restriction of phages is a property associated with
sorrte plasmids including the R-plasmids (Siccardi, 1966) .
Plasmids also interfere with phage propagation (Duckworth
et al., 1981). These two properties of phages with their
bacterial hosts have been exploited for the grouping of
the bacteria like E.coli (Meynell et al., 1968).
Phage typing of certain bacterial strains is some
times difficult and confusing for examples S_, aureus
strains are not typable by bacteriophages of international
typing set. Supplementary bacteriophages therefore have
been isolated to distinguish these non typable strains of
S. aureus (Vickery et al.., 1983; Richardson et al_,, 1988) ,
Antibiotic sensitivity patterns or antibiograms
have been used for the classification and grouping of
bacterial strains by a number of workers. This typing
32
method is faster and more cost effective than biochemical
analysis and bacteriophage typing (Gillespie et al.,
1990). E.coli isolated from porcine fecal origin, patients
of urinary tract infection and patients from Egypt have
been divided into various groups on the basis of their
multiple antibiotic resistance pattern or antibiograms
(Ohmae et al.., 1980; Elkhovly, 1981; Gees et al., 1982).
Molecular Genetic Approach to Bacterial Taxonomy :
The approaches like serotyping, biotyping, phage
typing and numerical taxonomy described for the classifi
cation have ordered bacteria rather tentatively, in terms
of phenotypic traits. The development of molecular gene
tics, however has now revolutionised taxonomy. This taxo
nomy is based on macromolecular sequence homology (Brenner
and Falkow, 1971) .
The sinplest comparison is the base conposition of
DNA, This can be estimated easily from melting tenperature
or buyont density. The base corrposition is essentially the
same in all vertebrates about 40 moles per cent guanine,
cytosine [G-^-C) and 60 per cent adenine, thymine, (A+T) .
But the base conposition among bacteria vary remarkably
from about 30-70 moles per cent (Marmur et al_., 1963).
DNA sequence homology (DNA-DNA homology) can be
measured quantitatively in terms of the ability of the
33
DNA strand from two different sources to form molecular
hybrids iji vitro. Among bacteria, DNA-DNA hybridisation
is useful only within closely related groups because it
quickly vanishes in the broad range of variation between
more distant organisms, Ribosomal RNA hybridisation to
DNA is useful for estimating more distant kinship among
bacteria. These processes have been employed in descrip
tive classification of bacterial strains (Brenner and
Falkow, 1971; Schiffer and Stackebrandt, 1983).
DNA-DNA hybridisation of R-plasmid harbouring
airpicillin resistant gram negative bacterial strains was
used for their characterisation and classification. All
of the anpicillin resistant gram negative strains demons
trated sequence homology (Foek-Ruediger and Rainer, 1982).
Since the antibiotic resistance in bacteria is
usually conferred by plasmids. Plasmid profiles have
therefore, been used for identification and corrparison
of bacterial strains in addition to antibiograms and phage
typing, Eiectrophoretic patterns of plasmid were used for
bacterial identification. 7 2 E.coli strains conferring
resistance to multiple antibiotics have been shown to
contain single type of plasmid by their eiectrophoretic
patterns (Ohmae et al ., 1980). Similarly 5-6 plasmids
ranging from 30-70 Mdal, have been isolated from 30 stra
ins of bacteria (Vakulenko et ad,, 1980) .
34
Antibiograms, plasmid profiles and phage typing
have been jointly used for the classification of E.coli,
Salmonella typhimurium and Staphylococcus aureus strains
from clinical origin (Frost et al., 1982; Martinez et
al., 1987; Gillespie et al,., 1990).
Restriction endonuclease fragmentation of DNA or
plasmids and molecuxar size differences have also been
used for taxonomic studies of various bacterial spp.
(Achtman and Plusckhe, 1986).
Hardman et_ al.. (1986) have used restriction frag
mentation and molecular size difference of plasmids for
the characterisation and classification of four Pseudomonas
and two Alcaliqenes species. Shinji and Yokiko (1988) have
classified E.coli strains by corrparing their plasmid pro
files and the restriction fragments of these plasmids.
Burnie et al, (1989) have used EcoRI restriction enzyme
fragmentation patterns and immunoblotting to differentiate
and classify methicillin resistant isolates of Staphylo
coccus aureus.
Objectives of the M.Phil work :
As we have already defined our problem in preface,
the present literature survey enlightened us to initiate
the work on the following lines j
1. Estimation and Enumeration of bacterial densities viz.
35
TBC, TEC, TC and FS in sewage and soil in the neigh
bourhood of Industrial area of Aligarh,
2. Correlation between the bacterial population from domes
tic sewage water and industrial effluents as well as
from soils of the same area,
3. Prevalence of antibiotic resistance if any, in entero
bacterial population of sewage and soil origin.
4. Prevalence of metal resistance in enterobacterial popu
lation derived from soil and sewage water.
5. Incidence of multiple antibiotic and metal resistance,
if any, in the enterobacterial population.
6. Evaluation of correlation between the drug and metal
resistant enterobacterial population in domestic sewage
water and industrial effluents as well as that between
soils and sewage.
7. Studies on the involvement of plasmids in mediating
such resistances and their possible transfer by con
jugation and transformation.
8. Confirmation of the role of plasmids in displaying the
test resistance characteristics, exploiting the pheno
menon of curing of harboured cells under certain
conditions,
9. Isolation and characterisation of R-plasmids from the
above mentioned isolates by agarose gel electrophoresis,
10. Determination of molecular size using known plasmid
markers and DNA fragments.
MATERIAL AND METHODS
36
MATERIALS
MEDIA
Azlde dextrose broth (pH 7.2)
Peptone i 15 g/1
Beet extract i4.5 g/1
Dextrose t7,5 g/1
Sodium chloride jO,2 g/1
Sodium azide »0.2 g/1
EMB agar (pH 7. 2)
Peptone t 10.0 g /1
Lactose : 5.0 g /1
Sucrose ; 5.0 g /1
Potass ium phospha te t 2.0 g /1
Agar X 13.5 g / 1
Eosin Y J 0.4 g / 1
Methylene Blue » 0 , 0 6 5 g / l
MacConkey's agar (pH 7.1)
p a n c r e a t i c d i g e s t of g e l a t i n t 17.0 g / 1
P a n c r e a t i c d i g e s t of ca se in j 1.5 g / 1
Tissue i 1.5 g / 1
Bi le s a l t s i 1.5 g /1
Sodium c h l o r i d e : 5.0 g / 1
Agar 1 15.0 g / 1
Neutra l red » 30.pmg/l
Crys t a l v i o l e t ; l.Omg/1
37
MacConkey's broth (Double Strength) (pH 7.4)
Peptone . 40.0 g/1
Lactose : 2O.O g/1
Bile s a l t s ; 10.0 g/1
Sodium chlor ide • 10.0 g/1
Neutral red ; 0 . i 5 g / l
MacConkey's borth (Single strength) (pH 7.4)
Peptone , 2O.O g/1
Lactose , 10.0 g / i
Bile s a l t s ; 5,0 g/1
Sodium chlor ide • 5.0 g/1
Neutral red » 0.075g/l
Nutr ient agar (pH 7.4)
Peptone , 5.0 g/1
Sodium ch lor ide $ 5,0 g/1
Beef ex t r ac t • 1.5 g / i
Yeast e x t r a c t • 1,5 g / i
Agar » 15.0 g/1
Nutr ient broth (pH 7.4)
Peptone j 5.0 g/1
Sodium chlor ide ; 5,0 g/1
Beef e x t r a c t . 1,5 g / i
Yeast e x t r a c t j I .5 g / i
38
SIM agar (pH 7.3)
Peptone
Beef extract
Ferrous ammonium sulfate
Sodium thiosulfate
Agar
Simmons citrate agar (pH 6.9)
Ammonium hydrogen phosphate
Dipotassium phosphate
Sodium chloride
Sodium citrate
Magnessium sulfate
Agar
Bromo thymol blue
Soft or Top agar
Nutrient broth
Agar powder
Triple sugar iron (pH 7.4)
Peptone
Tryptone
Yeast extract
Beef ex t r ac t
La'ctose
Sacchasose
30.0 g/1
3.0 g/1
0.2 g/1
0.2 g/1
3.0 g/1
: 1.0 g/1
: 1.0 g/1
J 5.0 g/1
; 2.0 g/1
i 0.2 g/1
. 15.0 g/1
: 0.08g/l
: 13.0 g/1
: 7.0 g/1
t 10.0 g/1
» 10.0 g/1
* 3.0 g/1
» 3.0 g/1
s 10.0 g/1
: 10.0 g/1
39
Dextrose i 1,0 g/1
Ferrous sulphate : 0.2 g/1
Sodium chloride j 5,0 g/1
Sodium thiosulphate j 0,3 g/1
Phenol red : 0,024g/l
Agar ; 12.0 g/1
Trypticase soy broth (pH 7. 2)
Trypticase : 15.0 g/1
Phytene x 50.0 g/1
Sodium chloride t 5,0 g/1
REAGENTS AND BUFFERS i
Alkaline SDS
SDS ; 1.0 %
NaOH I 0,2 N
Ammonium acetate Solution
Ammonium acetate i 0,1 K
Barrits reagent
Soln. A ; Alpha-naphthol : 5,0 gm
Ethanol (absolute) : 5.0 ml
Soln. B : Potassium hydroxide : 40 gm
Calcium chloride solution
Calcium chloride : O.l M
40
C r y s t a l v i o l e t
C r y s t a l v i o l e t ( 8 5 % d y e c o n t e n t ) ; 1,0 gm
D i s t i l l e d w a t e r : lOO ml
D e c o l o r l z e r
E t h a n o l (95%) ; 250 mi
A c e t o n e ; 250 ml
E l e c t r o p h o r e s i s b u f f e r ( T r l s - a c e t a t e , pH 8 , 0 )
T r l s J 9 . 7 gm
EDTA : 0 . 7 gm
A c e t i c a c i d ( g l a c i a l ) j 2 . 2 8 ml
D i s t i l l e d w a t e r j 197 ml
G r a m ' s I o d i n e
I o d i n e c r y s t a l s . 1,0 gm
P o t a s s i u m I o d i d e j 2 . 0 gm
D i s t i l l e d w a t e r , 30O ml
K o v a c ' s r e a g e n t
p - D l m e t h y l a m i n o b e n z a l d e h y d e ; 5 , 0 gm
Amyl a l c o h o l : 7 5 . 0 ml
HCl ( c o n c e n t r a t e d ) j 2 5 , 0 ml
L y s i s b u f f e r
T r i s - c l (pH 8 , 0 ) i 2 5 . 0 mM
EDTA i 10 mM
G l u c o s e . 50 mM
L y s o z y m e , 2 i r g /ml
41
Marker dye
Glycerol
Bromophenol blue
EDTA J
Methyl red s o l u t i o n
Methyl red ;
Ethyl a l coho l (95y«) :
Reagent A
TE buffer c o n t a i n i n g
5% dow co rn ing ant ifoam RD emuls ion .
Reagent B
1 M NaOH saturated at 20°C
with SDS. Distilled water ;
Safranin
Safranin 0
(25% solution in 95% ethyl alcohol);
Distilled water ;
Sodium acetate solution
Sodium acetate ;
CHEMICALS
The following chemicals were used
; 50.0 %
I 0.05 %
J 40 mM
; 0.1 gm
: 300 ml
200 ml
10 ml
100 ml
3.0 M
Chemicals Source
Acetic acid BLH, India
42
Agarose
Agar powder
Anpicillin
Ammonium acetate
Antifoam RD emulsion
Azide dextrose broth
Butanol
Calcium chloride
Chlorai tphenicol
Dipotassium phosphate
Ethylene d i a m i n e t e t r a a c e t a t e
Eosin methylene b lue agar
Glucose
Glycerol
Kanamycin
Lambda DNA
Lysozyme
KacConkey's agar
MacConkey's b r o t h
Kagnessium s u l p h a t e
N u t r i e n t agar
N u t r i e n t b r o t h
Potass ium a c e t a t e
Potass ium dihydrogen
Sigma, USA
Hi-Media, I n d i a
Ranbaxy, I n d i a
Hi-Media, I n d i a
Hopkins and Williams,
England
HI-Media, India
Sisco Laboratories, India
Sisco Laboratories, India
Ranbaxy, Indid
BDH, India
sd Fine Chemicals , Ind ia
Hi-Media, India
Hi-Media, India
Hi-Media, India
Ranbaxy, India
Sigma, USA •
Sisco L a b o r a t o r i e s , Ind ia
Hi-Media, I n d i a
Hi-Media, I n d i a
E. Merck, I n d i a
Hi-Media, I n d i a
Hi-Media. I n d i a
Ci ty Chemicals Corpn. , USA
BDH, I n d i a
43
ortho phosphate
Sodium acetate
Sodium citrate
Sodium chloride
Sodium hydroxide
Sodium thiosulphate
Simmons citrate agar
SIM agar
Sucrose
Streptomycin
Tetracycline
Tris HCl
Triple sugar i ron agar
Trypticase soy broth
BDH, India
Loba Chemicals, India
BDH, India
Sisco Laboratories, India
sd Fine Chemicals, India
Hi-Media, India
Difco, USA
Qualigens fine Chemicals,
India
IDPL, India
Pfizer Ltd., India
Sigma, USA
Hi-Media, India
Difco, USA
Note } The chemicals which have not been included in this
list were of analytical grade.
44
Following E .co l l s t r a in s have been used in t h i s study
S t ra in Relevant genet ic d e s i g n a t i o n marke r s
Source
AB1157
EcRp
SWi,
SWd.
swd
S S i
SSd.
SSd.
t h i - 1 , a r g E ~ 3 , t h r - 1 , l e u B 6 , p roA2 , h i s G 4 , l acYl ,F- , S^-, X
r T r r r r A^,B , C t ^ E ' - ^ P b ,R S ^ , S z ^ , H g ^ , C d ^ , 2 n ^ , P b ^ / l a c A ^ , B ^ , C t ^ , E ^ , F r ^ , K ^ R ^ , s r , S z ^ , H g ^ , C d ^ , Z n ^ , P b ^ , l a c
A ^ , A k ^ , B ^ , C t ^ , E ^ , F r ^ , N ^ R ^ , S z ^ , H g ^ , C d ^ , E n ^ , P b ^ , l a c
A ^ , B ^ , C ^ , C t ^ , E ^ , F r ^ , S z ^ H g r , c d ^ , Z n ^ , P b ^ . l a c
A ^ , B ^ , C t ^ , E ^ , F r ^ , S ^ , S z ^ H g ^ , C d ^ , Z n ^ , P b ^ , l a c
A ^ , B ^ , C t ^ , E ^ , F r ^ , K ^ , N ^ ,
l a c
Srivastava, B. S,
Srivastava, B.S.
Isolated from industrial effluents
Isolated from domestic sewage
Isolated from domestic sewage
Isolated from soils of industrial area
Isolated from soils in the proximity of domestic sewage drains.
Isolated from soils in the proximity of domestic sewage drains.
(r = resistant, s = sensitive)
45
Collection of Sanples
Water Sairples j
Water sanples were collected routinely from the
different drains of the Aiigarh city including industrial
estate (Table III), The sampling sites were severely
affected by human and industrial activities. Water sanp
les were collected in 250 ml sterilized glass bottles and
transferred to the laboratory immediately for initial
processing. The elapsed time between the collection of
sanple and initial processing did not exceed 2 h.
Soil samples :
The soil sanples were collected in sterilized
container with the help of sterilized scapula. The sar-ple
were taken at a depth of 6 inch from the upper surface of
soil (Table lH) .
Isolation and Identification of Enterobacteria from Water
and Soil
A variety of media were enployed. The isolation of
E.coli and Enterobacter aeroqenes was done according to
the standard methods described in APHA (1985). 10 ml ali-
quots was taken in double strength MacConkeys broth and
incubated at 37°C for 24 h. Furthermore the sanples indi
cating acid and gas production was spread on eosin methyl
ene blue (EMB) agar plates and incubated at 37°C for 24 h.
Some colonies were small showing green metallic sheen and
46
Table III
S.No. Test sairples Various sources of the sarrples collected for the isolation of E.coli and Enterobacter aeroqenes
Water Samples
1. SW Muddy and dirty water from the drain near V.M, Hall crossing,
2. SW Sewage water from the Jamalpur drain, 2 Aiigarh.
3. SW Sewage water from the Quwarsi drain, ^ Aiigarh.
4. SW. Blackish sewage water near Herkut 4 Udyog Industrial area, Aiigarh
5. SW Brownish dirty water near ITI, Industrial area, Aiigarh
6, SW Stagnant water from the Aiigarh drain,
near GT Road, Aiigarh.
Soil Sarrples
7. SS Soil from agricultural field near
V.M. Hall crossing, Aiigarh 8, SS Soil from agricultural field near 2 Jamalpur drain, Aiigarh.
9. SS Soil from agricultural field near • Quwarsi drain, Aiigarh.
10. SS Soil, near Herkut Udyog, Industrial area, Aiigarh.
11. SS Agricultural field soil near ITI, Industrial area, Aiigarh.
12. SSg Soil from agricultural field near GT Road in the vicinity of Aiigarh drain Aiigarh.
47
others were large pinkish mucoid colonies. The colonies were
picked and grown individually in nutrient broth at 37°c for
12 to 15 h (Martin and Washington, 1980).
100 gm of soil in a beaker were taken and suspended
in an equal amount of normal saline solution. The soil sanples
were homogenised in a waring blender for about 5 to 10 min,
and then 0.5 ml of the supernatent to culture media were
plated and incubated at 37 C for 12 to 16 h, the suspected
colonies were picked up and subcultured on different selec
tive media for the isolation of pure cultures of entero-
bacteria e.g. E.coli and Enterobacter aeroqenes (Martin and
Washington, 1980) .
MPN count from soil was done according to the method
of Topping (l9 38) .
Gram's Staining »
The gram's staining of all E.coli and Enterobacter
aerogenes was done according to the standard procedure of
Cappucino and Sherman (1987).
Biochemical Reactions ;
The isolated E.coli and Enterobacter aeroqenes were
finally identified on the basis of their biochemical proper
ties and enzymatic reactions m the presence of specific
substrates. The IMViC series of reactions were performed for
E.coli and Enterobacter aeroqenes according to the method of
Cappucino and Sherman (i987) ,
48
Determination of Minimum inhibitory Concentrations (MIC5)
of Heavy Metals
This was done by plate dilution method. The metals
of, Hg, Cd, Pb and Zn were used as HgCl , CdCl , Pb(CH-COO)
and ZnCl , varying in concentrations from 3.25 ug/ml to
6400 jug/ml and were supplemented in nutrient agar which were
then spot inoculated with overnight broth culture of different
test strains of E.coli and Enterobacter aeroqenes. The
plates were incubated at 37°C for 24 h. The sensitivity
and resistance criteria were established in accordance with
the data of Joly e^ al_, (1976), Austin e^ al.. (1977), Marques
et al . (1979) and Ahmad and Yadava (i988) .
Antibiotic Sensitivity test
All the isolates of E.coll and Enterobacter aeroqenes
were tested for sensitivity to antimicrobial agents by
means of disc diffusion method enploying multidiscs (Bauer
et al., 1966; Coleman et .., 1985), This test was carried
out by mixing 0,3 ml of fresh exponential culture of the
test strains with 3.0 ml of molten top agar (held at 45°C) .
This mixture was layered over a freshly prepared nutrient
agar plate and allowed to set for about half an hour. The
antibiotic discs were placed on the agar layer using steri
lised forceps and the plates were Incubated overnight at
37°C. The zone of growth inhibition around the antibiotic
discs were measured and the results recorded. The following
49
antibiotic discs (all from HI Media) were used. Concen
tration of the antibiotics used was in micrograms per disc,
except where otherwise is stated. The symbols and concen
trations of respective antibiotics are given in the paren
thesis I Amikacin (Ak 30) , Anpicillin (A 10), Bacitracin
(B 10 units), Chloranphenicol (C 30), Chlorotetracycline
(Ct 30) , Erythromycin (E 15) , Furazolidone (Fr 15) , Genta-
mycin (G 10) , Kanamycin (K 30) , Nalidixic acid (Na 30) ,
Neomycin (N 30), Peniciilin-G (P 10 units), Polymyxin B (Pb
300) , Rif anpicin (R 5) / Streptomycin (S 10) , Sulphadiazine
(Sz 30) , Tetracycline (T 30) , These antibiotics were selected
on the basis of their common use by the humans and domestic
animals.
Isolation of Plasmids
The isolation of plasmid from satisfactory cleared
lysate from any bacterial species is frequently a combina
tion of skill, luck and patience, especially if large plasmids
are to be isolated. Plasmids were routinely isolated from
E.coli isolates of soil and water. The mlniprep method was
followed essentially as described by Birnboim and Doly (l979),
Determination of Solecuia Weight
The plasmid size estimates of the isolated plasmid
were obtained by conparing their relative mobilities on
agarose gel with standard known molecular weight plasmid
50
and XDNA fragments. The standard curve was drawn by p l o t t i n g
r e l a t i v e mobil i ty Vs log of the molecular weight of the s t an
dard markers. The molecular weight of the unknown plasmids
was determined by p lo t t i ng the r e l a t i v e mobil i ty on the s t an
dard curve (Ohman, 1988) .
Agarose Gel Electrophoresis
The i so la ted plasmids were charac te r i sed by agarose
gel e lec t rophores i s according to the standard procedure of
Maniatis e t a l . (1982). About 30 to 40 Jul f rac t ion along
with g lycerol dye mixture (50% glycerol , 0.25% bromophenol
blue) were applied on the s l o t s . Horizontal s lab gel e l e c t r o
phoresis was car r ied out using 1% agarose. The gel was p r e -
electrophoresed a t 40 mA for 20 nriin and the normal run was
performed a t 20 mA in e lec t rophores i s buffer (0.004 M T r i s -
ace ta te , 0.00 2 M EDTA, pH 8.0) for 3 to 5 h. The DNA bands
were s ta ined with ethidium broniide and f luorescent p r o f i l e
was photographed by uv i l luminat ion through photodyne UV 300
t r ans i l l umina to r .
Plasmid Transfer
Conjugation ;
Standard method of conjugation was followed enploying
the donor, t e s t E .col i s t r a i n s and the r e c i p i e n t AB1157 (Ohman,
1988). An overnight cu l tu re of r ec ip ien t and donor E .co l i
s t r a in s were added to fresh nu t r i en t broth and incubated
a t 37 C for 30 mln and 60 mln respec t ive ly . For cont ro l , the
51
parental culture alone was added to fresh nutrient broth
and incubated at 37°C. These cultures were serially diluted
and spread on plain and antibiotic supplemented plates.
The selection was done on the basis of appearance
of plasmidial markers along with the original markers of
the parent recipient strain. The AB1157 strain is lac mutant
and also lacking antibiotic resistance markers except for
streptomycin resistance. The selection of transconjugants
was therefore based on appearance of the donor antibiotic
resistance plus non pink colonies on the MacConkey agar
plates.
Transformation t
The transfer of R-plasmid to recipient E,coli
AB1157 cells was also studied through transformation follow
ing the method of Lederberg and Cohen (1974), The overnight
grown AB1157 cells were made coirpetent by suspending in 0,1 M
MgCl for 30 min on ice and then 0,1 M CaCl under similar
conditions. To 1,0 ml cortpetent cells/ 2,0 ugm of plasmid
DNA, was added and left on ice for 30 min. Heat shock was
given to these cells at 42 C for 3 min. These cells were
then allowed to grow in nutrient broth for 2 h at 37°C,
The cells were spread on the antibiotic supplemented plates
after suitable dilutions. The plates were incubated at 37°c
for 16 h.
52
Plasmld Curing j
The curing of R-plasmids was performed by the method
described by Hirota (i960) and Hardman et al. (1986). Over
night cultures of E.coli were grown in the nutrient broth
in the presence of curing agent (ethidium bromide) at concen
tration ranging from 0.5 to 100 wgn/ml. A control tube lacking
the curing agent was also included. All the tubes were incu
bated overnight at 37 C. Contents of the tube were then
plated, after making required dilutions, on antibiotic supple
mented and plain nutrient agar plates. The plates were incu
bated at 37 c overnight and the number of colonies appeared
on the plates were then- recorded. The number of colonies
on antibiotic supplemented plates was subtracted trom that
on the plain plate.
The number of cured cells = number of colonies on
the plain plate at a specific concentration - number of
colonies on the antibiotic supplemented plate at the same
concentration.
RESULTS
53
Water samples for bacteriological analysis were
collected from six different locations. The results of
analysis in various seasons are shown in table 4. The
maximum TBC was observed during spring season in SW2
test samples while the lowest TBC was recorded in SW,
test samples during the spring season again. Total entero
bacterial count was highest (3.66x10 CFU/lOO ml) during
summer in SW^ test samples while the lowest enterobac
terial count (0.78x10^ CFU/lOO ml) was observed in SW^
test samples during winter season. Maximum number of
coliform (i.e. 1.6x10^ MPN/100 ml) was present at 3^^
location in all the seasons while the minimum counts (i.e.
0.24x10 MPN/lOO ml) was observed in SW^ test samples. In
terms of Fecal streptococci the SW and SW- locations
were maximally polluted.
Soil samples for bacteriological analysis were also
collected from agricultural fields in the proximity of
drains. The results of analysis in various seasons are
shown in table 5. A remarkably higher TBC compared with
sewage samples was invariably observed throughout the
period of study in all the locations. The total entero
bacterial counts were highest in SS. location during
spring season while it was lowest in SS. location during
winter season. As compared with sewage samples, the soil
sampling sites displayed relatively lower counts to the
54
TABLE 4 : S e a s o n a l v a r i a t i o n i n b a c t e r i o l o g i c a l p a r a m e t e r s i n t h e sewage w a t e r
P a r a m e t e r s S p r i n g Summer W i n t e r Mean sf % CV** 90 90 9 0 - 9 1
T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml
T o t a l e n t e r o b a c t e r i a l c o u n t s x l 0 3 CFU/100 ml
T o t a l c o l i f o r m x l 0 3 MPN/100 ml
F e c a l s t r e p t o c o c c i x l 0 3 MPN/lOOml
64800 72800 62400 66667 4446 6 .6
3080 3260 1360 2567 856 33 .4
1600 1600 900 1366 330 2 4 . 1
900 1600 500 1000 454 4 5 . 5
T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml
83200 69800 43800 65600 16356 24.9
Total enterobacterial counts xl03 CFU/lOO ml
2540 3660 2280 2827 599 21.1
Total coliform 1600 xl03 MPN/100 ml
Fecal streptococci 900 xl03 MPN/100 ml
1600
1600
1600 1600 0 0
500 1000 454 45.4
Total bacterial counts xl03 CFU/lOO ml
55800 58400 46800 53667 4970 9.3
Total enterobacterial counts xlO^ CFU/lOO ml
2180 2280 1580 2013 309 15.3
Total coliform xl03 MPN/100 ml
300 500 240 347 111 32.0
Contd,...
55
Fecal streptococci 130 220 130 160 42.4 26.5 xl03 MPN/lOG ml
SW. 4_
Total bacterial 12100 36200 17800 22033 10284 46.6 counts xl 03 CFU/lOO ml
Total entero- 980 1900 780 1220 488 39.9 bacterial coxints xl03 cFU/lOO ml
Total coliform 240 xlO^ MPN/lOO ml
Fecal streptococci 70 xl03 HPN/lOO ml
Total bacterial 31800 41800 39000 37800 4320 11.4 counts xl03
300
130
240
30
260
76.6
28
41
10.8
53.6
CFU/lOO ml
T o t a l e n t e r o b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml
1360 2260 1120 1580 491 3 1 . 0
T o t a l c o l i f o r m 300 300 220 2 7 3 . 3 38 13 .7 x l 0 3 MPN/100 ml
F e c a l s t r e p t o c o c c i 240 280 50 190 100 5 2 . 8 x l 0 3 MPN/lOO ml
SW, 6
T o t a l b a c t e r i a l 32600 42600 20800 32U00 8910 2 7 . 8 c o u n t s x l 0 3 CFU/lOO ml
T o t a l e n t e r o - ^^^^ 2100 176.0 1900 145 7 . 6 b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml
T o t a l c o l i f o r m x l 0 3 MPN/lOO ml
F e c a l s t r e p t o c o c c i x l 0 3 MPN/lOO ml
* Standard d e v i a t i o n
** Percent c o e f f i c i e n t of v a r i a t i o n
500
220
500
300
70
110
357
210
203
78
56.8
' 37.0
56
TABLE 5 ; S e a s o n a l v a r i a t i o n i n b a c t e r i o l o g i c a l p a r a m e t e r s i n t h e s o i l
P a r a m e t e r s S p r i n g 90
Summer 90
W i n t e r 9 0 - 9 1 Mean SD %cv
SS.
T o t a l b a c t e r i a l c o u n t s x l 03 CFU/lOO ml
T o t a l e n t e r o b a c t e r i a l c o u n t s x l 0 3 CFli/100 ml
T o t a l c o l i f o r m xlO^ MPN/lOO ml
1024000 8X8000 712000 851333 129536 15 .2
1960
24
2420
13
1860 2080
7 . 0 1 4 . 6
244 11 .7
7 .0 4 8 . 2
F e c a l s t r e p t o c o c c i x l 0 3 MPN/lOO ml
24 13 7 . 0 1 4 . 6 7 . 0 4 8 . 2
T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml
1064000 962000 886000 970667 72926 7 .5
T o t a l e n t e r o b a c t e r i a l counts x l 0 3 CFU/lOO ml
3320 3080 2460 2953 362 12 .2
T o t a l c o l i f o r m x l 0 3 MPN/lOO ml
50 160 13 74 62.4 84.0
Fecal streptococci xl03 MPN/lOO ml
SS.
2.3 5 . 1 2 . 0 3 9 . 6
T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml
698000 558 000 47600 577333 91656 15 .8
T o t a l e n t e r o b a c t e r i a l counts x l 0 3 CPU/100 ml
2280 2240 2180 2233 41 1.8
C o n t d . . .
57
T o t a l c o n f o r m x l03 MPN/100 ml
Fecal s t r e p t o c o c c i x l03 MPN/100 ml
SS,
28 30 13 23.6 7.5 32.2
3.4 4.1 0.6 15.7
Total bacterial counts xl03 CFU/100 ml
Total enterobacterial counts xl03 CFU/100 ml
Total coliform xl03 MPN/100 ml
Fecal streptococci xl03 MPN/100 ml
848000 624000 596000 689333 112775 16.4
1890
13
7
i860
7
2.3
1460 1737
4.1
19 6 11.2
2.8 31.4
2.0 50.4
Total bacterial counts xl 03 CFU/100 ml
Total enterobacterial counts Xl03 CFU/100 ml
Total coliform xl03 MPN/100 ml
Fecal streptococci xl03 MPN/100 ml
622000
244 0
742000
2680
340000 568000 168499 29.6
11
13
1520 2213
8.3
2.3 7.4
500 22.5
2.5 29.9
4.3 58.9
Total bacterial 728000 counts xl03 CFU/100 ml
Total entero- 2860 bacterial counts xl03 CFU/100 ml
Total coliform 11 xl03 MPN/100 ml
Fecal streptococci 7 xl03 MPN/100 ml
732000
3020
376000 612000 166885 27.2
22
11
2420 2767
4 12.3
7.6
254 9.2
7.4 60.0
2.5 32.0
58
t o t a l coliform in a l l the seasons. Fecal s t reptococci count
in the so i l sample was also invar iably low ra ther lowest in
a l l the t e s t samples comparing with other b a c t e r i a l counts .
Table 6 shows the loca t iona l va r i a t ion and average
bac t e r io log i ca l parameters in domestic sewage water . The
average TBC was highest in the spring season (67.9x10 CFU/
100 ml) while t o t a l en te robac te r i a l count was highest during
svimmer (3.1x10 CFU/100 ml) . The minimum TBC and TEC were 6 6
recorded in winter season i . e . 51x10 and 1.7x10 CFU/100 ml
r e spec t ive ly . As far as the en te robac te r i a l populat ion in
t h e t e s t sanples was concerned, i t was maximxjm (4.5%) in
summer and lowest (3.4%) in winter season. The proportion
of t o t a l conforms among the t o t a l b a c t e r i a l (TBC) was nearly
same (1.7%) in a l l the seasons. However the t o t a l coliforms
in the en te robac te r i a l population was maximim in winter-
seasons (52.4%) and was minimum in the summer (40.2%). The
average fecal s t reotpcocci were found t o be highest (1.1x10
MPN/lOO ml) during summer and lowest (0,3x10 MPN/100 ml)
in winter seasons.
Table 7 p resen ts the loca t iona l va r i a t i on and
average bac t e r io log ica l parameters in i n d u s t r i a l e f f l u e n t s .
The average TBC was highest in summer season (40.2x10^ CFU/
100 ml) and TEC was also maximum (2.1x10^ CFU/100 ml) in
t he same season again. As far as the r e l a t i v e proport ion
59
TABLE 6 : L o c a t i o n a l v a r i a t i o n a n d a v e r a g e b a c t e r i o l o g i c a l ~ p a r a m e t e r s i n t h e d o m e s t i c s e w a g e w a t e r
E a r a m e t e r SW. sw. SW. Mean SD
T o t a l b a c t e r i a l c o T i n t s X 10^ CFU/lOO m l
T o t a l e n t e r o b a c t e r i a l c o u n t s x l 0 3 CFU/iOO m l
T o t a l c o l i f o r m x l 0 3 MPN/100 ml
SPRING 9'0
6 4 8 0 0 8 3 2 0 0 5 5 8 0 0 6 7 9 3 3
3080 2540 ( 4 . 7 ) ( 3 . 0 5 )
1600^ 1600^ ( 2 . 4 ) ^ , ( 1 . 9 )
2180 ( 3 . 9 )
2 6 0 0 ( 3 . 8 )
F e c a l s t r e p t o c o c c i x l 0 3 MPN/lOO ml
( 5 1 . 9 )
9 0 0
* * ( 6 2 . 9 )
9 0 0
• *
SUMMER 9 0
300 1 1 6 6 . 6 , ( 0 . 5 ) ^ ^ ( l - 7 ) _
( 1 3 . 8 ) * * ( 4 4 . 8 )
130 64 3
11403
613
363
1 6 . 7
3 6 9 . 8 1 5 . 2
5 2 . 5
5 6 . 4
T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/IOO m l
T o t a l e n t e r o b a c t e r i a l c o u n t s x l 0 3 CFU/lOO m l
T o t a l c o l i f o r m x l 0 3 MPN/lOO ml
7 2 8 0 0 69800 5 8 4 0 0 6 7 0 0 0
3 2 6 0 3660 ( 4 . 4 ) ( 5 . 2 )
1 6 0 0 ^ 1 6 0 0 ^ / 2 . 1 ) ( 2 . 2 h ( 0 . 8 ) ; ( 1 . 8 ) " ( 4 9 . 0 ) ( 4 3 . 7 ) ( 2 1 . 9 ) ( 4 0 . 2 )
2280 ( 3 . 9 )
500 .
3 0 6 6 ( 4 . 5 )
1233 ,
F e c a l s t r e p t o c o c c i 1600 x l 0 3 MPN/lOO m l
1600 220
T o t a l b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml
T o t a l e n t e r o b a c t e r i a l c o u n t s x l 0 3 CFU/lOO ml
T o t a l c o l i f o r m x l 0 3 MPN/lOO m l
F e c a l s t r e p t o c o c c i x l 0 3 MPN/lOO m l
WINDIER 1 9 9 0 - 9 1
6 2 4 0 0 4 3 8 0 0 4 6 8 0 0
1360 ( 2 . 1 )
9 0 0 (1 .4):
2280 ( 5 . 2 )
1600
1580 ( 3 . 3 )
240 ,
1140
5 1 0 0 0
1 7 4 0 ( 3 . 4 )
9 1 3
( 6 6 . 2 ) * * ( 7 0 . 2 ) * * ( 3 . 6 ) ^ ^ ( 0 . 5 ) _ ( 1 . 7 )
500 500
( 1 5 . 2 )
130
* * ( 5 2 . 4 )
376
* *
6 2 0 3
580
5 1 8
65 0
8 1 5 3
392
555
174
9 . 2
1 8 . 9
42.U
5 7 . 0
15 .9
22.5
6 0 . 8
4 6 . 3
60
TABLE 7 : Locational variation and average bacteriological parameters in the industrial sewage water
P a r a m e t e r s
T o t a l b a c t e r i a l c o u n t s X 103 C F U / 1 0 0 m l
T o t a l e n t e r o b a c t e r i a l c o x i n t s x l 0 3 CFU/100 m l
T o t a l c o l i f o r m x l 0 3 MPN/100 m l
4 ^ 5
SPRING 90
1 2 1 0 0
9 8 0 ( 8 . 0 )
31800
1360 ( 4 . 2 )
2 4 0 ^ 300 .
( 2 4 . 4 ) ( 2 2 . 0 )
F e c a l s t r e p t o c o c c i 70 x l 0 3 MPN/lOO m l
T o t a l b a c t e r i a l c o i i n t s x l 0 3 CFU/100 ml
T o t a l e n t e r o b a c t e r i a l c o u n t s x l 0 3 CFU/100 ml
T o t a l c o l i f o r m
3 6 2 0 0
1900 ( 5 . 2 )
3 0 0 ^
240
SUMMER 90
4 1 8 0 0
2260 ( 5 . 4 )
, 300^
^^^6
3 2 6 0 0
1840 ( 5 . 6 )
5 0 0
( 2 7 . i f 220
4 2 6 0 0
2 1 0 0 ( 4 . 9 )
, 5°?*
Mean
2 5 5 0 0
1 3 9 3 . 3 ( 5 . 4 )
3 4 6 . 6 ^
( 2 4 . 8 )
1 7 6 . 6
4Q200
2087 ( 5 . 2 )
3 6 6 . 6 ^
SD
9 4 8 0 .
3 5 1 ,
1 1 1 ,
7 5 .
284 2
147
94
.8
, 8
,15
,8
%CV
3 7 . 1
2 5 . 2
3 2 . 0
4 2 . 9
7 . 0
- 7'°
2 5 . 7 xl03 MPN/lOO ml (0.8).. (0.7).. (1.2)_ (0.9)^^
( 1 5 . 7 ) ( 1 3 . 2 ) ( 2 3 . 8 ) ( 1 7 . 6 )
F e c a l s t r e p t o c o c c i 130 280 3 0 0 237 76 3 2 . 0 x l 0 3 MBN/100 m l
T o t a l b a c t e r i a l 17800 c o u n t s x 103 CFU/100 ml
T o t a l e n t e r o - 7 8 0 b a c t e r i a l coxonts ( 4 . 3 ) x l 0 3 CFU/100 ml
T o t a l c o l i f o r m 240 x l 0 3 MPN/lOO m l ( 1 - 3 ) ^ ^ ^ 0 . 5 ) " ^ ^ ( 0 . 3 ) " ( 0 . 7 ) '
( 3 0 . 7 ) * * ( 1 9 . 6 ) * ( 3 . 9 ) * * ( 1 4 . 4 ) * "
F e c a l s t r e p t o c o c c i 30 5 0 n o 63 3 3 . 9 5 3 . 7 x l 0 3 MPN/lOO m l
WINTER
39800
1120 ( 2 . 8 )
, 2 2 0 ^
9 0 - 9 1
2 0 8 0 0
1760 ( 8 . 4 )
,. 19*
2 6 1 3 3
1220 ( 4 i 6 )
, IZ?*
9 7 4 1
4 0 6 . 3
7 5 . 8
3 7 . 2
3 3 . 3
4 2 . 9
61
of en te robac te r i a l covint with respect t o TBC was concerned,
t he TEC was highest in the t e s t b a c t e r i a l populat ions
during spring (5.4%) and lowest in winter (4.6%). The
maximtun proportion of t o t a l colifoxm in the t o t a l bac
t e r i a l population was observed in spring (1.3%) while
t h e minimum was recorded in the winter (0.7%). The avera
ge proport ion of TC with respect to en te robac te r ia l count
was a l so maximum in spring (24.8%) and minimum in winter
again (14.4%). Fecal s t reptococci showed the maximum count
in summer (0.23x10 MPN/lOO ml) and lowest count was reco
rded in winter (0.063x10^ MPN/lOO ml) .
Table 8 gives the loca t iona l va r i a t ion and average
bac t e r io log i ca l parameters of s o i l in a g r i c u l t u r a l f i e ld s
in t h e proximity of domestic sewage d r a i n s . The average TBC
was maximum in spring (9 28.6x10 CFU/lOO ml) and minimum
in winter season (691.3x10 CFU/lOO ml) . As far as the
r e l a t i v e proport ion of en te robac te r ia in the t o t a l b a c t e r i a l
populat ion was concerned, i t was approximately same in a l l
seasons (0.3%). Total colifoxms in the b a c t e r i a l populat ions
(TBC) were maximum in suimier (0.0008%) and a minimxan during
winter (0.0001%). The r e l a t i v e proportion of t o t a l c o l i -
form out of en t e robac te r i a l counts was again maximum during
summer season (2.6%). Average fecal s t reptococci was maximum
during spring (0.01x10 MPN/lOO ml) but i t was minimiim
(0.004x10^ MPN/lOO ml) during winter season.
62
TABLE 8 : L o c a t i o n a l v a r i a t i o n and a v e r a g e b a c t e r i o l o g i c a l p a r a m e t e r s of s o i l xxi t h e v i c i n i t y of d o m e s t i c sewage w a t e r
P a r a m e t e r s SS SS SS Mean SD %CV
SPRING 90
T o t a l b a c t e r i a l 1024000 1064000 698000 928666 163921 1 7 . 6 c o u n t s x l 0 3 CFU/lOO ml
T o t a l e n t e r o - 1960 33 20 b a c t e r i a l c o u n t s ( 0 . 2 ) ( 0 . 3 ) x l 0 3 CFU/lOO ml
T o t a l c o l i f o r m 2% 50^ x l 0 3 MPN/lOO ml ( . 0 0 0 2 ) ( . 0 0 0 4 )
( 1 . 2 ) * * ( 1 . 5 ) * *
F e c a l s t r e p t o c o c c i 24 7 x l 0 3 MPN/lOO ml
SUMMER 9 0
2280 ( 0 . 3 )
28 ( . 0 0 0 4 ^
( 1 . 2 )
5
25 20 ( 0 . 3 )
34 * ( . 0 0 0 3 ^ ^
( 1 . 3 )
12
5 8 0
1 1 . 4
8 . 5
2 3 . 0
3 3 . 6
7 1 . 0
T o t a l b a c t e r i a l 818000 962000 558000 779333 167183 21 .4 c o u n t s x l 0 3 CFU/lOO ml
T o t a l e n t e r o - 2420 3080 2240 2580 361 13 .9 b a c t e r i a l c o u n t s ( 0 . 3 ) ( 0 . 3 ) ( 0 . 4 ) ( 0 . 3 ) x l 0 3 CFU/lOO ml
T o t a l c o l i f o r m 13 ^ 1 6 0 ^ 30 6 7 . 6 ^ 66 9 7 . 0 x l 0 3 MPN/lOO ml ( .OOOlsf ( . 0 1 6 ) ^ ^ ( . 0 0 0 5 L ( . 0 0 0 8 )
( 0 . 5 ) ( 5 . 1 9 ) ( . 1 3 ) ( 2 . 6 ) *
F e c a l s t r e p t o c o c c i 13 6 4 7 . 6 3 . 8 5 0 . 3 x l 0 3 MPN/lOO ml
WINTER 9 0 - 9 1
T o t a l b a c t e r i a l 712000 886000 476000 691333 168018 24 .3 coxints x 103 CFU/lOO ml
T o t a l e n t e r o - 1860 2460 2180 2166 .6 245 1 1 . 3 b a c t e r i a l c o u n t s ( 0 . 3 ) ( 0 . 3 ) ( 0 . 5 ) ( 0 . 3 ) x l 0 3 CFU/lOO ml
T o t a l c o l i f o r m 7 ^ 13 ^ 13 ^ 11 ^ 2 . 8 25 .7 x l 0 3 MPN/lOO ml ( . 0 0 0 0 4 ) ( . 00014) ( . 0 0 0 2 7 ) ( .OOOll)
( 0 . 3 7 ) * * ( 0 . 5 2 ) * * ( 0 . 5 9 ) * * ( 0 . 5 ) * *
F e c a l s t r e p t o c o c c i 7 2 . 3 3.4 4 . 2 2 .0 4 7 . 4 x l 0 3 MPN/lOO ml
63
Table 9 shows the loca t iona l va r ia t ion and average
bac t e r io log ica l parameters of so i l from a g r i c u l t u r a l f i e l d s
in the v i c i n i t y of i ndus t r i a l a rea . The average TBC was
maximum in spring season (732.7x10^ CFU/lOO ml) while i t
was minimum during winter (437.3x10^ CFU/iOO ml) . As far
as the r e l a t i v e proport ion of en te robac te r i a l counts with
TBC was concerned, i t was maximum (0.4%) in winter and mini
mum in spring season (0.3%). Total ooliform count with
respect t o t he TBC was lowest (0.0001%) in a l l the seasons
while the t o t a l coliform count with respect to en te robac te r i a l
population was maximum in summer (0.5%) and minimum in
winter (0.3%). In terms of fecal s t reptococci / t he average
FS count was nearly the same (0.008x10^ MPN/lOO ml) in
spring and summer seasons but r e l a t i v e l y lower in w in t e r
(0.003x10^ MPN/lOO m l ) .
A t o t a l 49 E.col l i s o l a t e s from sewage out of 5 3
were found t o be multiply r e s i s t a n t t o a n t i b i o t i c s . Among
the 15 commonly used a n t i b i o t i c s / d r u g s t e s t ed for s e n s i t i
v i t y , 9 d i f fe ren t mul t ip le a n t i b i o t i c s / d r u g s r e s i s t ance
p a t t e r n were observed. The number of a n t i b i o t i c s or drugs
against which res i s t ance was observed ranged from 3 t o 9 .
Nine d i f fe ren t E.col i i s o l a t e s were iden t i f i ed on the b a s i s
of antibiograms (Table lO). EigHt types of a n t i b i o t i c s /
drugs r e s i s t ance pa t t e rn s among the so i l i s o l a t e s were
64
TABLE 9 : L o c a t i o n a l v a r i a t i o n and a v e r a g e b a c t e r i o l o g i c a l p a r a m e t e r s of s o i l i n t h e v i c i n i t y of i n d u s t r i a l a r e a
P a r a m e t e r s SS^ SS^ SS^ Mean SD >iCV
SPRING 90
Total bacterial 848000 622000 728000 732667 92323 12.6 counts xl03 CFU/lOO ml
Total entero- 1890 2440 2860 2397 397 16.5 bacterial counts (0.2) (0.4) (0.4) (0.3) xl03 CFU/100 ml
Total ooliform 13 ^ 9 ^ 11 ^ H * 1'6 14.8 '^ "' " • 00015) (.000111 (.00015) (.00015:
(' x l 0 3 MPN/100 ml ( . 00015) ( . 00014) ( . 00015) ( . 0 0 0 1 5 )
( 0 . 7 8 ) * * ( 0 . 4 ) (0.41)^** ( 0 . 5 ) * *
F e c a l s t r e p t o c o c c i 7 7 7 7 0 0 x l 0 3 MPN/100 ml
SUM14ER 9 0
T o t a l b a c t e r i a l 624000 742000 732000 699333 53424 7 . 6 c o u n t s x l 0 3 CFU/lOO ml
T o t a l e n t e r o - 1860 2680 3020 2520 487 1 9 . 3 b a c t e r i a l c o u n t s ( 0 . 3 ) ( 0 . 4 ) ( 0 . 4 ) ( 0 . 4 ) x l 0 3 CFU/lOO ml
T o t a l c o l i f o r m 7 ^ 11 ^ 2 2 ^ 1 3 . 3 ^ 6 . 3 4 7 . 5 xlO 3 MPN/100 ml ( . O O O i n ( . 00014) ( . 0 0 0 3 ) ^ ( .OOOl) .
( 0 . 4 7 ) * * ( 0 . 4 ) * * ( 0 . 7 ) * * ( 0 . 5 )
F e c a l s t r e p t o c o c c i 2 . 3 13 11 8 . 7 4 . 6 5 2 . 9 x l 0 3 MPN/lOO ml
WINTER 9 0 - 9 1
Total bacterial 596000 340000 376000 437333 113153 25.8 counts xlo3 CFU/lOO ml
Total entero- 1460 1520 2420 1800 439 24.3 bacterial counts (0.2) (0.4) (0.6) (0.4) xl03 CFU/lOO ml
Total coliform xl03 MPN/lOO ml Total coliform 7 ^ 5^. 4^ 5 . 3 ^ 1.2 23.3
(.00011) (.00014) (.0001) (.0001) (0.5)** (0.3)** (0.2)** (0.3)**
Fecal s t reptococci 3 2.3 5 3.4 1.2 33.3 xl03 MPN/100 ml
* Total coliform counts expressed in terms of percentage of the t o t a l b a c t e r i a l count
** Total coliform expressed in terms of % of t o t a l en te robac te r i a l count.
65
TABLE 10 : A n t i b i o t i c r e s i s t a n c e p a t t e r n of E.co 1 i s t r a i n s i s o l a t e d from sewage w a t e r * ""
S .No. E . c o l i R e s i s t a n c e a g a i n s t No. of a n t i -a n t i b i o t i c s / d r u g s b i o t i c s
a g a i n s t w h i ch r e s i s t a n c e wag^ o b s e r v e d
Number of s t r a i n s abu-showing nda-r e s i s t a - n e e n e e
1 .
2 .
3 .
4 .
5 .
6 .
7 .
8 .
9 .
Wi.
Wi ,
Wi .
Wi .
Wi .
Wd.
Wd,
Wd,
Wd,
A, Ak, R 3
A,B,E ,Fr ,Na , .R 6
A , B , E , F r , P b / R , S z 7
A , A k , B , E , F r , P b , R , 8 Sz
A , B , C t , E , P b , R , S,Sz 8
A , A k , B , C , E , F r , P b , R 8
A , B ^ C t , E , F r / N a , R , 9 S/ Sz
A , B , C t , E , F r , K , R , S , 9 Sz
A , A k / B , C t , E , F r , N , R , 9 Sz
3
2
26
4
4
4
1
3
2
5 . 6
3 .7
4 9 . 0
7.5
7 .5
1.8
5 . 6
3 . 7
be T o t a l number of E . c o l i s t r a i n s found t o ^ s e n s i t i v e t o a l l a n t i b a c t e r i a l a g e n t s = 4 ( 5 . 6 2 % ) .
* T o t a l number of s t r a i n s employed f o r t h e s t u d y = 53
* * T o t a l number of a n t i b a c t e r i a l a g e n t s employed f o r t h e s t u d y = l 5
I n f a c t we had u s e d 17 a n t i b a c t e r i a l a g e n t s b u t t h e r e was no
e x c e p t i o n w i t h r e g a r d t o t h e r e s i s t a n c e a g a i n s t a m p i c i l l i n
and p e n i c i l l i n a s w e l l a s c h l o r o t e t r a c y c l i n e and t e t r a c y c l i n e .
T h e r e f o r e o p e r a t i o n a l l y we were work ing on 15 a g e n t s o n l y .
66
recorded (Table 11) . E ,col l i s o l a t e s of i n d u s t r i a l or ig in
(Wi ) were r e s i s t a n t to only 3 a n t i b i o t i c s whereas the
domestic sewage i s o l a t e s displayed res i s t ance t o 9 diff
erent a n t i b i o t i c s simultaneously (Table 10) .
Table 12 and 13 show the percent r e s i s t ance agains t
the individual a n t i b i o t i c s . Out of 15 an t ib io t i c s /d rugs
t e s t e d , the s e n s i t i v i t y to gentamycin was invar iably obser
ved in a l l the i s o l a t e s from water and soil whereas r e s i s
tance to ampici l l in and rifampicin was equally prevalent
(92.45%). Moreover, 86.7% of i s o l a t e s were r e s i s t a n t t o
both the Erythromycin and Baci t racin whereas 79.2% were
against furazolidone in t he sewage water samples. Further-
more, 95.5% E.co l i i s o l a t e s from so i l were at l e a s t r e s i s
t a n t to a m p i c i l l i n / p e n i c i l l i n and erythromycin whereas
88.8% were simultaneously r e s i s t a n t against furazolidone
and sulphadiazine (Table 13) .
A t o t a l of 30 E.col i i s o l a t e s from sewage were
t e s t e d from re s i s t ance against four metals eg. Hg, cd, Pb
and Zn. 66.6% of s t r a in s showed re s i s t ance t o Hg"*"*" of which
53.3% sanples were of i ndus t r i a l o r i g i n . Among the E.col i
s t r a i n s i so l a t ed from i n d u s t r i a l e f f luen t s , 13.3% showed
MIC of 200 ug/ml towards Hg"*""*" whereas 10% of the s t r a i n s
showing MIC at 12.5 ug/ml t o Hg" "*". 93.3% of E.col i i s o l a t e s
exhibi ted r e s i s t ance t o Pb" "*" and Zn"*" of which 66.6% and
67
TABLE 11 : Ant ib io t ic r e s i s t ance pa t te rn of E.col i s t r a i n s i so la t ed from soi l
S.No. E.col i s t r a i n s
Resistance against ant ib io t ic s/drug s
No. of a n t i - Number % b i o t i c s of s t r -abun-against a ins dance
which showing res i s t ance re s i s -was observed** tance
1.
2.
3 .
4 .
5.
6.
7 .
8 .
S i .
S i ,
S i .
S i ,
Sd.
Sd,
Sd.
Sd,
A /Ak ,E ,Pb ,R
A, A k , C , E , F r , Sz
A , B , E , F r , P b , R , Sz
A , B , C , C t , E , F r , Sz
A , B , C t , E , F r , S, Sz
A , B , C t , E , F r , K , N , R , Sz
A , B , C t , E , F r , N a , R , S, Sz
A , B , C t , E , F r , K , R , S, Sz
5
b
7
7
7
9
9
9
3
3
27
2
3
2
6 . 6
6 .6
6 0 . 0
4 . 4
6 .6
4 . 4
4 .4
2 . 2
Total number of E.col i s t r a i n s found t o ^ e n s i t i v e t o a l l a n t i b a c t e r i a l agents = 214,4%).
*Total number of s t r a i n s ertployed for the study = 45
**Total nxomber of a n t i b a c t e r i a l agents employed for the study = 15 .
68
TABLE 12 ; P e r c e n t r e s i s t a n c e aga ins t i n d i v i d u a l a n t i b a c t e r i a l agent moni tored in t h e sewage wa te r
T o t a l Number Number of i s o l a t e s r e s i s t a n t Pe rcen t (%) of E . c o l i t o t h e fo l lowing a n t i b i o t i c s / r e s i s t a n c e s t r a i n s i s o l a - drugs t e d from sewage wa te r
53 Ampic i l l i n -49 92.4
Amikacin - 13 24.5
B a c i t r a c i n - 46 86.7
Chloramphenicol - 4 7.5
C h l o r o t e t r a c y c l i n e - 1 0 18.8
Erythromycin - 46 86.7
Furazo l idone - 42 79.2
Gentamycin - 0 0
Kanamycin - 3 5.6
N a l i d i x i c acid - 3 5.6
Neanycin - 2 3.7
polymyxin B-41 77 .3
Rifampicin - 49 92.4
Streptomycin - 8 15.0
Su lphad iaz ine - 40 75.4
69
TABLE 13 : Percent r e s i s t ance against individual a n t i b a c t e r i a l agent monitored in the soi l
Total number Number of i s o l a t e s r e s i s t a n t Percent (%) of E .co l i s t r a - t o a n t i b i o t i c s / d r u g s res i s tance ins i s o l a t e d from so i l
45 A i n p i c i l l i n - 43
Amikacin - 6
B a c i t r a c i n - 37
C h l o r a m p h e n i c o l - 5
C J i l o r o t e t r a c y c l i n e - 1 0
E r y t h r o m y c i n - 43
F u r a z o l i d o n e - 40
Gentamycin - 0
Kanamycin - 3
N a l i d i x i c a c i d -2
Neomycin - 2
Po lymyxin B - 3 0
R i f a m p i c i n - 35
S t r e p t o m y c i n - 6
S u l p h a d i a z i n e - 40
9 5 . 5
13 .3
8 2 . 2
1 1 . 1
22 .2
9 5 . 5
8 8 . 8
0
6 . 6
4 . 4
4 . 4
6 6 . 6
7 7 . 7
1 3 . 3
8 8 . 8
70
7 3.3% were derived from i n d u s t r i a l or ig in r e spec t ive ly .
The highest MIC of 3200 ug/ml was observed in both cases .
89.9% of t he i s o l a t e s showed re s i s t ance t o Cd"*"*" of which
73.3% of the i s o l a t e s were from i n d u s t r i a l e f f l u e n t s . The
maximum MIC at a concentration of 3200 ug/ml was exhib i
t ed by these s t r a i n s t owards Cd"*""*" (Table 14).
Table 15 descr ibes the incidence of r e s i s t ance t o
var ious metals and t h e i r MIC value in the t e s t E .col i i s o
l a t e s frcxn so i l o r i g i n . 46.7% of the t e s t s t r a i n s showed
r e s i s t ance t o Hg"*""*" of which 26.6% of the s t r a i n s were i s o
l a t ed from i n d u s t r i a l a rea . The highest MIC of 100 ug/ml
t o Hg"*""*" was observed in 6.7% of t he s t r a i n s i s o l a t e d from
i n d u s t r i a l area whereas the minimum MIC t o Hg' "*" was 12.5
ug/ml. 100% of the i s o l a t e s exhibi ted res i s t ance t o Pb" "*",
Cd"*"'" and Zn"*"*" of which 5 3.3% Pb"*"*" r e s i s t a n t , 70% Cd'*"''
r e s i s t a n t and 63.3% 2n"*""*" r e s i s t a n t E.col i s t r a i n s had been
i s o l a t e d from the i n d u s t r i a l a r ea . The maximum MIC of 3200
ug/ml was observed for these meta ls , which were mostly of
i n d u s t r i a l i s o l a t e s . The minimum MIC of 800 ug/ml was
displayed for Pb"*""*" (Table 15).
Table 16 shows the MIC values in the 18 s t r a i n s
of Enterobacter aeroqenes against four heavy metals
i s o l a t e d from sewage. 61% of the s t r a i n s were r e s i s t a n t
t o mercury whereas 100% of the s t r a i n s showed re s i s t ance t o
TABLE 14 i i n c i d e n c e of meta l r e s i s t a n c e and t h e i r MIC^ v a l u e in E . c o l i i s o l a t e d from sewage water
71
Heavy Me ta l s
Minimal I n h i b i t o r y concent r a t i o n s (MICs) (iig/ml)
No. of s t r a i n s showing r e s p e c t i v e MIC v a l u e I
Domestic sewage
Indust r i a l e f f l u e n t s
Inc idence of a b s o l u t e r e s i s t a n c e agai n s t t h e r e s p e c t i v e me ta l i
I n d u s t r i a l e f f l u e n t s
T o t a l
Frequency
Mercury (Hg)
12.5 25 50
100
200
1 1 1 1 0
2 4 1 5 4
53.3% 66.6%
10.0%
16.7%
6.7%
20.0%
13 .3%
Lead (Pb)
800
1600
3200
3
3
2
0
9
11
66.6% 93.3%
10.0%
40.0%
44 .3%
Cadmium 1600 (Cd)
3200
11
11
73.3% 89.9%
4 3 .3%
4 6.6%
Zinc (Zn)
1600
3200 15
7 3 .3% 9 3 .3%
36.6%
5 6.6%
*Total number of isolates « 30
72
TABLE 15 t I nc idence of meta l r e s i s t a n c e and t h e i r MIC va lue in E . c o l l i s o l a t e d from s o i l
Heavy M e t a l s
Mercury (Hg)
Lead (Pb)
Cadmium (Cd)
Z inc (Zn)
Min ima l I n h i b i t o r y con-r c e n t r a t i on (MICa) u g / m l
12 .5
25
5 0
100
8 0 0
1600
3200
1600
3200
1600
Number o: showing . t i v e MIC
1 D o m e s t i c sewage a r e a
3
2
1
0
6
7
1
6
3
5
f s t r a i n s r e s p e c -v a l u e
I n d u s t r i a l a r e a
0
3
3
2
0
11
5
13
8
7
I n c i d e n c e of a b s o l u t e r e s i s t a n c e a g a i n s t t h e r e s p e c t i v e m e t a l ]
1 I n d u s - T o t a l t r i a l a r e a
26.6% 46.7%
53 .3% 100%
70% 100%
Frequency
10.0%
16.7%
13.3%
6.7%
20.0%
60.0%
20.0%
63 .3%
36.6%
40.0%
3200 63.3% 100%
11 60.0%
* T o t a l number of i s o l a t e s = 30
73
TABLE 16 : I n c i d e n c e of m e t a l r e s i s t a n c e and t h e i r MIC v a l u e i n E n t e r o b a c t e r a e r o q e n e s i s o l a t e d from sewage w a t e r
Heavy M e t a l s
Mercury (Hg)
Min ima l I n h i b i t o r y c o n c e n t r a t i o n s (MICs) u g / m l
12 .5
25
50
No. of s t r a i n s showing t h e i r MIC v a l u e
5
4
2
I n c i d e n c e of a b s o l u t e r e s i s t a n c e a g a i n s t t h e r e s p e c t i v e m e t a l
F requency
6 1 . 1 %
27.7%
22.2%
1 1 . 1 %
Lead (Pb)
1600 11
100%
6 1 . 1 %
3200 38.8%
Cadmium (Cd)
1600
100%
33 .3%
3200 12 66.6%
Z i n c (Zn)
1600 13
100%
72.2%
3200 27.7%
* Total number of i s o l a t e s = 18
74
lead, cadmixjm and z inc . Highest MIC of 3200 ug/ml was
observed agains t Pb"*""*", Cd"*" and Zn'*"''" and minimum was at
1600 iig/ml ^Table 16) .
All the 18 i s o l a t e s of E.aeroqenes were a l so t e s t ed
for t h e i r r e s i s t ance against 15 a n t i b a c t e r i a l agents and
these were invar iably found to be multiply r e s i s t a n t . The
nxjmber of ant ib iot ics /dr^igs against which r e s i s t ance was
found ranged from 6 t o 8 a n t i b i o t i c s / d r u g s . Thus out of
18 E.aeroqenes i s o l a t e s , 5 subtypes were i den t i f i ed on the
b a s i s of antibiograms as shown in t a b l e 17.
Although the aforementioned parameters s trongly
suggested for the presence of plasmids, yet a d i r ec t
experiment was requi red . All the aforementioned E .co l i
and E,aeroqenes i s o l a t e s were found to harbour plasmids
based on the agarose gel e lec t rophores i s (data not shown).
Fig . 1 shows the gel e l ec t rophore t i c p r o f i l e s of plasmid
i so l a t ed from 5 d i s t i n c t E.col i i s o l a t e s from s o i l . Only
a very s l i gh t difference was observed in t h e i r r e l a t i v e
mobi l i ty . Fig . 2 shows the agarose gel e l ec t rophore t i c
p r o f i l e s of plasmids in the 8 E .col i s t r a i n s i so l a t ed
from the sewage water . Most of the s t r a i n s show mul t ip le
copies of plasmids in the sewage i s o l a t e s whereas s ingle
plasmid appeared in s o i l i s o l a t e s IFig . l ) .
75
TABLE 17 t A n t i b i o t i c r e s i s t a n c e p a t t e r n of 18 E n t e r o b a c t e r a e r o q e n e s s t r a i n s i s o l a t e d from sewage w a t e r
S .No. E , a e r o q e n e s R e s i s t a n c e a g a i n s t No. of Nxoraber of % s t r a i n =„+. v ^ ^.^ ^ ^ / H V . , , ^ „ a n t i b i o - s t r a i n s a b u -
a n t i b i o t i c s / u r u g s . . T_ j j ' ^ t i c s a g a - s h o w i n g ndan-
i n s t r e s i s t a - ce which n e e r e s i s t a n c e was o b s e r v e d *
A , B , E , R , S , S z
A , B , E , K , R , S z
A , B , E , F r , R , Sz
A,B , C t , E , F r , R , SZ
A,B , C t , E , F r , R , S, Sz
T o t a l number of E n t e r o b a c t e r a e r o q e n e s s t r a i n s foxond t o b<£
s e n s i t i v e t o a l l a n t i b a c t e r i a l a g e n t s = 2 ( 1 1 . 1 % ) .
* T o t a l niamber of a n t i b a c t e r i a l a g e n t s employed f o r t h e s t u d y
= 1 5 .
In fact we had used 17 antibacterial agents but there was
no exception with regard to the resistance against ampicillin
and penicillin as well as chlorotetracycline and tetracycline.
Therefore operationally we were working on 15 agents only.
1 .
2 .
3 .
4 ,
5 .
EA^
EA^
EA3
EA4
EA5
6
6
6
7
8
3
1
7
3
2
1 6 . 6 %
5 . 5 %
3 7 . 7 %
1 6 . 6 %
1 1 . 1 %
Fig. 1. Agarose gel e lec t rophores i s p a t t e r n s of
plasmid DNA i so l a t ed from 5 E.col l Si and Sd
i s o l a t e s ( lanes a to e ) .
*arrow ind ica tes t h e pos i t ion of the wel ls
( s t a r t i n g point) .
Fig, 2. Agarose gel electrophoresis pat terns of
plasmid DNA isolated from 8 E.coli sewage
water Isolates (lanes a to h ) .
*arrow indicates the position of the wells
(s tar t ing po in t ) .
a b c d e f g h
78
Transfonaation : To ascer ta in whether the drug re s i s t ance
in the aforementioned E«coli s t r a i n s Ci .e . Wi, Wd, Si and
Sd) was a plasmid mediated character , tne transformation
of E»coli AB115 7 with R-plasmids i so la t ed from these s t r a i n s
was performed. Tne transformation frequency of AB1157 with
these four R-plasmids i . e . Wx , Wd-, Wd., Si . as well as
two more plasmids i so l a t ed from soi l* Sd. and Sd^ i s shown
in Table 18. The transformation frequency of r ec ip i en t c e l l s
with R-plasmids Wi^/ Wd_, Wd., a l l derived from sewage was -3 -3 -3
ca lcu la ted t o be 1.7x10 , 3.83x10 and 2.96x10 •" respec
t i v e l y , on the o ther hand, t he plasmids of so i l o r i g i n . S i . ,
Sd , Sd- displayed t h e transformation frequencies of 2.54x
10"-^, 2.46x10"'^ and 3.29x10-3 respec t ive ly (Table 18) .
Plasmid curing t Further confirmation in favour of t he
plasmid mediated res i s t ance charac ter was ca r r i ed out by
means of curing experiment. For t h i s purpose we made use
of two E.col i s t r a i n s i . e . Si . and Wd. los t the res i s tance
character on treatment with ethidium bromide. Fig . 3 shows
a curve of percent curing Vs ethidixom bromide concentra t ions .
Curing was observed at the minimum concentration of 0.5
ug/ml and increased with t h e increasing concentrat ion of
ethidixjm bromide. One E.col i s t r a i n s i so l a t ed from so i l
(S i . ) displayed 7.4% curing while other from water i s o l a t e
(Wd.) displayed 37.7% curing on treatment with 0.5 ug
79
TABLE 18 ; T r a n s f o r m i n g a b i l i t y of AB1157 w i t h t h e s i x t e s t p l a s m i d s
S.No. T e s t T o t a l n o . No. of t r a n s - T r a n s f o r m - M a r k e r s P l a s m i d s of c e l l s f o r m a n t s a t i o n t r a n s f e r -
f r e q u e n c y r e d
1 .
2 .
3 .
4 .
5 .
6 .
pWis
pWd3
pWd^
p S i 4
pSd^
pSd_
1.80x10"^ 3.07x10'^
LOSxlo"^ 4 . 1 4 x 1 0 ^
1.68x10"^ 4 .98x10^
1.27x10*^ 3 .77x10*
1.48x10*^ 3 .13x10*
l .OSxlo '^ 3 .46x10*
1.70x10
3.83x10"^
2.96x10"^
2.54x10"^
2.46xl0~^
3.29x10"^
A,Ct,Hg, Cd,Zn
A, Ct,Hg, Cd
A,Ct,N,R, Cd
Cd,Zn
A,Ct,Hg, Cd
Hg
Fig . 3 . Plasmid curing and survival of plasmid
harbouring £.co 1 i Si. and Wd. s t r a i n s
by treatment with ethidium bromide.
Symbols - curing ( so l id l i n e ) ; survival (broken l i n e ) ;
E .col i Si^' ( © ) ; g . co l i Wd ( O ) .
100
CT C
cr D o •«—>
c 0 u
a.
-vQO (i> - 1
o (V> ^ ^ ^ r 60-CO c -^ <
< 40-
2o|
60 80 100 Ethidium Bromide(ug/m()
81
ethidium bromide per ml of cu l tu re on l6 h of t rea tment .
A maximum curing of 92% was observed with maximum dose of
(80 Aig/ml) of ethidium bromide (Fig. 3 ) .
Lethal e f fec t s of curing agent : The curing agent ethidium
bromide was a lso observed t o be l e t h a l a t the concentrat ions
used (Fig . 3 ) . The survival was decreased by 3% in Si^
s t r a i n of E .col i on 0.5 aig/ml ethidium bromide treatment
and was only 7.0% at the concentrat ions of 100 ug/ml
(Fig . 3 ) . In E .co l i Wd s t r a in t he survival was decreased
by 7% at 0.5 ug/ml ethidium bromide treatment and was
reduced to a mere 4,6% at t he concentrat ions of 100 ug/ral
(Fig . 3 ) .
Conjugation : Table 19 gives the conjugative p o t e n t i a l
of the four t e s t E .col i s t r a i n s . E .col i Wi_ demonstrated
58.7% conjugation when mated for 60 min with r ec ip i en t
E .co l i AB115 7 s t r a i n . The E.col i Wd. and Wd exhibi ted
5 2.8 and 63.15 per cent conjugation respect ive ly under the
same experimental condi t ions . The conjugation was observed
t o be 62.9 per cent in another E .co l i s t r a i n . Si . which
was i so la t ed from s o i l .
Molecular weight of R-plasmids : F ig . 4 shows the r e l a t i v e
mobi l i ty of R-plasmids on agarose gel i so l a t ed from E.col i
Wi2# Wi-# Wi- and Wic s t r a i n s in lane d,e/,f and g respec
t i v e l y . Other lanes carr ied t he marker DNAs of known
molecular weight.
8 2
TABLE 19 : Conjugative p o t e n t i a l of mult iply r e s i s t a n t E.col i i s o l a t e s with rec ip ien t AB1157 s t r a in
Donor Time Colonies on Colonies on Percent s t r a i n (min) p la in p l a t e s A+T+Cd conjugation
p l a t e
28.1%
58.7%
22.6%
5 2.8%
26.4%
63.1%.
31.3%
62.9%
SWi^
SWd3
SWd^
SSi^
30
60
30
60
30
60
30
60
194
228
146
176
121
152
204
232
54
134
33
93
32
96
64
146
Fig. 4 . Agarose ge l e lec t rophores i s of the plasmid
species i so l a t ed from E.cxjli i s o l a t e s from
water.
lane a - Plasmid pBR322
lane b - R-plasmid i so la ted from marker Ec Rp4.
lane c - I n t a c t A DNA
lane d - R-plasmid i so l a t ed from E.col i Wi2 s t r a i n . ""
lane e- R-plasmid i so l a t ed from E.col i Wi s t r a i n ~
lane f- R-plasmid i so la ted from E.col i Wi. s t r a i n .
lane g -R-plasmid i so la ted from E.col i Wic s t r a i n .
*arrow ind ica t e s t he pos i t ion of the wells ( s t a r t i n g p o i n t ) .
a b c d e f g
84
Fig. 5 gives the ca l ib ra t ion curve for the molecular
weight determination making use of the data from Fig . 4 .
The molecular weight of t he plasmids i so l a t ed from the four
aforementioned E.col i s t r a i n s ca lcula ted on the b a s i s of
t h i s c a l i b r a t i o n p lo t was found t o be 46 and 50 Kb (Kilo-
base p a i r s ) .
Fig. 6 shows the r e l a t i v e mobil i ty of mult iple
plasmids i so l a t ed from E,co1i Wi, and Wd s t r a i n s . The
s t r a i n s Wi appears to harbour two plasmids (lane c ) .
The molecular weight ranged from 17,5 t o 50 Kb (Fig. 7 ) .
The s t r a in v;d- a l so appear to harbour two d i s t i n c t plasmids
(lane d) and the molecular s ize of the two plasmids
ranged from 22.0 t o 51 Kb. In con t ras t , the s t r a i n s Wd.
seems t o harbour four plasmids (lane e) and the molecular
s ize of these plasmid DNA calcula ted t o be carying from
5.6 t o 52 Kb.
Fig . 5 . Molecular .size Vs mobil i ty p lo t of the t e s t
species ,4nd the standard DNA.
Symbols - R-pLasmids i s o l a t e d from Wi„ and Wi_ (O )
a ;^rox. s i ze i s 52 Kb.
asmid i so l a t ed from Wi and Wi (©)
approx. s ize i s 50 Kb.
- Standard DNA markers ( • )
60
50
N
I 30 o
20
IHO-I± i baJ -Bloa i abiiTTaXlq-/!
, a.'i £cf 3± aJTia . x o a t
10
0 10 20 30
Relative Mobility (mm)
W
Fig . 6. Agarose gel e lect rophoresis of mult iple
plasmid species i so la ted from E.col i (Wij-,
Wd^, Wd.) s t r a i n s .
lane a - Plasmid pBR3 2 2
lane b - R-plasmid i so la ted from -marker Ec%>4.
lane c - Plasmid i so la ted from E.col i Wij. s t r a i n .
lane d - Plasmid i so la ted from E. co 1 i Wd_ s t r a i n .
lane e - Plasmid i so la t ed from E . co l i Wd. s t r a i n . 4
lane f - Intact A DNA
*arrow indicates the position of the wells
(starting point).
Fig. 7. Molecular size Vs mobility plot of the t e s t
(multiple species of the R-plasmids isolated
froB Wi,
Symbols - Wij. p
- Wi^ p2
- " S f>l -^.Wd3 P2
- Wd^ p ^
' - Wd^ P3
- w d ^ p .
Wd and Wd strains) and standard DNAr*
0 ) approx. size 50 Kb
Q ) approx. size 17.5 Kb
L ) approx. size 51 Kb
A ) approx. size 22.0 Kb
n ) approx. size 5 2 Kb
01 ) approx. size 4 3.5 Kb
a ) approx. size 32.5 Kb
• ) approx. size 5.6 Kb
Standard DNA markers - ( • )
60
50
40
N
in 30
Z3 O
o
20
W
0' 10 20 30 Relative Mobility (mm)
40
DISCUSSION
88
Seasonal and loca t iona l va r i a t ions in TBC, TEC, TC
and FS in sewage water and so i l s are shown in t a b l e 4 - 9 .
The average t o t a l b a c t e r i a l covmts were found t o be retnark-
ably higher in domestic sewage water (679x10 CFU/100 ml)
as compared with t h e i n d u s t r i a l e f f luen t . Almost s imi la r
r e s u l t s were repor ted e a r l i e r by Biemond (1963). The t o t a l
en t e robac t e r i a l coxints and t o t a l coliforms were always
much l e s s than the t o t a l bac t e r i a l coionts. This i s q u i t e
obvious in nature because t o t a l b a c t e r i a l counts represent
a l l b a c t e r i a including TEC and TC groups of microorganisms.
The mean populat ions of t o t a l colifozm and feca l 4
s t rep tococc i in danes t i c sewage were 12.3x10 MPN/ml and 4
0.7x10 MPN/ml r e s p e c t i v e l y . The r e s u l t s were almost simil a r with the f indings of Al-Shahwani et a l . (1986) who
repor ted t o t a l coliform and fecal s t reptococci counts to 4 4
be 20.9x10 ce l l s /ml and 8.0x10 cel ls /ml in sewage water .
Bel l et_ al_. (1980) a lso reported nearly s imi la r d e n s i t i e s
of t he se b a c t e r i a l f l o ra in the sewage system.
Higher c e l l densi ty as well as a high propor t ion
of coliforms with respect to t o t a l b a c t e r i a l populat ion
was a l so observed in domestic sewage and in i n d u s t r i a l
e f f l uen t s as compared to s o i l s . Remarkably high t o t a l
coliforms and fecal coliform count in sewage compared
with s o i l a l so reported by several authors (Geldreich
e t a l . , 1964; Gordon, 1972; Cohen and Shuval, 1973).
89
In the present inves t iga t ion , t he average d e n s i t i e s
of t o t a l en te robac te r ia l counts, t o t a l coliforms and fecal
s t reptococci obviously including the t o t a l bac t e r i a l
counts were found t o be more in domestic sewage water
than the i ndus t r i a l e f f luen t s . This i s q u i t e ovbious in
na tura l system due to contamination of more animal and
human excreta t o domestic sewage whereas i n d u s t r i a l e f f lu
ents are expected t o contain t o x i c heavy metals which
would ra ther inh ib i t the growth and k i l l the bac te r i a at
high concentra t ions . Jana and Bhattacharya (1988) studied
the inhib i tory ef fect of various heavy metals l i ke Hg, Cd,
Pb, As, Cu and Cr on the growth of feca l E .col i and demo
ns t r a t ed a gradual decl ine in i t s growth with the increase
of exposure time as well as concentrat ion of meta l s . More
over, several authors have reported t h a t heavy metals
introduced into the water not only br ing about several
chemical changes and high degree of v a r i a t i o n in metal
concentration but a lso affect the e n t i r e ecosystem inc lu
ding the bac t e r i a l population (Fostner and Wittman, 19 79;
Vinkour et al_., 1980; Moore and Ramamoorthy, 1984) .
I t i s a well deocumented fac t t ha t t he Aligarh c i ty
not only possesses the large scale lock manufacturing
i n d u s t r i e s but also provides s h e l t e r t o innumerable num
ber of small scale lock manufacturing p l an t s re leas ing
t h e i r e f f luents to domestic d ra ins (Ajmal et al.»# 1980) .
90
In our system t h e average t o t a l b a c t e r i a l counts
which largely included t h e aerobes and facu l t a t ive anae
robes was found to be higher during spring season in dome
s t i c sewage water . Reddy e t al^. (1986) also reported a
s ign i f ican t ly higher count of t o t a l aerobic bac te r i a
during spring season in po l lu ted water .
The average t o t a l en t e robac t e r i a l count, t o t a l c o l i -
forms and fecal s treptococ 'ci were recorded to be higher
in our study during summer and spring season in sewage
water . I t i s cons is ten t t o the findings reported by seve
r a l authors who obtained r e l a t i v e l y more en te robac te r i a l
populat ions during t h e warm season (Rudolf et a_l., 1950;
Sommers and Nelson, 197 6) . Such seasonal va r i a t ion has
been a t t r i bu t ed to c e r t a i n environmental fac tors l i k e
temperature, so la r r a d i a t i o n , seasonal v a r i a b i l i t y and
concentration of n u t r i e n t s .
The present study a lso c lear ly ind ica tes tha t sewage
system was not a good r e se rvo i r for the b a c t e r i a l f lora
(TBC) compared with s o i l both in terms of locat ion as well
as of season (Table 4-9) . Such a lower t o t a l b a c t e r i a l
counts may be due t o t h e presence of t ox i c metals and o ther
undesirable substances in the sewage samples which would
often be inh ib i to ry t o b a c t e r i a . This i s qu i te reasonable
because of the lack of p a r t i t i o n i n g between the i n d u s t r i a l
and domestic sewage.
91
Our contention gains fu r ther support from the
s imi la r data on TBC derived from s o i l s in the v i c i n i t y
of i n d u s t r i a l as well as domestic d ra ins thereby sugg
es t ing a common type of n u t r i t i o n a l and physiological
s t a t u s of the two l o c a t i o n s .
Similar t o the f indings obtained with the sewage in
the seasonal v a r i a t i o n , we found a comparatively lower
t o t a l b a c t e r i a l counts in s o i l during winter season. This
may be largely due t o t he overflow of sewage carrying
the t o x i c metals and o ther undesirable substances during
the post monsoon season extending upto the win te r . Fur
thermore the environmental fac to rs l i k e temperature, so la r
r ad ia t ion , seasonal v a r i a b i l i t y and concentration of nut
r i e n t s would also s i g n i f i c a n t l y cont r ibute to such v a r i a
t i on (Santo-Domingo et_ al . . , 1989).
Comparing the r e l a t i v e proport ion of en te robac te r i a l
count within the t o t a l b a c t e r i a l f lo ra , i t was qu i t e
obvious t ha t the so i l did not contain impressive q u a n t i
t i e s of enterobacter ia r a the r ce r ta in nonenterobacter ia l
f lora would be p r e sen t . This was t r u e not only in terms
of aforesaid r e l a t i v e proport ion but also in terms of
absolute number of e n t e r o b a c t e r i a l populat ion. Contrary
t o t h i s sewage systan ca r r i ed a lo t of en te robac te r ia
presumably due t o t h e feca l contamination.
92
Another s ign i f i can t obsejrvation t o be mentioned
here was regarding the seasonal va r i a t i on which was much
more pronounced in case of t o t a l b a c t e r i a l f lora but such
a systematic v a r i a t i o n was absent in the en te robac te r ia l
count. This pa t t e rn might r e f l e c t a sor t of p ro tec t ive
effect on the en te robac te r i a but not on other b a c t e r i a l
f lora brought about by ce r t a in mechanism of res i s tance
against the undesi rable substances l i k e metals and a n t i
b a c t e r i a l agen ts .
The r e l a t i v e propor t ion of t o t a l coliform within
the en te robac te r i a l populat ion appears t o be s i gn i f i can t ly
reduced in the s o i l sartples (0.00 2%) compared t o t h a t in
sewage (1.3%). This probably r e f l e c t not only the presence
of such organisms because of the la rge amount of intake
into the sewage system but a l so due to the capacity t o
withstand under t h e unfavourable condit ions resu l t ing
out of the tox ic metal contamination.
The r e l a t i v e propor t ion of t o t a l coliform with
respect to e n t e r o b a c t e r i a l count did not indicate t he
expected reduction in winter season in the sewage system
ra the r the opposite t r end was recorded. The aforementioned
and other parameters d i r ec t ed us t o in tens ively work
upon the following two l i n e s : -
i ) The s t a t u s of metal and a n t i b i o t i c res i s tance in po
l l u t i o n ind ica to r coliform bacterium of fecal or ig in
93
i . e . E . c o l i .
i i ) The s t a tu s of t h e metal and a n t i b i o t i c res i s tance in
the enterobacterium of nonfecal o r ig in i . e . Enterobac-
t e r aeroqenes.
Almost a l l L .co l i and Enterobacter aeroqenes i s o
l a t e s from sewage water and s o i l s were found to be mult iply
r e s i s t a n t t o an t imic rob ia l agents (Table 10,11 and 17) .
The organisms were mostly r e s i s t a n t t o ampic i l l in , amika-
cin, b a c i t r a c i n , chloramphenicol, ch lo ro te t racyc l ine ,
erythromycin, furazol idone, kanamycin, na l id ix i c acid,
neomycin, polymyxin B, r i fampicin , streptomycin and su l -
phadiazine, con f l i c t ing data have been reported from v a r i
ous p a r t s of the world as f a r as the percentage of r e s i s
tance character i s concerned (Feary et_ al_,, 19 72; Niemi
et a l . , 1983; Sameer et_ a l , . , 1988).
A high l eve l of r e s i s t a n c e against p e n i c i l l i n
(9 2.45%) was observed in t h e sewage water i s o l a t e s and
86.6% of i s o l a t e s were r e s i s t a n t t o rifampicin and 81.1%
to polymyxin B. Shears e t al_. (1988) studied the prevalence
of res i s tance t o s ix commonly used ant imicrobial agents
in fecal coliform from chi ldren in Khartoum (Sud<S-"n) and
he found r e l a t i v e l y very high frequency of r e s i s t ance
against ampic i l l in (96%) and 705'o t o chloramphenicol.
94
Baya et^ al_. (1986) i s o l a t e d E.col i from sewage
e f f luen ts t h a t were r e s i s t a n t t o a combination of a n t i
b i o t i c s including kanamycin, chloramphenicol, and t e t r a
cyc l ine .
In the same year , Aljebouri and Meshhadoni examined
po l lu ted water samples from r i v e r T i g r i s m the v i c i n i t y
of raw sewage o u t f a l l for the incidence of a n t i b i o t i c
r e s i s t ance among E .co l i and found t h a t 70% or more of '
t hese organisms were r e s i s t a n t t o one or more a n t i b i o t i c s
(Aljebouri and Meshhadoni, 1986) . Vinayagamoorthy et_ a l .
(1986) found t r a n s f e r a b l e r e s i s t a n c e to ampic i l l in ,
chloramphenicol, t e t r a c y c l i n e and sulphamethoxazole, among
1207 c l i n i c a l i s o l a t e s . Further more, Chong (1986) i s o
l a t ed 25% s t r a i n s of en te robac te r ia from man in penin
su la r , Malaysia of which 7 were found t o be Enterobacter
spp.and 5 were E . c o l i . They were a l l sens i t ive t o n a l i
d ix i c acid but r e s i s t a n t t o more than 3 other a n t i b i o t i c s .
The nvimber of r e s i s t a n c e charac ter ranged from 3 t o 19
a n t i b i o t i c s .
In our study, E.col i i s o l a t e s frcxn so i l s a lso
showed a renarkably high l eve l of r e s i s t ance t o ampici
l l i n (95.5%). Frederickson (1988) a lso found t h a t E .col i
i s o l a t e s from s o i l s were r e s i s t a n t t o ampici l l in with
maximum frequency and a lso showed t h a t these i s o l a t e s
were mul t ip ly r e s i s t a n t to ant imicrobia l agents l i ke
95
ampic i l l in , p e n i c i l l i n , c a r b e n i c i l l i n , streptomycin,
kanainycin and t e t r a c y c l i n e .
The higher l e v e l of r e s i s t a n c e as conferred by
these E.col i i s o l a t e s agains t ampici l l in and some other
ant imicrobial agents (Table 12 and 13) seems t o r e f l e c t
wide spread chemotherapeutic use of these drugs in the
v i c i n i t y of t e s t r eg ion . The indiscr iminate use i s one of
t he major f ac to r s t h a t r e su l t ed in the energence of a n t i
b i o t i c r e s i s t a n t b a c t e r i a ( an i th , 1970; Levy, 1983; Al-
Doori et_ a l . , I9b6) .
The E .co l i and K.aeroqenes isola ted from sewage
water and s o i l s have been c l a s s i f i e d into many d i f fe ren t
an t ib io types on t h e b a s i s of antibiograms. The E.col i
i so l a t ed frcxn sewage have been c lass i f i ed in to 9 groups
and from s o i l s i n t o 8 group on the basis of antibiograms
obtained against d i f f e ren t a n t i b i o t i c classes (Table 10
and 11). The E.aeroqenes i s o l a t e s have been c l a s s i f i e d
in to 5 biotypes on t h e b a s i s of antibiograms (Table 17) .
Among the var ious methods of taxonomic s tud ies , the
bac te r i a have been commonly c l a s s i f i ed on the b a s i s of
antibiograms alongwith t h e i r plasmid prof i l es (Gi l lesp ie
et a l . , 1990). The E .co l i i so l a t ed from sewage samples
were multiply r e s i s t a n t t o t he antimicrobial agents
ranging from 3 to 9 . These findings are in agreement with
those of Bell et a]^. (1980) and Al-Doori et a l . (1986).
96
The p r e s e n t i n v e s t i g a t i o n i n d i c a t e d t h a t 66.6% of
E . c o l i s t r a i n s i s o l a t e d from sewage harboured r e s i s t a n c e
t o Hg" "*" whereas 93.3% i s o l a t e s were r e g i s t a n c e t o Pb"*""*"
and Zn++ both and 89.9% t o Cd"*" o n l y . Nakahara and Yone-
kura (1987) found t h e f requency of meta l r e s i s t a n c e in t h e
a q u a t i c E . c o l i i s o l a t e s t o be 49% fo r Hg"*""*", 74% for Cd"*"*"
and 84% for As"*"*"*".
In t h e p r e s e n t s t u d y , E . c o l i and E .aeroqenes
i s o l a t e d from sewage and s o i l were a l s o t e s t e d f o r t h e
r e s i s t a n c e a g a i n s t f o u r heavy m e t a l s i . e . Hg, pb , Cd and
Zn, Major i ty of E . c o l i i s o l a t e s from sewage and s o i l s sho
wed MIC of more t h a n 25 ug/ml t o Hg"*""*". F u r t h e r , MIC v a l u e s
upto 3 200 iig/ml were r e c o r d e d f o r Cd"*"*", Pb"*"*" and Zn"*""*•
(Tgble 14-16) whxch were e x c e p t i o n a l l y high compared t o
p r e v i o u s s t u d i e s (Summers and S i l v e r , 1972; Austin et_ a l . ,
1977; Clark e t a l . . , 1977 ; Marques e t a l . , 1979; and
Hor i t su et. a l . , 1985) . Summers and S i l v e r (1972) and
Clark et. al. . (1977) r e p o r t e d t h e maxiravim i n h i b i t o r y
concen t ra t ion t o be on ly 10 ug/ml fo r Hg . Moreover,
H o r i t s u et. aul- (1986) r e p o r t e d t h e E . c o l i s t r a i n s r e s i s
t a n t t o as high c o n c e n t r a t i o n s of Cd"*"" , Pb"*"*" and Zn"*""
a s 1283 iig/ml, 650 jug/ml and 3 22 ug/ml r e s p e c t i v e l y . In
our study most of t h e t e s t b a c t e r i a d i s p l a y i n g t h e h i g h
e s t MIC had been i s o l a t e d from i n d u s t r i a l e f f l u e n t s . T h i s
may be due t o t h e s t r a t e g i c p o s i t i o n of t h e area of ou r
97
study namely t he o u t s k i r t s of Aligarh c i ty which houses
many lock manufacturing fac to r i e s and t h e i r e f f luents
would e s s e n t i a l l y contain q u i t e a large amount of these
meta l s .
According t o some authors , the emergence of
m e t a l l i c r e s i s t a n c e could have been due to the exposure
of these t o x i c metals t o human and animals (Allen et_ a l » ,
1977; Clark e t a l . , 1977; Dutta e t al^., 1980). However
such a p o s s i b i l i t y is not f ea s ib l e in our case because
t he to l e rance l i m i t s in human and animals for Hg' '*', Cd" "
Pb" "*" and Zn" "*" have been reported t o be 0,001 ug/ml, 0.005
ug/ml, 0,05 ug/ml and 5.0 ug/ml respec t ive ly . The l eve l s
exceeding the se value i s not expected t o be present in
animal and host (WHO, 1985).
On the contrary , we have found the en terobac ter ia
r e s i s t a n t t o 20 0 ug/ml for Hg"*" , 3 200 ug/ml for Cd"*"*",
3 200 ug/ml t o Pb" "*" and 3200 ug/ml for Zn" " (Table No. 14-
16). The incidence of highly m e t a l l i c r e s i s t a n t en tero
bac t e r i a t he re fo re r e f l e c t such a high concentrat ions of
these metals i n t h e environment o ther than the hos t . The
only poss ib le explanation the re fore i s the presence of
high concentrat ion of t o x i c metals in our t e s t systems
i . e . sewage and s o i l .
This s u b s t a n t i a l l y supports our contention tha t
the coliform populat ion present in the sewage could be
98
r e l a t i v e l y more due t o both the reasons : -
i) The la rge in take of mul t ip le drug r e s i s t an t coliform
into t he sewage system,
i i ) The acqu i s i t i on of metal r e s i s t a n c e of these coliforms
within the sewage a f t e r the entry in to the system.
The f i r s t point c a r r i e s weight because the sewage
i s not ej<pected t o carry a s ign i f i can t amount of the drugs/
a n t i b i o t i c s t o acqui re mul t ip le r e s i s t ance unless t h e host
i s exposed t o a high concentrat ions of these cnemothera-
peu t ic agent for q u i t e a long t ime . tttJweVer t he second
point could be proved on the b a s i s of the presence of the
large q u a n t i t i e s of t o x i c metals (Ajmal et al_., 1980) which
would be l e t h a l t o t h e host and thus a acquis i t ion of metal
r e s i s t ance in b a c t e r i a ins ide the host i s most un l ike ly .
To a sce r t a in whether or not the res i s tance was
plasmid mediated, we fur ther extended our s tud ies in t h i s
d i r e c t i o n . 5 s t ra ins from 8 ant ib io types i so la ted from so i l
and 8 s t r a i n s from sewage were screened for plasmid i s o l a
t i o n and found t o harbour plasmids (Fig. 1 and 2) . These
r e s u l t s were almost s imi la r to t h a t of vakulenko and h i s
coworkers who found 30 out 32 a n t i b i o t i c r e s i s t a n t E.col i
s t r a i n s were carrying plasmids (Vakulenko et_ al^., 1980).
Hada and Sizemore (1981) suggested t h a t t h e i n c i
dence of p lasnid bear ing s t r a in s i s more in po l lu ted s i t e
99
than in the unpolluted zone. The incidence of high metal
r e s i s t a n t populat ion in E.col i and E.aeroqenes from sewage
and s o i l , suggest t h e p o s s i b i l i t y of increasing environ
mental po l lu t ion t o these me ta l l i c s a l t s .
As f a r as t h e number of plasmids in the plasmid
harbouring s t r a in i s concerned most of t he s t r a i n s from
sewage were carrying mul t ip le copies of plasmids (Fig . 2
and 6)but so i l i s o l a t e s harboured s ingle plasmid. Baya
et a l . (1986) i s o l a t e d E.col l from sewage e f f luen ts r e s i s
tance to 9 d i f f e ren t a n t i b i o t i c s and these s t r a i n car r ied
mul t ip le bands of plasmid. Schuett (1988) also showed multi
p l e bands of plasmid in t he s t r a i n i s o l a t e d from Sksshuts-
zyoen lake , whereas i s o l a t e s from brown water lake in south
Sweden showed t o harbour single plasmid of high molecular
weight O 3 0 Mdal) .
In t he present i nves t iga t ion , one of the s t r a in s
appears t o carry 4 types of plasmids ranging in molecular
weight from 5.6 t o 52 Kb whereas two other s t r a i n s ca r r ied
2 plasmids varying in molecular s ize from 17.5 t o 50Kb and
22.0 t o 51 Kb r e spec t ive ly .
Vinayagamoorthy et al^. (1986) i so la ted E.col i from
drinking water and demonstrated t h i s bacteriiom t o harbour
5 p lasn ids one of which having a molecular s ize of 2.4 Kb
ca r r i ed res i s t ance t o sulphonamide. He has also found the
100
E.col i from c l i n i c a l i s o l a t e s exh ib i t ing t r ans fe rab le
res i s tance to ampic i l l i n , t e t r a c y c l i n e , streptomycin,
chloramphenicol and tr imethoprim. These markers were
present in the s ing le plasmids of 60-120 Kb.
To provide evidence i f the r e s i s t a n c e markers were
ac tua l ly on the plasmid DNA, we ca r r i ed out the t r a n s
formation experiment employing plasmids i so la t ed from
t e s t E.ooli s t r a i n s . The expression of r e s i s t ance markers
in a l l the transformants suggested t h a t the a n t i b i o t i c
as well as metal r e s i s t a n c e was plasmid mediated. The
transformation frequency obtained with the rec ip ien t
s t r a in i . e . E.col i AB1157 (Table 18) was comparable t o
t h a t obtained by Bopp _et a_l. (1983) . He found t h a t E.cioli .
P.put lda and P. f luorescens s t r a i n s could be transformed
with P .putida derived RP. plasmid DNA at frequencies
ranging from l.SxlO" t o 3.5xlO" transformants per r e c i
p i e n t . All the t e s t E.col i s t r a i n s t r a n s f e r r e d 4 or more
res i s tance markers out of 7 t o 13 markers (Table 10 and
11) . Tantulivanchi et_ al_. (1981) had shown t h a t 9 out of
15 res is tance markers were t r a n s f e r a b l e in E .co l i •
The final proof in favour of t h e presence of
R-plasmids in t h e t e s t E .co l i s t r a i n s was provided
through the curing experiment. A s ign i f i can t f ract ion of
t e s t s t r a i n s was found t o lose t h e t e s t r es i s tance markers
101
in presence of curing agent i . e . ethidium bromide (Fig. 3 ) .
Our r e s u l t s of curing with ethidium bromide were even
b e t t e r than those obtained by El Syed and h i s coworkers
(1988) in case of c l i n i c a l i s o l a t e s of i^ .col i .
The t e s t E .co l i s t r a i n s were able t o t r a n s f e r some
of t h e i r r e s i s t ance markers t o standard E.col l r ec ip ien t
s t r a i n by conjugation CTable 1-9) . Moreover t h e percent
conjugation was much higher than those observed by Bell
et a l . (1980) in feca l coliform i so l a t ed from sewage e f f l
uen ts , but coincides with t he r e s u l t s obtained by Genthner
e t a l . (1988) in aqua t i c gram negative b a c t e r i a . This
t r a n s f e r of mul t ip le r e s i s t ance markers strongly suggests
t h a t t h i s property of a n t i b i o t i c as well as metal r e s i s
tance i s plasmid mediated (Summers, 1986).
Cavalcante et_ al. . (1988) i so l a t ed E.col l s t r a i n s
harbouring the conjugative plasmids from waste treatment
p lan t and demonstrated t h a t the i s o l a t e s were carrying
r e s i s t ance t o both an t imicrobia l agents and heavy meta ls .
Several workers have demonstrated the t r a n s f e r of
plasmids through conjugation in various systems including
the s o i l , water and o the r environmental condi t ions (Trevors
and Oddie, 1986> Trevors and Starodxib, 1987) .
We had performed our conjugation experiments in
l iqu id which might not be a b e t t e r environment as compared
t o so l id media. Since Genthner et a l , (1988) demonstrated
10 2
t h a t t h e c o n j u g a t i o n was much e f f i c i e n t on s o l i d media
r a t h e r t h a n i n l i q u i d i n c a s e o f a q u a t i c gram n e g a t i v e
o r g a n i s m s .
I n c o n c l u s i o n , we can s u g g e s t t h a t t h e s o i l and
sewage sys tem i n t h e v i c i n i t y of A l i g a r h c i t y i s h e a v i l y
p o l l u t e d w i t h s e v e r a l t y p e s o f t o x i c m e t a l s a s w e l l a s
t h e p o t e n t i a l l y h a z a r d o u s m i c r o b i a l f l o r a b e c a u s e of t h e i r
c a p a c i t y t o r e s i s t one o r t h e o t h e r w e l l known c h e m o t h e r a -
p e u t i c a g e n t s . The p r o b l e m t a k e s t h e s e r i o u s t u r n i n v i ew
of t h e r e s i s t a n c e t o m u l t i p l e a n t i b i o t i c s / d r u g s wh ich
would b e i n e f f e c t i v e even i f t h e y a r e g iven i n c o m b i n a t i o n ,
One s t e p a h e a d o f t h e a b o v e , we can e n v i s a g e o n l y t h e
a l a r m i n g s i t u a t i o n p r e v a i l i n g i n o u r system and s u r r o u n d
i n g s i n t h e l i g h t of t r a n s m i s s i b l e n a t u r e of R - p l a s m i d s .
T h i s s t u d y c a l l s f o r p a r t i c u l a r a t t e n t i o n t o w a r d s t h i s
p o t e n t i a l h a z a r d of i n c r e a s i n g i n c i d e n c e of t r a n s m i s s i b l e
m u l t i p l e d r u g r e s i s t a n c e . One s o o t h i n g a s p e c t of t h e
p r o b l e m however , i s t h e h i g h i n c i d e n c e of m e t a l r e s i s
t a n c e which wou ld somehow d e t o x i f y t h e e f f e c t of m e t a l l i c
p o l l u t a n t s and t h e s e b a c t e r i a can s e r v e a s n a t u r a l p u r i
f i e r t o t h e t o x i c i t y p r o d u c e d b y t h e t e s t m e t a l s i . e .
Hg"^" , Cd^''", Pb++ and Zn'^'^.
B e s i d e s t h e a p p l i e d a s p e c t of t h e p r o b l e m , one
can n o t even i g n o r e t h e s i g n i f i c a n c e of t h e m o l e c u l a r
103
taxonomy of plasmid han^ouring bac te r i a which would
provide a quick and r a t i o n a l approach t o c lass i fy then
on the b a s i s of antibiograms and plasmid p r o f i l e s . This
study i s one s tep forward towards t h i s end.
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