Laboratory diagnosis of infections produced by germs of the family

Enterobateriaceae

Family Enterobacteriaceae- Clinical significance -

• Intestinal and extraintestinal infections

• Highly pathogenic species of the genera:– Yersinia– Salmonella– Shigella

• Facultatively pathogenic species of the genera:– Escherichia coli– Klebsiella– Enterobacter– Proteus– Serratia– Citrobacter

Family Enterobacteriaceae- Common characters -

• Gram negative bacilli, nonsporulating, non-fastidious• Glucose-fermenters; • Lactose is only fermented by some genera – good

differential criterion• Oxidase-negative

• Catalase-positive

• Habitat: – soil, plants, human & animal intestines, mucous membranes; – Particular situation: Salmonella typhi (causative agent of typhoid

fever) – only present in humans (diseased / asymptomatic carriers)

Family Enterobacteriaceae- Collection of specimens -

• Extraintestinal infections:– urine, respiratory/digestive samples, wound secretions, blood,

CSF, etc)

• Intestinal infections:– Faeces: collection close to onset / depending on pathogenesis of

infection – Transport media: Stuart, Cary-Blair, Amies

Collection of urine

When?:

- in the morning (first miction)

How?: - clean uro-genital area

- eliminate first flow

- collect middle flow in

sterile container

Send to lab immediately or store

at 2-8°C

Collection of stool (faeces)

• Disposable stool collection containers (simple / with transportation medium Carry Blair: non-nutritive medium which prevents overgrowth of Enterobacteriaceae but preserves viable enteric pathogens (Salmonella, Shigella, etc)

Family Enterobacteriaceae- Isolation (inoculation of culture media) -

• Extraintestinal specimens from normally sterile sites: – Blood agar

• Extraintestinal specimens with moderate bacterial load (e.g. pus, sputum):– Blood agar + MacConkey

• Highly contaminated specimens (faeces):– MacConkey (low selectivity) – ADCL, Hektoen agar, XLD agar (medium selectivity) – High selective media e.g. S-S (for Salmonella and Shigella),

Wilson-Blair (for Salmonella)

e.g. Hektoen agar(developed at the Hektoen Institute in Chicago)

• indicators of:– lactose fermentation – H2S production;

• inhibitors (bile salts) to prevent the growth of Gram positive bacteria

Lactose H2S

Salmonella (-); alkaline reaction: blue-green colonies

(+); black centre colonies

Shigella (-); alkaline reaction: blue-green colonies

(-)

E.coli and others

(+); acid reaction: yellow-orange colonies

(-)

Left: lactose (+) = yellow-orangeRight: lactose (-) = blue-green, H2S (+) = black

centre

E.coli / others (definitely not Salmonella, not Shigella)

• Salmonella (not Shigella because of H2S production – black centre colonies)

Family Enterobacteriaceae- Identification -

• Biochemical tests:– TSI (triple sugar iron) agar– MIU (motility, indol, urea) agar– Simmons agar (use of citrate as unique carbon source)– PAD (phenylalanine deaminase) test– Fermentation of sugars

• Antigenic structure-based identification:– Agglutination with antisera

Family Enterobacteriaceae Biochemical tests = testing for enzyme systems

• characterization of bacterial isolate by testing for characteristic enzyme systems

• Method: re-inoculation of isolated colony (primary culture) into a series tubes with culture media containing specific substrates and chemical indicators

• Principle: detection of – pH changes produced by utilization of substrates / – colour / other changes produced by specific by-

products

Family Enterobacteriaceae Biochemical tests (continued)

TSI (triple sugar iron) agar – assessment of bacterial capacity to:

a. metabolize lactose and/or sucroseb. conduct fermentation to produce acidc. produce gas during fermentation

d. generate H2S

TSI agar: sucrose, lactose, glucose + mehyl red + ferrous

sulfateIF:• only glucose fermented →acid production in the butt of tube →

yellow, but insufficient acid to affect the methyl red in the slant

• either sucrose or lactose fermented → sufficient fermentation products → both the butt and the slant yellow

• gas during fermentation → gas bubbles/cracking of agar

• no fermentation → slant and butt remain red

• If bacterium forms H2S, this chemical will react with the iron to form ferrous sulfide = black precipitate in the butt (black butt)

TSI agar (continued)

• R = red = no fermentation (obligate aerobe)

• Y = yellow = some fermentation (facultative anaerobe)

• YG = fermentation + gas

• ”+” = Black = H2S

Family EnterobacteriaceaeIdentification – biochemical tests

• API (Analytical Profile Index)

Escherichia coli

• Gram negative, short bacilli, rounded ends, nonsporulating, motile (peritrichous cilia)

• Normal microbial flora of human and animal intestines; involved in vitamin synthesis and balance of intestinal microbiota

• Facultatively pathogenic

Escherichia coli- Clinical significance -

• Enteral infections (5 groups):– EPEC (enteropathogenic E.coli)– ETEC (enterotoxigenic E.coli)– EIEC (enteroinvasive E.coli)– EHEC (enterohemorrhagic E.coli) – produce verotoxins* (bloody

severe diarrhoea resembling dysentery!)– EAEC (enteroadherent E.coli)

• Extraenteral infections:– Urinary, respiratory, wounds & burns, sepsis , meningitis, etc.

*Verotoxins (Shiga-like toxins)

• toxins produced by certain strains of E.coli (EHEC) which disrupt the function of the ribosome

• Action similar to the toxin produced by Shigella disenteriae strains (Sh.shiga) – see below

• causes the hemolytic uremic syndrome• Term “verotoxin” related to the effect on “vero” cell

cultures (lineages of kidney epithelial cells extracted from African green monkeys; name: abbreviation from “verda reno” = green kidney in Esperanto)

E.coli – Enteral infectionsSpecimen: stool

• Culture media: MacConkey: pH indicator = neutral red (red in acid medium; colourless in basic medium):

• RED colonies (lactose-positive), round, shiny, 2-3 mm

E.coli colonies on blood agar

E.coli colonies on MacConkey: lactose positive (red) colonies

E.coli on medium containing lactose and bile salts: lactose positive (red) colonies

E.coli on Hektoen agar: yellow-orange colonies (lactose acidification)

E.coli – Enteral infectionsIdentification: biochemical tests

E.coli – Enteral infectionsIdentification on antigenic structure

• Slide agglutination with Ab against O and B antigens:– 5 lactose-positive colonies –

pick up with loop and emulsify with polyvalent anti-EPEC serum

– If positive test (agglutination) continue with monovalent

antisera (standard set) • Similar procedure for EIEC,

ETEC, EHEC (antisera for antigens O and H)

Escherichia coli- Clinical significance -

• Enteral infections (5 groups):– EPEC (enteropathogenic E.coli)– ETEC (enterotoxigenic E.coli)– EIEC (enteroinvasive E.coli)– EHEC (enterohemorrhagic E.coli) – produce verotoxins (bloody

severe diarrhoea resembling disenteria!)– EAEC (enteroadherent E.coli)

• Extraenteral infections:– Urinary, respiratory, wounds & burns, sepsis , meningitis, etc.

E.coli – Urinary tract infections (UTI)

• Collection of urine for bacterial culture (see above)

• Gram stained smear from urine sediment (after centrifugation): high no of PMNs + Gram negative bacilli

• Quantitative urine culture:– A. Dilutions technique– B. Calibrated loop technique

E.coli – Gram stained smear

E.coli – Urinary tract infections (continued)Quantitative urine culture

• Colony counts: method to determine number of viable bacterial cells in urine specimen

• 1 colony = 1 viable bacterial cell (colony-forming unit =

CFU) → after inoculation: division by binary fission i.e. 1 cell to 2; 2 to 4; 4 to 8; 8 to 16....and so on (at least a million cells must be present in order to be seen as a colony with the naked eye!)

• Results reported as: number of bacterial cells per mL of urine

Quantitative urine culture (continued)

A. The dilutions technique:• 2 dilutions (1/100 and 1/1000) in sterile saline solution • Inoculate 0.1 mL of each dilution onto blood agar and

MacConkey (lactose containing medium)• Spread inoculum to obtain isolated colonies (L-shaped

loop); Incubate overnight at 37°C

• Calculate the no of germs / mL urine i.e. multiply:– No of colonies (CFU) x – dilution factor (100 or 1000) x – 10 (we inoculated 0.1 mL of each dilution)

Quantitative urine culture (continued)

B. The calibrated loop technique: (nondiluted sample)• 5 mm loop → 0.01 mL urine (dilution: 1/100) →

MacConkey• 2.5 mm loop → 0.001 mL urine (dilution: 1/1000) →

blood agar• Incubate overnight at 37°C

• Calculation: (for each plate)• No. of colonies (CFU) x dilution = no of germs / mL• Calculate the arithmetic mean between the 2 counts

(on the 2 plates)

Quantitative urine culture (continued)

• Interpretation of results:• Under 10,000 germs/mL = nonsignificant bacteriuria

(probably contamination from lower urethra)• 10,000–100,000 germs/mL = nonconclusive; repeat test• Over 100,000 germs/mL = UTI

• Next steps: identification of causative agent i.e. – colonial characters, biochemical tests– Antimicrobial susceptibility testing

Genus Salmonella

• The most complex of all Enterobacteriaceae • over 2400 serotypes – Classification: Kauffmann-White

scheme based on bacterial antigens: – O (somatic)– H (flagellar)– Vi (virulence) – derived from the K (capsular) antigen

• Clinical significance:– Food poisoning– Systemic infections (the germs cross the intestinal barrier) –

”enteric fevers”

• Transmission: fecal-oral (via contaminated water, foods)

Genus Salmonella - The Kauffmann-White classification -

• E.g. Based on the “O” antigens: groups A – I (+ others)

• some examples given in

table →

“O” group A S.paratyphi A

“O” group B S.paratyphi BS.typhimurium

“O” group C S.paratyphi C

“O” group D S.typhiS.enteritidis

Salmonella typhi

• Prototype agent of ”enteric fevers” (other enteric fevers caused by Salomnella paratyphi A, B, C – less severe)

• Typhoid fever - fever in plateau (39-40°C), headache, muscle ache, vomit, diarrhoea /constipation, skin rush (lenticular maculae; “rose spots”), mental confusion, hepato-splenomegaly

• Complications: internal bleeding, intestinal perforation + peritonitis

• Laboratory diagnosis:– Bacteriology

– Serology

Salmonella typhi (+ paratyphi)- Bacteriological diagnosis -

• Collection of specimens depends on:– Patient status: diseased / chronic carrier– Clinical stage (time from onset)

• Secimens:– Blood for blood culture (best collected during 1st week; percent

of isolation decreases to 25% in the 4th week)– Bone marrow (in late stages) – lower patient compliance– Faeces for coproculture (low isolation during 1st week; percent

increases progressively to ~75% in 4th week); also collected from suspected chronic carriers

– Urine for culture (same isolation curve as for faeces)

Salmonella typhi (+ paratyphi)- Bacteriological diagnosis - continued

Blood culture: • inoculated media examined daily for 7-10 days; negative

result only if liquid media remain sterile for 10 days!• Gram stained smear from turbid tubes: Gram negative

bacilli• Subculture on agar slant

• Identification based on:– colonial characters, – biochemical tests, – antigenic structure – slide agglutination with anti-O and anti-H

sera (Kauffmann-White classification)

Salmonella typhi (+ paratyphi)- Bacteriological diagnosis - continued

Coproculture:• Both in diseased patients and in chronic carrires• Inoculation in liquid enrichment media (favour

multiplication of salmonellae and inhibit other microbial flora) e.g. – Leifson (nutrient broth with acid sodium selenite); – Muller-Kauffmann (broth with tetrationate and bile)

• Incubate overnight at 37°C

• Reinoculate on selective solid media (nutrients + sugars + pH indicator + substances which inhibit other germs)

Salmonella typhi (+ paratyphi)- Bacteriological diagnosis - continued

Coproculture: (continued)

Colonial characters on selective solid media:• Wilson-Blair (high selectivity; indicator: brilliant green)

– Black, flat colonies, 1-2 mm, metallic halo

• S-S (selective for Salmonella and Shigella):– Fine, semitransparent colonies, with black centre (H2S)

• Hektoen enteric agar :– Fine, green colonies, with black centre (H2S)

• MacConkey (medium selectivity):– Semitransparent, lactose-negative (colourless) colonies

Salmonella on S-S agar: fine, semitransparent colonies, black centre (H2S)

Salmonella on MacConkey agar

• Semitransparent, lactose-negative (colourless) colonies

Salmonella on agar with lactose and bile salts: lactose negative (colourless) colonies with black

centre (H2S)

Salmonella on Hektoen agar

• black centre colonies (H2S)

Salmonella – Coproculture - continued

• Identification based on:– colonial characters (see above), – biochemical tests (API 20E),

– antigenic structure – slide agglutination with anti-O and anti-H sera (Kauffmann-White classification)

– Phage typing – see next slide

(Bacterio)phage typing for Salmonella

• Bacteriophage = virus which specifically attacks bacteria• Banks of phages developed for Salmonella serotypes

Procedure: • agar plates flooded with liquid culture of the bacterial

isolate; remove excess liquid; leave culture film to dry;• inoculate set of phage suspensions onto plate surface;

incubate overnight at 37°C• Interpretation: phage lysis reactions recorded and

compared to a set of standards (developed by Public Health England, formerly PHLS, Colindale, England)

Phage typing Salmonella enteritidis

Salmonella typhi - Serological diagnosis -

The Widal test: • Principle: reaction between antibodies in patient serum

and specific antigens of S. typhi → clumping (agglutination) visible to the naked eye

• easy to perform BUT less reliable than bacteriology:– cross-reactivity with other Salmonella species,– the test cannot distinguish between a current infection

and a previous infection or vaccination status

• Still used in low resource areas with high prevalence of typhoid fever (endemic areas e.g. India, Pakistan)

Genus Shigella

Clinical significance: • dysentery – diarrhoea with multiple stools with mucus

and blood + general symptoms: dehydration, fever, abdominal pain + neurologic symptoms (neurotoxin secreted by Shigella shiga)

Common characters:

• Gram negative bacilli, nonmotile, nonsporulating

Genus Shigella - classification

4 serological subgroups (based on biochemical characters and antigenic structure):

• Subgroup A: Shigella dysenteriae– Types: Sh. shiga, Sh. Schmitzi, Sh. Large-Sachs

• Subgroup B: Shigella flexneri• Subgroup C: Shigella boydii

• Subgroup D: Shigella sonnei

_______________• Shigella shiga: the most severe disease (secretion of

neurotoxin)

Kiyoshi Shiga (1871-1957)

• Discovered Shigella dysenteriae during severe epidemic in 1897: over 90,000 cases

• “shiga” toxin named after him

Genus Shigella – Bacteriological diagnosis

• Collection of specimens: faeces (especially portions with mucus and blood)

• Transport: Cary Blair medium • Cultivation and isolation: endo-agar, MacConkey, S-S,

Hektoen, XLD• Colonial characters:

– small, 1-2 mm, transparent, round / irregular contour, convex, lactose-negative (colourless i.e. colour of the culture medium)

– No H2S production

Hektoen agar: Shigella – colourless colonies;

Salmonella - green colonies with black centre

Shigella – identification, continued

• Biochemical characters: API 20E

Genus Shigella – Bacteriological diagnosis- continued -

• Antigenic structure based identification:– Agglutination with sets of anti-

sera (polyvalent + monovalent: subgroups + typing)

Genus Klebsiella

• Comensal/Facultatively pathogenic: Colonizes the respiratory mucosa and the intestine

• In immunosuppressed patients (premature infants, elderly people) – potential for severe infections (pneumonia, sepsis, meningitis)

• Hospital acquired infections: surgical wound infections, urinary infections, sepsis

• Involvement in diarrhoeic diseasae - debated

Genus Klebsiella

4 species important for human pathology:• Klebsiella pneumoniae – comensal of human airways

and intestinal mucosa; facultatively pathogenic• Klebsiella oxytoca - idem• Klebsiella ozenae – ozena = chronic inflammatory

infection of the nasal mucosa; mucosal atrophia, crusts & purulent secretions with unpleasant odour

• Klebsiella rhinoscleromatis – rhinoscleroma = chronic hypertrophic rhinitis with granulomatous lesions

Genus Klebsiella

Common characters:• Gram negative, short bacilli,

rounded ends, nonsporulating, arranged in diplo (in pairs) on the long axis, enacpsulated

• Sometimes bipolar staining (ends more intensly stained than middle of rod)

Genus Kelbsiella

Isolation:• Specimens from normally sterile sites (blood, CSF, etc):

– Nutrient broth + reinoculation on blood agar

• Specimens from highly contaminates sites (e.g. faeces):– Media for enterobacteria: MacConkey, XLD

• Identification:– Blood agar – large, white-grey colonies, mucous, aspect of

”pouring culture” – in time the colour changes to brown (”chameleoning” phenomenon)

– MacConkey – large, mucoid colonies, red/pink (lactose positive) – in time colour changes to yellow (lactose negative) – “chameleoning” by alkalinisation of the medium

Klebsiella – blood agar (non hemolytic mucoid colonies)

Klebsiella: Mucous colonies

Klebsiella – pink colonies (Lactose positive) on MacConkey agar

Klebsiella colonies on MacConkey: colour starts to change - Lactose positive (red/pink) colonies start

to change to lactose negative (yellow) – alkalinisation

Klebsiella – identification – continuedBiochemical characters

Genus Proteus

• 4 species: – P.vulgaris, P.mirabilis, P.penneri, P. myxofaciens

• Common characters: – Gram negative, short bacilli, rounded ends, high polymorphism,

high motility (peritrichous cili), nonsporulating, nonencapsulated

• Habitat: – soil, trash, sewage, altered meat, etc. – involved in putrefaction

processes

• Clinical significance: – comensal of human and animal digestive flora;

– facultatively pathogenic: UTI, otitis, synusitis, meningitis, sepsis (community or hospital acquired infections)

Genus Proteus

• Collection of specimens: – urine, faeces, pus, sputum, CSF, blood, etc

• Direct microscopy: – only for naturally sterile specimens e.g. CSF– PMNs + Gram negative bacilli, noncharacteristic arrangement +

filamentous bacilli (high polymorphism of Proteus spp)

• Isolation and identification:– Blood agar: swarming phenomenon (concentric growth waves

invading the entire plate after overnight incubation); invades other bacterial colonies; no isolated colonies

– Selective media: round colonies, same colour as the medium/transparent, black centre (”cat‘s eye”) – H2S production

Proteus

• Swarming phenomenon on tryptic soy agar

Genera Morganella and Providencia

• Previously classified as species of the genus Proteus• Morganella morganii (formerly: Proteus morganii)• Providencia (formerly: Proteus rettgeri)

• Involved in UTI especially in urinary catheterized patients

Genera Proteus, Morganella, Providencia

Genus Yersinia

3 species important for human pathology:

1. Y. enterocolitica: – intestinal pathogen (some strains produce an enterotoxin similar

to E.coli; may infect abdominal lymph nodes – apendicitis-like symptoms)

• Isolation: faeces inoculated on selective media:– MacConkey – colonies much smaller than of other

enterobacteria– CIN (cefsulodin, irgasan, novobiocin): overnight incubation at

32°C/48 hours at 25°C: transparent colonies→ (at 48 hours): larger, pink colonies (increased motility at room temperature)

Genus Yersinia

3 species important for human pathology (continued):

2. Y.pseudotuberculosis• Enteric infection involving also abdominal lymph vessels

and nodes• Collection of specimens, Isolation and identification

similar with Y.enterocolitica

• Differential diagnosis based on biochemcal tests

Genus Yersinia

3 species important for human pathology (continued):

3. Y.pestis – Plague:– reservoir of germs: rodents (rats) – interpersonal transmission (human to human)

– Routes of infection:

• Vectors: Flea bites →skin lesions (inflammation, necrosis, purulent secretion) + swollen lymph nodes (buboes) = bubonic plague →sepsis

• Airborne: Inhalation →pneumonia = pulmonary plague

Left: Oriental rat flea (vector of Y.pestis)Right, upper image: Y.pestis infected flea bite

Right, lower image: swollen lymph nodes (buboes)

Yersinia on blood agar

Yersinia on MacConkey

Yersinia – medium with lactose and bile salts

Yersinia on Hektoen agar

Yersinia agar

Yersinia – identification: API 20E gallery

Hektoen agar inoculated with stool sample

• E.coli – red arrow• Salmonella – blue

arrow• Proteus – yellow

arrow

S-S agar:A = Klebsiella; B = E.coli; C = Salmonella; D = Proteus;

E = Ps.aeruginosa

• Klebsiella and E.coli – ferment sugars (red colonies)

• Salmonella and Proteus – H2S production (black centre)

• Pseudomonas aeruginosa – colourless colonies