Research Article Microbiological Quality Assessment by PCR ...

Research ArticleMicrobiological Quality Assessment by PCR andIts Antibiotic Susceptibility in Mangrove Crabs (Ucidescordatus) from Guanabara Bay Rio de Janeiro Brazil

M C N Carvalho1 M M Jayme2 G S Arenazio3 F V Arauacutejo3

S G F Leite4 and E M Del Aguila1

1 Instituto de Quımica Universidade Federal do Rio de Janeiro Avenida Athos da Silveira 149 Bloco A Cidade Universitaria21949-909 Rio de Janeiro RJ Brazil2Faculdade de Ciencias Medicas Universidade do Estado do Rio do Janeiro (UERJ) Avenida Professor Manoel de Abreu 4442∘ andar Vila Isabel 20550-170 Rio de Janeiro RJ Brazil3Faculdade de Formacao de Professores Universidade do Estado do Rio de Janeiro (FFP-UERJ) Rua Dr Francisco Portela 1470Patronato 24435-005 Sao Goncalo RJ Brazil4Escola de Quımica Universidade Federal do Rio de Janeiro (UFRJ) Avenida Athos da Silveira 149 Bloco E Cidade Universitaria21949-909 Rio de Janeiro RJ Brazil

Correspondence should be addressed to E M Del Aguila emdaiqufrjbr

Received 15 November 2015 Accepted 10 February 2016

Academic Editor Giuseppe Comi

Copyright copy 2016 M C N Carvalho et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The bacteriological quality of crabs from three different mangroves (Itaoca Suruı and Piedade) from Rio de Janeiro state Brazilwas investigated using conventional and molecular methods The results revealed high counts for total coliforms in meat andhepatopancreas samples PCR analyses identified 25 Escherichia coli colonies in the Itaoca Piedade and Suruı samples detecting13 enterotoxigenic colonies and 9 enteroaggregative colonies Respectively 12 11 and 21 Vibrio parahaemolyticus strains weredetected in the Itaoca Piedade and Suruı samples Two V cholerae strains were detected in the Piedade samples The E coli strainsisolated in the present study showed resistance to gentamicin E coli strains from the Piedade samples showed 33 resistance tochloramphenicol and the strains also showed multiresistance to several antimicrobial agents with a MAR index ranging from 012to 031Vibrio strains from Piedade Itaoca and Suruı showed 86 78 and 85 resistance respectively to ampicillinThe isolatedVibrio strains showed multiresistance to several antimicrobial agents with a MAR index ranging from 012 to 025 The presenceof these organisms in crab meat is an indication of microbial contamination which may pose health risks to consumers whenimproperly cooked

1 Introduction

Brazil has ca 8500 km of coastline with the second largestmangrove area on Earth [1] These mangroves have suf-fered extensively with urbanization and industrialization incoastal regions and over the years extensive ecosystemshave disappeared ending many of their important functionssuch as being buffers against coastal erosion retaining somepollutants and being fishery areas [2]

Crabs are decapod crustaceans rich in sodium potas-sium and phosphorus with high amounts of iron zinc

copper and manganese They also present high concentra-tions of vitamins A C B6 thiamine and riboflavin andare considered a delicacy in several parts of the world [3]Along the Brazilian coast crabs are one of themost importantnatural resources in estuarine regions and can be intenselyexploited without reaching an overfishing threshold mainlybecause the picking method allows for the identification ofthe female individuals which are of a different size comparedto the males and their release back into the environment [4]

Among the large and diverse range of mangrove prod-ucts in the Brazilian north and north-eastern estuaries the

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2016 Article ID 7825031 9 pageshttpdxdoiorg10115520167825031

2 International Journal of Microbiology

mangrove crab Ucides cordatus is the most harvested withthe highest commercial and subsistence importance to ruralhouseholds of the coastal population [5] Environment qual-ity as well as the mode of collection and processing of prod-ucts may affect the quantity and diversity of the microorgan-isms present on the surface of seafood and fishery productswhich may cause increases in microbial contamination [6]

Beside the concern regarding the fecal contamination ofhuman foods from marine ecosystems starting in the late1960s various indigenous bacteria from estuarine andmarinewaters were also recognized as potential human pathogensThey can be concentrated in shellfish presenting humanhealth risks [7] The main concern is with regard to severalspecies of Vibrio such as Vibrio parahaemolyticus Recentstudies have also identified shellfish as sources of Vibriocholerae Vibrio vulnificus and other Vibrio species in casesof human infections [8] Some of these human pathogens cansurvive and grow at the low temperatures that characterizemarine ecosystems

Vibrios are Gram-negative bacteria that are primarilyassociated with estuarine and coastal marine environmentsA number of species have been associated with intestinalor extraintestinal infections in humans All Vibrios have anabsolute requirement of Na+ for growth although some suchas V cholerae only require trace amounts Only a small pro-portion of the Vibrios belong to species potentially patho-genic in humans and of these only a small proportion maypossess the pathogenicity traits that enable them to colonizeand cause disease in the human body [9]

Marine Vibrios naturally contaminating bivalve molluskshave been shown to be harder to remove by depuration thanfecal bacterial indicators such as E coli [10] Such processingmethods may therefore not provide the necessary level ofpublic health protection if significant levels of pathogenicVibrios are present in the harvested product

Escherichia coli is a commensal microorganism whoseniche is the mucous layer of the mammalian colon It is themost abundant facultative anaerobe of the human intestinalmicroflora [11] Furthermore E coli is widely distributed inthe intestinal tracts of warm-blooded animals [12] E coli isoften nonpathogenic although different strains may causediseases in the gastrointestinal urinary or central nervoussystems [13] Currently six categories of diarrheagenic Ecoli have been acknowledged enterotoxigenic E coli (ETEC)[14] enteropathogenic E coli (EPEC) [15] enteroinvasive Ecoli (EIEC) [16] enterohemorrhagic E coli (EHEC Shigatoxin-producing E coli or STEC) [17 18] enteroaggregativeE coli (EAEC or EAggEc) [19] and diffusely adherent E coli(DAEC) [20] Despite not being very common the isolationof diarrheagenic E coli from seafood has been reportedIn Brazil Ayulo et al (1994) [21] isolated only one strainof STEC from shellfish and gave evidence that preventivemeasures especially during harvest and postharvest are ofmajor importance to avoid contamination of any nature

Detection of pathogenic bacteria in seafood is essentialto ensure safe products for consumers sustainable fish andshellfish growing activities Molecular diagnostic methodshave evolved significantly in the last few years and arenow established as useful and reliable methods to allow the

rapid detection and identification of pathogens Moleculardetection identification and enumeration of Vibrio spp arelargely based on PCR amplification following purification ofnucleic acids from the samples Although less sensitive andmore time consuming DNA or oligonucleotide probe-basedhybridization methods have been proposed for the detectionof Vibrio spp in food [22]

Herein the presence of potentially pathogenic isolates(Vibrio and Escherichia coli strains) from Ucides cordatuscrabs from the Guanabara Bay Rio de Janeiro Brazil isreported using both conventional (biochemical identifica-tion) and molecular (PCR) methods The antibiotic suscep-tibility of the isolates was also evaluated

2 Material and Methods

21 Study Area The mangroves selected for this study arelocated in Itaoca (Sao Goncalo) Piedade (Mage) and Suruı(Mage) in Guanabara Bay Rio de Janeiro Brazil where thegathering of this crustacean for marketing is more intense

22 Sample Collection Thirty live crabs (Ucides cordatus)were collected between March 2012 and June 2014 in eachmangrove studied These samples were analyzed at the Lab-oratory of Environmental Microbiology at the University ofthe State of Rio de Janeiro (UERJ) The crabs were washedto remove any excess sediment and other impurities presenton their bodies The viscera and meat were removed witha sterile forceps and a scalpel and placed into sterile Petridishes Twenty-five grams of each sample were mixed with225mL of buffered peptone water and the suspensions weretransferred to homogenizer bags (Interscience Saint NomFrance) and coupled to a Stomacher 400 circulator (SewardWorthing West Sussex UK) at 260 rpm for 1min [23] Thesuspensions were serial-diluted from 10minus6 to 100 and 100 120583Lof each dilution was transferred onto specific broths

23 Microbiological Analyses of Crab Samples The tests usedfor the determination of E coli andVibrio spp are establishedin the Methods for the Microbiological Examination ofFoods The reference strains used as controls were providedby the Oswaldo Cruz Foundation Rio de Janeiro Brazil

231 Fecal Coliforms Analyses Twenty-five grams of tis-sue were immersed in 225mL of lactose broth (HimediaMumbai India) for 48 hours at 35∘C Subsequently 10minus1 to10minus4 dilutions were carried out with 9mL of saline solutionfor posterior inoculation in lauryl sulfate broth (HimediaMumbai India) at 35∘C for 24 h An 100 120583L aliquot of eachpositive tube lauryl sulfate broth (Himedia Mumbai India)was transferred to a corresponding tube containing 3mL ofEC broth (HimediaMumbai India) with 5Durham tubes for24 hours with a series of dilutions and replicates in a waterbath at 445∘C [23] to determine the MPN (most probablenumber) coliform bacteria by counting

232 Escherichia coli Detection An 100 120583L aliquot wasremoved from the tube containing 3mL of positive EC broth(Merck Darmstadt Germany) and transferred to agar plates

International Journal of Microbiology 3

containing EMB (Merck Darmstadt Germany) The plateswere incubated for 24 hours at 37∘C The presumptive Ecoli spp colonies were submitted to biochemical tests SIM(Sulfide-Indole-Motility) (BioBras Minas Gerais Brazil)citrate (Citrate of Simmons) (Difco Sparks MarylandUSA) and MRVP Broth (methyl redVoges-Proskauer)(Merck Darmstadt Germany) [24]

233 Vibrio spp Detection Twenty-five grams of crab meatand viscera were immersed in 225mL of lactose broth (Hime-dia Mumbai India) for 48 hours at 35∘C and transferred to1mL tubes containing BHI (Heart Brain Infusion) (HimediaMumbai India) with 1 and 3 of NaCl and incubated for24 h at 37∘C A 100 120583L aliquot was transferred to plates con-taining TCBS agar (Himedia Mumbai India) and were incu-bated for 24 h at 37∘C The presumptive Vibrio spp colonieswere submitted to biochemical characterization tests oxidasetest Oxidation-Fermentation (OF) (Difco SparksMarylandUSA) inositol (Difco Sparks Maryland USA) and O129(Celon-Lab Madhapur Hyderabad India) [25]

24 Molecular Analyses

241 DNA Extraction DNA preparation was carried out bythe thermal shock method from all the harvested coloniesThe colonies were grown in 3mL of BHI broth harvested after24 h at 37∘C OnemL of themediumwas transferred to sterileEppendorf tubes and centrifuged for 10min at 12000 g Thesupernatant was discarded and the pellet was resuspendedin 400 120583L of pure sterile water After homogenization thesupernatant was boiled for 10min cooled on ice for 5minand then collected and used for the PCR analyses [26]

242 PCR Amplification for the E coli Virulence GenePCR was performed using multiplex JMS1 LT VirA andEAE oligos and PCR-uniplex for AggRks and EAST1 oligos(Table 1) The reactions contained a final volume of 25 120583Lcontaining 5 120583L of template DNA buffer (10x) 10mM dNTP25mMMgCl

2 2 U Taq polymerase (Invitrogen Technolo-

gies Sao Paulo Brazil) and 10mM of each primer (Invit-rogen Technologies Sao Paulo Brazil) The conditions ofreaction were 94∘C for 5min 30 cycles of 1min at 94∘C 1minat 58∘C 2min at 72∘C and a final cycle of 72∘C for 10min forall reactions PCR amplicons were visualized on 2 agarosegels stained with 3 120583L of ethidium bromide (05mgmLminus1)visualized on a UV light transilluminator (Uvitec Cam-bridge UK) and photodocumented by ldquoPolaroidrdquo (CanonSao Paulo Brazil)

243 PCR Amplification for the Vibrio sp Gene The reactionwas performed using multiplex oligos in a final volume of20120583LThemixture contained 2UTaqpolymerase (InvitrogenTechnologies Sao Paulo Brazil) 10mM dNTPs buffer (10x)25mMMgCl

2 3 120583L of template DNA and 10mM primers

(sodB sodB flaE hsp and 16S) (Table 2) [28] The conditionsof reaction were 5min at 93∘C followed by 35 cycles of 92∘Cfor 40 s 57∘C for 1min and 72∘C for 15min and a finalcycle at 72∘C for 7min for all reactions PCR amplicons werevisualized on 2 agarose gels stained with 3120583L of ethidium

bromide (05mgmLminus1) visualized on a UV light transillu-minator (Uvitec Cambridge UK) and photodocumented byldquoPolaroidrdquo (Canon Sao Paulo Brazil)

25 Antibiotic Susceptibility Test The microorganisms wereinoculated at a concentration equivalent to 05McFarlandunits (Probac Durban South Africa) onto a Muller Hintonagar plate (Difco Sparks Maryland USA) The antibioticdiscs were placed on the plates and incubated overnightat 37∘C The inhibition zone was interpreted according tothe Clinical Laboratory StandardsM100-S22 Guidelines [29]formerly known as the National Committee for Clinical Lab-oratory StandardsThe tested antibiotics were chlorampheni-col (30 120583g) tetracycline (30 120583g) gentamicin (10 g) amikacin(30 120583g) tobramycin (10 g) trimethoprim-sulfamethoxazole(1252375 120583g) cephalothin (30 120583g) ampicillin (10 g) cef-tazidime (30 120583g) cefotaxime (30 120583g) cefepime (30 120583g) aztre-onam (30 120583g) cefoxitin (30 120583g) imipenem (10 g) ampicillin-sulbactam (10 120583g-10 120583g) and ciprofloxacin (5 g) For qualitycontrol E coli ATCC 25922 and E coli ATCC 35218 weretested under the same conditions

For strains confirmed as Vibrio spp the test was per-formed according to the standard document M45-A2 [29]with the same antibiotic disks used for E coli (OxoidHampshire UK) with the exception of tobramycin (10 g) andaztreonam (30 120583g) and with the addition of levofloxacin (5 g)and ofloxacin (5 g)

The inhibition halos were measured with the aid of amillimeter ruler

3 Results

31 Fecal Coliforms High concentrations of fecal coliforms(62 times 102 and 72 times 102NMPgminus1) were found in the meatand hepatopancreas samples from the Itaoca mangroverespectivelyThe samples from Piedade and Suruı mangrovesshowed concentrations of 24 times 102 and 32 times 102NMPgminus1in meat samples respectively and 25 times 102 and 35 times102NMPgminus1 in hepatopancreas samples respectively Nosignificant difference was observed among the thermotoler-ant coliform values found in the meat and hepatopancreassamples between the mangroves (119901 lt 005)

32 Escherichia coli Detection Multiplex PCR enabled theidentification of 4 virulence genes (eaeA stx1 lt and virA)in single reaction (Figure 1)

Forty-six E coli colonies isolated from the crab samplesof the different mangroves (21 from meat and 25 coloniesfrom hepatopancreas) were confirmed by biochemical testsAfter biochemical characterization the molecular test (PCR)revealed that 25 (543) were positive for the researchedvirulence genes 9 presenting eastA (36) 13 presenting lt(52) and 3 presenting stx (12) No colonies presentingvirA eaeA st and agg genes were detected (Table 3)

Fourteen E coli strains were isolated from Itaoca withthe presence of virulence genes 2 presenting stx1 (hepatopan-creas) 7 presenting lt (4 in meat and 3 in hepatopancreas)and 5 presenting eastA (4 in meat and 1 in hepatopancreas)


Table 1 Primer sequences used for the identification of Escherichia coli virulence

Serotypes Oligonucleotides Gene Sequences (51015840-31015840) Fragment size (bp)

EPEC EAE-a eaeA ATG CTT AGT GCT GGT TTA GG 248EAE-b GCC TTC ATC ATT TCG CTT TC

EHEC JMS1-F stx1 GTC ACA GTA ACA AAC CGT AAC A 95JMS1-R TCG TTG ACT ACT TCT TAT CTG GA

ETEC

LT-1 lt AGC AGG TTT CCC ACC GGA TCA CCA 132LT1-2 GTG CTC AGA TTC TGG GTC TCSta-F st GCT AAT GTT GGC AAT TTT TAT TTC TGT A 190Sta-R AGG ATT ACA ACA AAG TTC ACA GCA GTA A

EAEC

Aggrks-1 aggR GTA TAC ACA AAA GAA GGA AGC 254Aggrks-2 ACA GAA TCG TCA GCA TCA GCEast1s astA GAG TGA CGG CTT TGT AGT CC 106East1sa GCC ATC AAC ACA GTA TAT CC

EIEC VirA-F virA CTG CAT TCT GGC AAT CTC TTC ACA 215VirA-R TGA TGA GCT AAC TTC GTA AGC CCT CC

The pathotypes and virulence genes for the E coli detected in this study are EPEC enteropathogenic E coli EHEC enterohemorrhagic E coli ETECenterotoxigenic E coli EAEC enteroaggregative E coli and EIEC enteroinvasive E coli (adapted from Bisi Johnson et al 2011 [27])

Table 2 Oligonucleotide sequences used for the identification of the Vibrio genus and serotypes


Vibrio spp V16S-700F 16S CGG TGA AAT GCG TAG AGA T 663V16S1325R TTA CTA GCG ATT CCG AGT TC

V cholerae VcsodB-F sodB AAG ACC TCA ACT GGC GGT A 248VcsodB-R GAA GTG TTA GTG ATC GCC AGA GT

V mimicus VmsodB-F sodB CAT TCG GTT CTT TCG CTG AT 121VmsodB-R2 GAA GTG TTA GTG ATT GCT AGA GAT

V parahaemolyticus VpflaE-79F flaE GCA GCT GAT CAA AAC GTT GAG T 897Vpflae-934R ATT ATC GAT CGT GCC ACT CAC

V vulnificus Vvhsp-326F hsp GTC TTA AAG CGG TTG CTG C 410Vvhsp-697R CGC TTC AAG TGC TGG TAG AAG

500pb

200pb100pb

215pb 248pb

132pb95pb

254pb

106pb190pb

LD virA eaeA lt stx1 eastA stagg

Figure 1 Specific amplicons of E coli virulence genes E coli viru-lence gene (virA) enteroinvasive E coli (eaeA) enteropathogenic Ecoli (lt and st) enterotoxigenic E coli (stx1) enterohemorrhagic Ecoli (astA) and enteroaggregative E coli (agg)

Eight strains were detected in samples from the Suruı man-grove where 4 strains showed the lt virulence gene (2 inmeat and 2 in hepatopancreas) and 4 strains showed the eastvirulence gene (2 in meat and 2 in hepatopancreas) Thirteen

Table 3 Expression of E coli virulence genes by PCR distributed bymangrove

Strains Gene Itaoca(119899 = 14)

Piedade(119899 = 3)

Suruı(119899 = 8)

Enteropathogenic eaeA 0 0 0Enterohemorrhagic stx1 2 1 0

Enterotoxigenic lt 7 2 4st 0 0 0

Enteroaggregative agg 0 0 0eastA 5 0 4

Enteroinvasive virA 0 0 0

strains were detected in the samples from the Piedade man-grove by means of the biochemical test but only one showedthe presence of the stx1 virulence gene (meat) while 2 showedthe presence of the lt virulence gene (hepatopancreas)

33 Vibrio spp Detection Suruı mangrove samples showedthe highest incidence of isolated Vibrio (46) followed by


Table 4 Distribution of Vibrio strains by mangrove

Strains Gene MangroveItaoca (119899 = 33) Piedade (119899 = 40) Suruı (119899 = 46) Total (119899 = 119)

Vibrio spp 16S 21 27 25 73V cholerae sodB 0 02 0 02V parahaemolyticus flaE 12 11 21 44V mimicus sodB1 0 0 0 0V vulnificus hsp 0 0 0 0119899 number of strains

V16S500pb

100pb121pb

248pb

663pb

410pb

897pb

LD Vm Vc Vv Vp

Figure 2Amplicons of specificVibrio genes LDmolecularmarkerVm V mimicus Vc V cholerae Vv V vulnificus and Vp V para-haemolyticus

Piedade (40) and Itaoca (33) One hundred and nineteenVibrio strains were confirmed by PCR in 90 samples (meat68 and hepatopancreas 51) The present study identified 5different genes one for theVibrio spp genus and 4 for species(Figure 2) A similar study was carried out by Teh et al (2010)[30] using multiplex PCR (identifying the gyrB and pntAgenes) to differentiate V parahaemolyticus V cholerae Vvulnificus and other Vibrio spp from fish

Among the researched Vibrio genus 613 (73119) ofthe samples were detected using only the 16S gene forthe Vibrionaceae family Pathogenic strains V cholerae andV parahaemolyticus were found with a frequency of 17(02119) and 37 (44119) respectively (Table 4) V choleraewas only detected in the crab samples from Piedade man-grove The highest incidence of V parahaemolyticus wasobserved in samples from the Suruı mangrove (21) followedby Itaoca (12) and Piedade (11)

No V mimicus and V vulnificus were detected in the pre-sent study (Table 4)

34 Antimicrobial Susceptibility Test The resistance resultsare displayed in Table 5 Twenty-six E coli strains showedsome resistance to the tested antimicrobials with a high indexof resistance E coli strains isolated from the Itaoca samplesshowed high resistance (63) against gentamicin (CN) andtobramycin (TOB)

E coli strains found at Piedade Itaoca and Suruı showedresistance to gentamicin (66 63 and 22 resp) TheE coli isolates from the Piedade samples showed 33 resis-tance to chloramphenicol (C) Only strains found in crabs

from the Piedade mangrove showed resistance (16) toampicillin (AMP) No resistance to amoxicillin + clavulanicacid (AMC) levofloxacin (LEV) cefoxitin (CTX) ofloxacin(OFX) and ciprofloxacin (CIP) was observed

Among the 26 resistant E coli strains 12 were resistantto two or more antibiotics (Table 6) This pattern is mainlydue to the indiscriminate use of antimicrobials and maycause serious impacts on human health [31 32] The Ecoli strains showed multiresistance to several antimicrobialagents with MAR indices ranging from 012 to 031 whereas3 strains showedMAR indexes from 012 to 025 and 3 strainspresentedMAR indexes of 018 (Table 6)The resistance of the26 E coli strains was distributed as follows 12 strains wereresistant to gentamicin and tobramycin 4 were resistant toamikacin and cephalothin 3 were resistant to ciprofloxacintetracycline ceftazidime and cefoxitin and one strain wasresistant to ampicillin

When 119 Vibrio strains were analyzed only 72 isolates(605) showed resistance to some of the tested antimicro-bials with higher rates in those isolated from crabs samplesfrom Piedade (29) followed by Itaoca (23) and Surui (20)(Table 5) The Vibrio strains from Piedade Itaoca and Suruıshowed resistance to ampicillin (86 78 and 85 resp)The strains from Surui showed 5 resistance to amoxicillin+ clavulanic acid (AMC) ampicillinsulbactam (SAM) andchloramphenicol (C) The Piedade strains showed resistanceto levofloxacin (LEV) and ciprofloxacin (CIP) No resistanceto cefoxitin (CTX) ceftazidime (CAZ) tobramycin (TOB)and tetracycline (TE) was observed

Vibrio strains isolated from crabs showed multiresistanceto several antimicrobial agents presenting a MAR indexranging from 012 to 025 24 strains presented MAR indicesof 012 (Table 6) 5 strains showed MAR indices of 018 andtwo strains showed MAR indices of 025 MAR indexes

The resistance of the 72 strains was distributed as follows60 strains were resistant to ampicillin 14 were resistant toamikacin (AK) 10 were resistant to cephalothin (KF) 8 wereresistant to cefoxitin (CTX) 5 were resistant to gentamicin(CN) 3 were resistant to ciprofloxacin (CIP) and 1 strainwas resistant to amoxicillin + clavulanic (AMC) ampicillin +sulbactam (SAM) levofloxacin (LEV) ofloxacin (OFX) andchloramphenicol (C)

4 Discussion

The thermotolerant coliforms found in the present study areabove the maximum permissible limit (maximum tolerance


Table 5 E coli and Vibrio resistance of strains isolated from crab to the tested antimicrobials

Antimicrobialagent resistance

E coli VibrioItaoca (119899 = 11) Piedade (119899 = 06) Suruı (119899 = 09) Itaoca (119899 = 23) Piedade (119899 = 29) Suruı (119899 = 20)

AMP mdash 16 mdash 78 86 85AMC mdash mdash mdash mdash mdash 5SAM mdash 16 11 mdash mdash 5KF 19 16 11 8 17 15CTX mdash mdash mdash 13 17 mdashCN 63 66 22 4 6 10CFO 9 16 mdash mdash mdash mdashCAZ 9 16 mdash mdash mdash mdashLEV mdash mdash mdash mdash 3 mdashOFX mdash mdash mdash 4 mdash mdashTOB 63 66 22 mdash mdash mdashAK mdash 33 22 17 24 15TE mdash 16 11 mdash mdash mdashCIP mdash mdash mdash mdash 3 10C 9 33 mdash mdash mdash 5119899 number of tested strains AMP ampicillin AMC amoxicillin + clavulanic acid SAM ampicillinsulbactam KF cephalothin CTX cefotaxime CFOcefoxitin CAZ ceftazidime LEV levofloxacin CIP ciprofloxacin OFX ofloxacin CN gentamicin TOB tobramycin AK amikacin TE tetracycline Cchloramphenicol

of 5 times 101NMPgminus1 for coliforms at 45∘C) in bivalve molluskscrab meat and similar samples according to laws from theBrazilian Sanitary Vigilance Agency (Agencia Nacional deVigilancia Sanitaria (ANVISA)) [33] Similar results werefound with regard to the microbiological quality of Uca crabmeat in 3 different points at Praia do Futuro located inFortaleza CE Brazil where thermotolerant coliforms weredetected ranging from 30 to 1100NMPgminus1 in 90 analyzedcrabs [34] According to de Lima Grisi and Gorlach-Lira(2010) [35] the presence of this group of bacteria is associ-ated to the dumping of fecal material in the environmentGuanabara Bay receives effluents without treatment daily andhas become bacteria reservoir which in turn has caused thecontamination of fish and other biota in this region [36]

The expression of E coli virulence genes is a public healthrisk since these genes characterize the presence of toxinsable to cause disease E coli cells are the main pathogensassociated to gastroenteritis of food origin in humans pro-voking diarrhea hemorrhagic colitis and hemolytic-uremicsyndrome [37] However some studies reporting humaninfection by E coli due to crab consumption are available[38] Despite the absence of the virA eae st and agg genes inthe present study the confirmation of E coli strains indicatesrecent fecal contamination in crabs and this indicates thatmajor care in the preparation of this type of food is required

The results regarding the presence of Vibrio can beexplained by the salinity and temperature of the studiedmangroves [39] Many studies show the presence of Vibrio inaquatic animals such as fish [40] shrimp [41] and mussels[42] but despite the importance of crabs only some studieshave been conducted on crab contamination byVibrio How-ever its occurrence inmarine food is pointed as amajor causeof gastroenteritis in the United States and Europe [43] and

associated with cases in Brazil and Chile [44] These resultssuggest a probable health risk for people that consume rawand undercooked seafood According to Alam et al (2012)[45] V vulnificus and V mimicus are most commonly foundin coccoid viable but not culturable form while anotherstudy confirmed the presence of Vibrio in crabs marketed inFortaleza Brazil where only 10 strains were identified up tothe species level 2 V alginolyticus and 8 V parahaemolyticusbut not anyV vulnificus andVmimicus [4] Abd-Elghany andSallam (2013) [46] detected 10 V parahaemolyticus isolates incrab bymolecular identification in Egypt and highlighted thatreliable molecular detection methods should be included inroutine seafood examinations in addition to the conventionalbacteriological methods

These findings of antimicrobial susceptibility are in agree-ment with data from previous studies which found thatresistance to aminoglycosides 120573-lactamase and penicillin iscommon among E coli isolates from food of animal origin[47ndash49] However the resistance frequency in E coli isolatedin the present study was lowwhen compared to other studieswhere a resistance of 58 and 42 in raw fish samplesfromKenya andVietnam respectively was observed [48 50]Mussel samples fromNiteroi (Brazilian Southeastern oceanicregion) showed 29 resistance to at least one antimicrobial[31] and strains isolated from mussels from the GuanabaraBay Rio de Janeiro showed 40 to 85 resistance to testedantimicrobials [51] indicating the intense presence of domes-tic and industrial effluents The percentage of high sensitivityto these antibiotics was also observed by Reboucas et al(2011) [41] in strains isolated from shellfish and is associatedwith various resistance mechanisms found in Gram-negativeorganisms Over time Vibrio strains exposed to antibi-otics through the environment can acquire antimicrobial


Table 6 Multiple antimicrobial resistance of E coli and Vibriostrains found in crab

Antimicrobial resistance MAR indexE coli(3) CN TOB 012(1) CFO TOB 012(1) CN KF TOB 018(2) AK CN TOB 018(1) C CAZ CN TOB 025(1) C CN KF SAM 025(1) CN CFO KF TOB 025(1) AK AMP C CAZ CN TOB 031(1) AK CN KF SAM TE TOB 031Vibrio(14) AK AMP 012(6) AMP KF 012(2) AK KF 012(2) CTX KF 012(2) AK CN CTX 018(1) AK CTX KF 018(2) CN CTX KF 018(1) AK CIP KF LEV 025(1) AK CTX CN KF 025The MAR (multiple antimicrobial resistance) index of an isolate is definedas 119886119887 where 119886 represents the number of antibiotics to which the isolatewas resistant and 119887 represents the number of antibiotics to which theisolate was subjected AK amikacin AMP ampicillin AMC amoxicillin+ clavulanic acid C chloramphenicol CAZ ceftazidime CFO cefoxitinCIP ciprofloxacin CN gentamicin CTX cefoxitin KF cephalothin LEVlevofloxacin OFX ofloxacin SAM ampicillinsulbactam TOB tobramycinTE tetracycline

resistance transferable by mobile genetic elements and hor-izontal gene transfer [52] Thus due to the presence of R-factors in the population resistance developed through generegulation of plasmids and chromosomes may be transferredvertically (by heredity) or horizontally [53] In the presentstudy ampicillin was an antibiotic that showed low efficiencyagainst the 60 testedVibrio spp strains (833) According tothe standard CLSIM45-A2 [54] species belonging to theVib-rio genus have intrinsic resistance to ampicillinThis data wasconfirmed in another study where from 169 Vibrio strainsisolated from shrimp only 3 were sensitive to ampicillin [55]The high percentage of pathogenic Vibrio with reduced sus-ceptibility to ampicillin suggests a potential for the low effi-ciency of ampicillin in the treatment ofVibrio infections [56]

Many cases ofmultiple antimicrobial resistance have beenreported from shellfish farms in countries where the activityis well developed such as China [57] Korea [58] and Chile[59] According to theWorldHealthOrganization changes inthe microbiota can induce the evolution of new pathogenicmicroorganisms and the development of new virulencefactors in ancient pathogens such as the development ofresistance to antimicrobials or changes in their survival abilityin adverse environmental conditions [60]

5 Conclusions

Several E coli and Vibrio isolates were found in crabs(Ucides cordatus) from different mangroves in the state ofRio de Janeiro Brazil Considering the current legislation thepresence of these pathogens in crab indicates contaminationinfluenced by mangrove pollution by using newer molecularmethods and thus contributing to seafood safety Some iso-lated strains showed differential resistance to antimicrobialsThe analyzed samples presented unsuitable hygienic-sanitaryconditions which can be considered a warning to theMunic-ipal Health Surveillance Agency since seafood is many timesconsumedwithout any subsequent thermal treatment or evensufficient thermal treatment able to eliminate pathogenicmicroorganisms causing disorders to consumer health

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

M C N Carvalho carried out the laboratory study andthe interpretation of the data under the supervision of F VAraujo S G F Leite and E M Del Aguila M M Jayme andG S Arenazio collected the samples and participated in themicrobiological analyses E M Del Aguila edited the paperAll authors read and approved the final paper

Acknowledgments

Theauthors acknowledge the financial support from theCon-selho Nacional de Desenvolvimento Cientıfico e Tecnolo-gico (CNPq Brasılia Brazil) and Coordenacao de Aper-feicoamento de Pessoal de Nıvel Superior (CAPES BrasıliaBrazil)

References

[1] B Kjerfve Mangrove Ecosystem Studies in Latin America andAfrica UNESCO Paris France 1997

[2] U Cotano and F Villate ldquoAnthropogenic influence on theorganic fraction of sediments in two contrasting estuaries abiochemical approachrdquoMarine Pollution Bulletin vol 52 no 4pp 404ndash414 2006

[3] R H S F Vieira E A De Lima D B R Sousa E F DosReis R G Costa and D D P Rodrigues ldquoVibrio spp and Sal-monella spp presence and susceptibility in crabs Ucides corda-tusrdquo Revista do Instituto de Medicina Tropical de Sao Paulo vol46 no 4 pp 179ndash182 2004

[4] P Nishidha ldquoNutritional Values of Crabsrdquo 2016 httpwwworganicfactsnet

[5] G Dallabona J de Loyola e Silva and M A A PinheiroldquoSize at morphological maturity of Ucides cordatus (Linnaeus1763) (Brachyura Ocypodidae) in the Laranjeiras Bay SouthernBrazilrdquo Brazilian Archives of Biology and Technology vol 48 no1 pp 139ndash145 2005

[6] C A Ristori S T Iaria D S Gelli and I N G Rivera ldquoPath-ogenic bacteria associated with oysters (Crassostrea brasiliana)


and estuarine water along the south coast of Brazilrdquo Interna-tional Journal of Environmental Health Research vol 17 no 4pp 259ndash269 2007

[7] D L Tion and R J Seidler ldquoVibrio aestuarianus a new speciesfrom estuarine waters and shellfishrdquo International Journal ofSystematic Bacteriology vol 33 no 4 pp 699ndash702 1983

[8] J M Hughes D G Hollis E J Gangarosa and R E WeaverldquoNon-cholera Vibrio infections in the United States clinicalepidemiologic and laboratory featuresrdquo Annals of InternalMedicine vol 88 no 5 pp 602ndash606 1978

[9] R J Lee and R E Rangdale Improving Seafood Products for theConsumer vol 158 of Woodhead Publishing Series in Food Sci-ence Technology and Nutrition Woodhead Publishing Cam-bridge UK 1st edition 2008 edited by T Borresen

[10] G E Rodrick and K R Schneider ldquoVibrios in depurationrdquo inMolluscan Shellfish Depuration W S Otwell G E Rodrick andR E Martin Eds pp 115ndash125 CRC Press Boca Raton FlaUSA 1991

[11] J B Kaper J P Nataro andH L TMobley ldquoPathogenic Escher-ichia colirdquo Nature Reviews Microbiology vol 2 no 2 pp 123ndash140 2004

[12] S Ishii andM J Sadowsky ldquoEscherichia coli in the environmentimplications for water quality and human healthrdquoMicrobes andEnvironments vol 23 no 2 pp 101ndash108 2008

[13] J P Nataro and J B Kaper ldquoDiarrheagenic Escherichia colirdquoClinical Microbiology Reviews vol 11 no 1 pp 142ndash201 1998

[14] C B Dalton E D Mintz J G Wells C A Bopp and RV Tauxe ldquoOutbreaks of enterotoxigenic Escherichia coli infec-tion in American adults a clinical and epidemiologic profilerdquoEpidemiology and Infection vol 123 no 1 pp 9ndash16 1999

[15] M-H Ruchaud-Sparagano M Maresca and B Kenny ldquoEnter-opathogenic Escherichia coli (EPEC) inactivate innate immuneresponses prior to compromising epithelial barrier functionrdquoCellular Microbiology vol 9 no 8 pp 1909ndash1921 2007

[16] M M Levine ldquoEscherichia coli that cause diarrhea enterotox-igenic enteropathogenic enteroinvasive enterohemorrhagicand enteroadherentrdquoThe Journal of Infectious Diseases vol 155no 3 pp 377ndash389 1987

[17] Y Takeda ldquoVibrio parahaemolyticus enterotoxigenic Escheri-chia coli enterohemorrhagic Escherichia coli and Vibrio cho-leraerdquo Proceedings of the Japan Academy Series B vol 87 no1 pp 1ndash12 2011

[18] H S Kumar S K Otta I Karunasagar and I KarunasagarldquoDetection of Shiga-toxigenic Escherichia coli (STEC) in freshseafood and meat marketed in Mangalore India by PCRrdquoLetters in Applied Microbiology vol 33 no 5 pp 334ndash338 2001

[19] C S Beauchamp and N J Sofos ldquoDiarrheagenic Escherichiacolirdquo in Pathogens and Toxins in Foods V K Juneja and J NSofos Eds pp 71ndash94 ASM Press Washington DC USA 2010

[20] I C A Scaletsky S H Fabbricotti R L B Carvalho et alldquoDiffusely adherent Escherichia coli as a cause of acute diarrheain young children in northeast Brazil a case-control studyrdquoJournal of Clinical Microbiology vol 40 no 2 pp 645ndash6482002

[21] A M R Ayulo R A Machado and V M Scussel ldquoEntero-toxigenic Escherichia coli and Staphylococcus aureus in fish andseafood from the southern region of Brazilrdquo International Jour-nal of Food Microbiology vol 24 no 1-2 pp 171ndash178 1994

[22] R A Amin and A M Salem ldquoSpecific detection of pathogenicVibrio species in shellfish by using multiplex polymerase chainreactionrdquo Global Veterinaria vol 8 no 5 pp 525ndash531 2012

[23] APHA (American Public Health Association) Standard Meth-ods for the Examination of Water and Wastewater APHAWashington DC USA 21th edition 2005

[24] P Feng S DWeagant M A Grant andW Burkhardt ldquoEnum-eration of Escherichia coli and the Coliform bacteriardquo in Bac-teriological Analytical Manual US Food Drugs 2002 httpwwwfdagovFoodFoodScienceResearchLaboratoryMethodsucm064948htm

[25] A De Paola and C Kysner ldquoVibriordquo in Bacteriological Ana-lytical Manual FDA 2011 httpwwwfdagovFoodFoodSci-enceResearchLaboratoryMethodsucm070830htm

[26] L R Santos ldquoIdentificacao de Salmonella atraves da reacao emcadeia pela polimerase (PCR)rdquo Arquivos da Faculdade Veteri-naria de UFRGS vol 28 pp 87ndash92 2001

[27] M A Bisi-Johnson C L Obi S D Vasaikar K A Baba andT Hattori ldquoMolecular basis of virulence in clinical isolates ofEscherichia coli and Salmonella species from a tertiary hospitalin the Eastern Cape South Africardquo Gut Pathogens vol 3 no 1article 9 8 pages 2011

[28] C L Tarr J S Patel N D Puhr E G Sowers C A Bopp andN A Strockbine ldquoIdentification of Vibrio isolates by a mult-iplex PCR assay and rpoB sequence determinationrdquo Journal ofClinical Microbiology vol 45 no 1 pp 134ndash140 2007

[29] CLSI Performance Standards for Antimicrobial SusceptibilityTesting Twenty-Second Informational Supplement CLSI Doc-ument M100-S22 Clinical and Laboratory Standards InstituteWayne Pa USA 11th edition 2012

[30] C S J Teh K H Chua and K L Thong ldquoSimultaneous dif-ferential detection of human pathogenic and nonpathogenicVibrio species using a multiplex PCR based on gyrB and pntAgenesrdquo Journal of AppliedMicrobiology vol 108 no 6 pp 1940ndash1945 2010

[31] M M A Jayme Avaliacao microbiologica deteccao e suscep-tibilidade a antimicrobianos de potenciais enteropatogenos dasfamılias Enterobacteriaceae e Vibrionaceae em mexilhoes Pernaperna da praia de Itaipu Niteroi-RJ [MS thesis] Universidadedo Estado do Rio de Janeiro Faculdade de Ciencias Medicas2014

[32] N S Evangelista-Barreto A F Pereira R A R Da Silva and LT B Ferreira ldquoCarne de siri como veıculo na disseminacao deenteropatogenos resistentes aos antimicrobianosrdquo Acta Pescavol 1 pp 45ndash56 2013

[33] Agencia Nacional de Vigilancia Sanitaria (ANVISA) Resolu-caomdashRDC n∘12 de 02 de Janeiro de 2001 Regulamento Tecnicosobre PadroesMicrobiologicos para Alimentos Agencia Nacionalde Vigilancia Sanitaria (ANVISA) Brasılia Brazil 2001

[34] A J G Araujo C O Brandao F C T Carvalho and R H S FVieira ldquoQualidade microbiologica do caranguejo-uca expostoa venda em tres pontos na orla da Praia do Futuro FortalezamdashCEmdashBrasilrdquo Boletim do Instituto de Pesca vol 37 pp 409ndash4162011

[35] T C S de Lima Grisi and K Gorlach-Lira ldquoThe abundance ofsome pathogenic bacteria in mangrove habitats of paraiba donorte estuary and crabmeat contamination of mangrove crabucides cordatusrdquo Brazilian Archives of Biology and Technologyvol 53 no 1 pp 227ndash234 2010

[36] J A Neto and EM Fonseca ldquoVariacao sazonal espacial e com-posicional de lixo ao longo das praias da margem oriental daBaıa de Guanabara (Rio de Janeiro) no perıodo de 1999ndash2008rdquoJournal of Integrated Coastal ZoneManagement vol 11 no 1 pp31ndash39 2011


[37] O E Ojo A T P Ajuwape E B Otesile A A Owoade MA Oyekunle and A I Adetosoye ldquoPotentially zoonotic shigatoxin-producing Escherichia coli serogroups in the faeces andmeat of food-producing animals in Ibadan Nigeriardquo Interna-tional Journal of FoodMicrobiology vol 142 no 1-2 pp 214ndash2212010

[38] P Matulkova M Gobin J Taylor F Oshin K OrsquoConnor andI Oliver ldquoCrab meat a novel vehicle for E coli O157 identifiedin an outbreak in SouthWest Englandrdquo Epidemiology and Infec-tion vol 141 no 10 pp 2043ndash2050 2013

[39] B Austin ldquoVibrios as causal agents of zoonosesrdquo VeterinaryMicrobiology vol 140 no 3-4 pp 310ndash317 2010

[40] U Messelhausser J Colditz D Tharigen W Kleih C Hollerand U Busch ldquoDetection and differentiation of Vibrio spp inseafood and fish samples with cultural andmolecularmethodsrdquoInternational Journal of Food Microbiology vol 142 no 3 pp360ndash364 2010

[41] R H Reboucas O V De Sousa A S Lima F R VasconcelosP B De Carvalho and R H S Dos Fernandes-Vieira ldquoAnti-microbial resistance profile of Vibrio species isolated frommarine shrimp farming environments (Litopenaeus vannamei)at Ceara Brazilrdquo Environmental Research vol 111 no 1 pp 21ndash24 2011

[42] S K Lhafi and M Kuhne ldquoOccurrence of Vibrio spp in bluemussels (Mytilus edulis) from the German Wadden Seardquo Inter-national Journal of Food Microbiology vol 116 no 2 pp 297ndash300 2007

[43] C A M L D Santos and R H S F Vieira ldquoBacteriologicalhazards and risks associated with seafood consumption inBrazilrdquo Revista do Instituto de Medicina Tropical de Sao Paulovol 55 no 4 pp 219ndash228 2013

[44] E Harth L Matsuda C Hernandez et al ldquoEpidemiology ofVibrio parahaemolyticus outbreaks southern Chilerdquo EmergingInfectious Diseases vol 15 no 2 pp 163ndash168 2009

[45] MAlamM T Islam SM Rashed et al ldquoVibrio cholerae classi-cal biotype strains reveal distinct signatures in Mexicordquo Journalof Clinical Microbiology vol 50 no 7 pp 2212ndash2216 2012

[46] SMAbd-Elghany andK I Sallam ldquoOccurrence andmolecularidentification of Vibrio parahaemolyticus in retail shellfish inMansoura Egyptrdquo Food Control vol 33 no 2 pp 399ndash4052013

[47] A Jouini K B Slama Y Saenz et al ldquoDetection of multiple-antimicrobial resistance and characterization of the implicatedgenes in Escherichia coli isolates from foods of animal origin inTunisrdquo Journal of Food Protection vol 72 no 5 pp 1082ndash10882009

[48] T T H Van J Chin T Chapman L T Tran and P J ColoeldquoSafety of raw meat and shellfish in Vietnam an analysis ofEscherichia coli isolations for antibiotic resistance and virulencegenesrdquo International Journal of Food Microbiology vol 124 no3 pp 217ndash223 2008

[49] A Vasilakopoulou M Psichogiou L Tzouvelekis et al ldquoPreva-lence and characterization of class 1 integrons in Escherichiacoli of poultry and human originrdquo Foodborne Pathogens andDisease vol 6 no 10 pp 1211ndash1218 2009

[50] AW Sifuna NM Njagi P Okemo AMunyalo G O Orindaand S Kariuki ldquoMicrobiological quality and safety of Rastri-neobola argentea retailed in Kisumu townmarkets Kenyardquo EastAfrican Medical Journal vol 85 pp 509ndash513 2009

[51] AM Valente Efeito da irradiacao sobre mexilhoes [Perna perna(Linnaeus 1758)] coliformes termotolerantes e enterococcus acao

antimicrobiana e analise sensorial das amostras [Master Disser-tation] Universidade Federal Fluminense Niteroi Brazil 2004

[52] K Kummerer ldquoAntibiotics in the aquatic environmentmdashareviewmdashpart IIrdquo Chemosphere vol 75 no 4 pp 435ndash441 2009

[53] M T Madigan J M Martinko J Parker M G Fernandez CR Fernandez andM S Perez Brock Biology ofMicroorganismsPearson Education Upper Saddle River NJ USA 2003

[54] CLSIMethods for Antimicrobial Dilution andDisk SusceptibilityTesting of Infrequently Isolated or Fastidious Bacteria ApprovedGuideline CLSI Document M45-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2nd edition 2010

[55] H Ji Y Chen Y Guo X Liu J Wen and H Liu ldquoOccurrenceand characteristics of Vibrio vulnificus in retail marine shrimpin Chinardquo Food Control vol 22 no 12 pp 1935ndash1940 2011

[56] S Sudha C Mridula R Silvester and A A M HathaldquoPrevalence and antibiotic resistance of pathogenic Vibrios inshellfishes from Cochin marketrdquo Indian Journal of Geo-MarineSciences vol 43 no 5 pp 815ndash824 2014

[57] H Dang X Zhang L Song Y Chang and G Yang ldquoMolecularcharacterizations of oxytetracycline resistant bacteria and theirresistance genes from mariculture waters of Chinardquo MarinePollution Bulletin vol 52 no 11 pp 1494ndash1503 2006

[58] H Y Kang Y S Jeong J Y Oh et al ldquoCharacterization of anti-microbial resistance and class 1 integrons found in Escherichiacoli isolates from humans and animals in Koreardquo Journal ofAntimicrobial Chemotherapy vol 55 no 5 pp 639ndash644 2005

[59] C D Miranda and R Rojas ldquoOccurrence of florfenicol resis-tance in bacteria associatedwith twoChilean salmon farmswithdifferent history of antibacterial usagerdquo Aquaculture vol 266no 1ndash4 pp 39ndash46 2007

[60] World Health Organization (WHO) ldquoFood Safety and Food-borne Illnessrdquo 2016 httpwwwwhointmediacentrefact-sheetsfs399en

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


mangrove crab Ucides cordatus is the most harvested withthe highest commercial and subsistence importance to ruralhouseholds of the coastal population [5] Environment qual-ity as well as the mode of collection and processing of prod-ucts may affect the quantity and diversity of the microorgan-isms present on the surface of seafood and fishery productswhich may cause increases in microbial contamination [6]

Beside the concern regarding the fecal contamination ofhuman foods from marine ecosystems starting in the late1960s various indigenous bacteria from estuarine andmarinewaters were also recognized as potential human pathogensThey can be concentrated in shellfish presenting humanhealth risks [7] The main concern is with regard to severalspecies of Vibrio such as Vibrio parahaemolyticus Recentstudies have also identified shellfish as sources of Vibriocholerae Vibrio vulnificus and other Vibrio species in casesof human infections [8] Some of these human pathogens cansurvive and grow at the low temperatures that characterizemarine ecosystems

Vibrios are Gram-negative bacteria that are primarilyassociated with estuarine and coastal marine environmentsA number of species have been associated with intestinalor extraintestinal infections in humans All Vibrios have anabsolute requirement of Na+ for growth although some suchas V cholerae only require trace amounts Only a small pro-portion of the Vibrios belong to species potentially patho-genic in humans and of these only a small proportion maypossess the pathogenicity traits that enable them to colonizeand cause disease in the human body [9]

Marine Vibrios naturally contaminating bivalve molluskshave been shown to be harder to remove by depuration thanfecal bacterial indicators such as E coli [10] Such processingmethods may therefore not provide the necessary level ofpublic health protection if significant levels of pathogenicVibrios are present in the harvested product

Escherichia coli is a commensal microorganism whoseniche is the mucous layer of the mammalian colon It is themost abundant facultative anaerobe of the human intestinalmicroflora [11] Furthermore E coli is widely distributed inthe intestinal tracts of warm-blooded animals [12] E coli isoften nonpathogenic although different strains may causediseases in the gastrointestinal urinary or central nervoussystems [13] Currently six categories of diarrheagenic Ecoli have been acknowledged enterotoxigenic E coli (ETEC)[14] enteropathogenic E coli (EPEC) [15] enteroinvasive Ecoli (EIEC) [16] enterohemorrhagic E coli (EHEC Shigatoxin-producing E coli or STEC) [17 18] enteroaggregativeE coli (EAEC or EAggEc) [19] and diffusely adherent E coli(DAEC) [20] Despite not being very common the isolationof diarrheagenic E coli from seafood has been reportedIn Brazil Ayulo et al (1994) [21] isolated only one strainof STEC from shellfish and gave evidence that preventivemeasures especially during harvest and postharvest are ofmajor importance to avoid contamination of any nature

Detection of pathogenic bacteria in seafood is essentialto ensure safe products for consumers sustainable fish andshellfish growing activities Molecular diagnostic methodshave evolved significantly in the last few years and arenow established as useful and reliable methods to allow the

rapid detection and identification of pathogens Moleculardetection identification and enumeration of Vibrio spp arelargely based on PCR amplification following purification ofnucleic acids from the samples Although less sensitive andmore time consuming DNA or oligonucleotide probe-basedhybridization methods have been proposed for the detectionof Vibrio spp in food [22]

Herein the presence of potentially pathogenic isolates(Vibrio and Escherichia coli strains) from Ucides cordatuscrabs from the Guanabara Bay Rio de Janeiro Brazil isreported using both conventional (biochemical identifica-tion) and molecular (PCR) methods The antibiotic suscep-tibility of the isolates was also evaluated

2 Material and Methods

21 Study Area The mangroves selected for this study arelocated in Itaoca (Sao Goncalo) Piedade (Mage) and Suruı(Mage) in Guanabara Bay Rio de Janeiro Brazil where thegathering of this crustacean for marketing is more intense

22 Sample Collection Thirty live crabs (Ucides cordatus)were collected between March 2012 and June 2014 in eachmangrove studied These samples were analyzed at the Lab-oratory of Environmental Microbiology at the University ofthe State of Rio de Janeiro (UERJ) The crabs were washedto remove any excess sediment and other impurities presenton their bodies The viscera and meat were removed witha sterile forceps and a scalpel and placed into sterile Petridishes Twenty-five grams of each sample were mixed with225mL of buffered peptone water and the suspensions weretransferred to homogenizer bags (Interscience Saint NomFrance) and coupled to a Stomacher 400 circulator (SewardWorthing West Sussex UK) at 260 rpm for 1min [23] Thesuspensions were serial-diluted from 10minus6 to 100 and 100 120583Lof each dilution was transferred onto specific broths

23 Microbiological Analyses of Crab Samples The tests usedfor the determination of E coli andVibrio spp are establishedin the Methods for the Microbiological Examination ofFoods The reference strains used as controls were providedby the Oswaldo Cruz Foundation Rio de Janeiro Brazil

231 Fecal Coliforms Analyses Twenty-five grams of tis-sue were immersed in 225mL of lactose broth (HimediaMumbai India) for 48 hours at 35∘C Subsequently 10minus1 to10minus4 dilutions were carried out with 9mL of saline solutionfor posterior inoculation in lauryl sulfate broth (HimediaMumbai India) at 35∘C for 24 h An 100 120583L aliquot of eachpositive tube lauryl sulfate broth (Himedia Mumbai India)was transferred to a corresponding tube containing 3mL ofEC broth (HimediaMumbai India) with 5Durham tubes for24 hours with a series of dilutions and replicates in a waterbath at 445∘C [23] to determine the MPN (most probablenumber) coliform bacteria by counting

232 Escherichia coli Detection An 100 120583L aliquot wasremoved from the tube containing 3mL of positive EC broth(Merck Darmstadt Germany) and transferred to agar plates
















3 Results










ETEC


EAEC











500pb

200pb100pb

215pb 248pb

132pb95pb

254pb

106pb190pb






Piedade(119899 = 3)

Suruı(119899 = 8)











V16S500pb

100pb121pb

248pb

663pb

410pb

897pb

LD Vm Vc Vv Vp












4 Discussion

















5 Conclusions


Competing Interests




Acknowledgments


References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















3 Results










ETEC


EAEC











500pb

200pb100pb

215pb 248pb

132pb95pb

254pb

106pb190pb






Piedade(119899 = 3)

Suruı(119899 = 8)











V16S500pb

100pb121pb

248pb

663pb

410pb

897pb

LD Vm Vc Vv Vp












4 Discussion

















5 Conclusions


Competing Interests




Acknowledgments


References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






ETEC


EAEC











500pb

200pb100pb

215pb 248pb

132pb95pb

254pb

106pb190pb






Piedade(119899 = 3)

Suruı(119899 = 8)











V16S500pb

100pb121pb

248pb

663pb

410pb

897pb

LD Vm Vc Vv Vp












4 Discussion

















5 Conclusions


Competing Interests




Acknowledgments


References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





V16S500pb

100pb121pb

248pb

663pb

410pb

897pb

LD Vm Vc Vv Vp












4 Discussion

















5 Conclusions


Competing Interests




Acknowledgments


References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















5 Conclusions


Competing Interests




Acknowledgments


References








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Microbiological Quality Assessment by PCR ...

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Transcript of Research Article Microbiological Quality Assessment by PCR ...