these mangroves have suffered extensively with urbanization and industrialization in coastal regions , and , over the years , extensive ecosystems have disappeared , ending many of their important functions , such as being buffers against coastal erosion , retaining some pollutants , and being fishery areas .
crabs are decapod crustaceans rich in sodium , potassium , and phosphorus with high amounts of iron , zinc , copper , and manganese .
they also present high concentrations of vitamins a , c , b6 , thiamine , and riboflavin and are considered a delicacy in several parts of the world . along the brazilian coast ,
crabs are one of the most important natural resources in estuarine regions and can be intensely exploited without reaching an overfishing threshold , mainly because the picking method allows for the identification of the female individuals , which are of a different size compared to the males , and their release back into the environment . among the large and diverse range of mangrove products in the brazilian north and north - eastern estuaries , the mangrove crab , ucides cordatus , is the most harvested , with the highest commercial and subsistence importance to rural households of the coastal population .
environment quality , as well as the mode of collection and processing of products , may affect the quantity and diversity of the microorganisms present on the surface of seafood and fishery products , which may cause increases in microbial contamination . beside the concern regarding the fecal contamination of human foods from marine ecosystems , starting in the late 1960s various indigenous bacteria from estuarine and marine waters
the main concern is with regard to several species of vibrio , such as vibrio parahaemolyticus .
recent studies have also identified shellfish as sources of vibrio cholerae , vibrio vulnificus , and other vibrio species in cases of human infections . some of these human pathogens can survive and grow at the low temperatures that characterize marine ecosystems . 

vibrios are gram - negative bacteria that are primarily associated with estuarine and coastal marine environments .
all vibrios have an absolute requirement of na for growth although some , such as v. cholerae , only require trace amounts .
only a small proportion of the vibrios belong to species potentially pathogenic in humans and , of these , only a small proportion may possess the pathogenicity traits that enable them to colonize and cause disease in the human body .
marine vibrios naturally contaminating bivalve mollusks have been shown to be harder to remove by depuration than fecal bacterial indicators , such as e. coli .
such processing methods may , therefore , not provide the necessary level of public health protection if significant levels of pathogenic vibrios are present in the harvested product . 

escherichia coli is a commensal microorganism whose niche is the mucous layer of the mammalian colon .
furthermore , e. coli is widely distributed in the intestinal tracts of warm - blooded animals .
e. coli is often nonpathogenic , although different strains may cause diseases in the gastrointestinal , urinary , or central nervous systems .
currently , six categories of diarrheagenic e. coli have been acknowledged : enterotoxigenic e. coli ( etec ) , enteropathogenic e. coli ( epec ) , enteroinvasive e. coli ( eiec ) , enterohemorrhagic e. coli ( ehec , shiga toxin - producing e. coli or stec ) [ 17 , 18 ] , enteroaggregative e. coli ( eaec or eaggec ) , and diffusely adherent e. coli ( daec ) . despite not being very common ,
( 1994 )   isolated only one strain of stec from shellfish and gave evidence that preventive measures , especially during harvest and postharvest , are of major importance to avoid contamination of any nature .
detection of pathogenic bacteria in seafood is essential to ensure safe products for consumers , sustainable fish , and shellfish growing activities .
molecular diagnostic methods have evolved significantly in the last few years and are now established as useful and reliable methods to allow the rapid detection and identification of pathogens . molecular detection , identification , and enumeration of vibrio spp .
although less sensitive and more time consuming , dna or oligonucleotide probe - based hybridization methods have been proposed for the detection of vibrio spp . in food .
herein , the presence of potentially pathogenic isolates ( vibrio and escherichia coli strains ) from ucides cordatus crabs from the guanabara bay , rio de janeiro , brazil , is reported , using both conventional ( biochemical identification ) and molecular ( pcr ) methods .
the mangroves selected for this study are located in itaca ( so gonalo ) , piedade ( mage ) , and suru ( mage ) , in guanabara bay , rio de janeiro , brazil , where the gathering of this crustacean for marketing is more intense .
thirty live crabs ( ucides cordatus ) were collected between march 2012 and june 2014 in each mangrove studied .
these samples were analyzed at the laboratory of environmental microbiology at the university of the state of rio de janeiro ( uerj ) .
the crabs were washed to remove any excess sediment and other impurities present on their bodies . the viscera and meat were removed with a sterile forceps and a scalpel and placed into sterile petri dishes .
twenty - five grams of each sample were mixed with 225  ml of buffered peptone water , and the suspensions were transferred to homogenizer bags ( interscience , saint nom , france ) and coupled to a stomacher 400 circulator ( seward , worthing , west sussex , uk ) at 260  rpm for 1  min .
the suspensions were serial - diluted from 10 to 10 and 100  l of each dilution was transferred onto specific broths .
the tests used for the determination of e. coli and vibrio spp . are established in the methods for the microbiological examination of foods .
the reference strains used as controls were provided by the oswaldo cruz foundation , rio de janeiro , brazil .
twenty - five grams of tissue were immersed in 225  ml of lactose broth ( himedia , mumbai , india ) for 48 hours at 35c .
subsequently , 10 to 10 dilutions were carried out with 9  ml of saline solution for posterior inoculation in lauryl sulfate broth ( himedia , mumbai , india ) at 35c for 24  h. an 100  l aliquot of each positive tube lauryl sulfate broth ( himedia , mumbai , india ) was transferred to a corresponding tube containing 3  ml of ec broth ( himedia , mumbai , india ) with 5 durham tubes for 24 hours with a series of dilutions and replicates in a water bath at 44.5c   to determine the mpn ( most probable number ) coliform bacteria by counting .
an 100  l aliquot was removed from the tube containing 3  ml of positive ec broth ( merck , darmstadt , germany ) and transferred to agar plates containing emb ( merck , darmstadt , germany ) .
the plates were incubated for 24 hours at 37c . the presumptive e. coli spp .
colonies were submitted to biochemical tests : sim ( sulfide - indole - motility ) ( biobrs , minas gerais , brazil ) , citrate ( citrate of simmons ) ( difco , sparks , maryland , usa ) , and mr / vp broth ( methyl red / voges - proskauer ) ( merck , darmstadt , germany ) .
twenty - five grams of crab meat and viscera were immersed in 225  ml of lactose broth ( himedia , mumbai , india ) for 48 hours at 35c and transferred to 1  ml tubes containing bhi ( heart brain infusion ) ( himedia , mumbai , india ) with 1% and 3% of nacl and incubated for 24  h at 37c .
a 100  l aliquot was transferred to plates containing tcbs agar ( himedia , mumbai , india ) and were incubated for 24  h at 37c .
colonies were submitted to biochemical characterization tests : oxidase test , oxidation - fermentation ( of ) ( difco , sparks , maryland , usa ) , inositol ( difco , sparks , maryland , usa ) , and o129 ( celon - lab , madhapur , hyderabad , india ) .
dna preparation was carried out by the thermal shock method from all the harvested colonies .
the colonies were grown in 3  ml of bhi broth harvested after 24  h at 37c .
one  ml of the medium was transferred to sterile eppendorf tubes and centrifuged for 10  min at 12,000  g. the supernatant was discarded and the pellet was resuspended in 400  l of pure sterile water .
after homogenization , the supernatant was boiled for 10  min , cooled on ice for 5  min , and then collected and used for the pcr analyses .
pcr was performed using multiplex jms1 , lt , vira , and eae oligos and pcr - uniplex for aggrks and east1 oligos ( table 1 ) .
the reactions contained a final volume of 25  l containing 5  l of template dna , buffer ( 10x ) , 10  mm dntp , 25  mm  mgcl2 , 2  u taq polymerase ( invitrogen technologies , so paulo , brazil ) , and 10  mm of each primer ( invitrogen technologies , so paulo , brazil ) .
the conditions of reaction were 94c for 5  min , 30 cycles of 1  min at 94c , 1  min at 58c , 2  min at 72c , and a final cycle of 72c for 10  min , for all reactions .
pcr amplicons were visualized on 2% agarose gels stained with 3  l of ethidium bromide ( 0.5  mg  ml ) , visualized on a uv light transilluminator ( uvitec , cambridge , uk ) , and photodocumented by  polaroid 
the reaction was performed using multiplex oligos in a final volume of 20  l .
the mixture contained 2  u taq polymerase ( invitrogen technologies , so paulo , brazil ) , 10  mm dntps , buffer ( 10x ) , 25  mm  mgcl2 , 3  l of template dna , and 10  mm primers ( sodb , sodb flae , hsp , and 16s ) ( table 2 ) . the conditions of reaction were 5  min at 93c followed by 35 cycles of 92c for 40  s , 57c for 1  min , and 72c for 1.5  min and a final cycle at 72c for 7  min , for all reactions .
pcr amplicons were visualized on 2% agarose gels stained with 3  l of ethidium bromide ( 0.5  mg  ml ) , visualized on a uv light transilluminator ( uvitec , cambridge , uk ) , and photodocumented by  polaroid 
the microorganisms were inoculated at a concentration equivalent to 0.5  mcfarland units ( probac , durban , south africa ) onto a muller hinton agar plate ( difco , sparks , maryland , usa ) .
the inhibition zone was interpreted according to the clinical laboratory standards m100-s22 guidelines , formerly known as the national committee for clinical laboratory standards .
the tested antibiotics were chloramphenicol ( 30  g ) , tetracycline ( 30  g ) , gentamicin ( 10  g ) , amikacin ( 30  g ) , tobramycin ( 10  g ) , trimethoprim - sulfamethoxazole ( 1.25/23.75  g ) , cephalothin ( 30  g ) , ampicillin ( 10  g ) , ceftazidime ( 30  g ) , cefotaxime ( 30  g ) , cefepime ( 30  g ) , aztreonam ( 30  g ) , cefoxitin ( 30  g ) , imipenem ( 10  g ) , ampicillin - sulbactam ( 10  g-10  g ) , and ciprofloxacin ( 5  g ) . for quality control , e. coli atcc 25922 and e. coli atcc 35218
, the test was performed according to the standard document m45-a2 , with the same antibiotic disks used for e. coli ( oxoid , hampshire , uk ) , with the exception of tobramycin ( 10  g ) and aztreonam ( 30  g ) and with the addition of levofloxacin ( 5  g ) and ofloxacin ( 5  g ) .
high concentrations of fecal coliforms ( 6.2  10 and 7.2  10  nmp  g ) were found in the meat and hepatopancreas samples from the itaoca mangrove , respectively .
the samples from piedade and suru mangroves showed concentrations of 2.4  10 and 3.2  10  nmp  g in meat samples , respectively , and 2.5  10 and 3.5  10  nmp  g in hepatopancreas samples , respectively .
no significant difference was observed among the thermotolerant coliform values found in the meat and hepatopancreas samples between the mangroves ( p < 0.05 ) .
multiplex pcr enabled the identification of 4 virulence genes ( eaea , stx1 , lt , and vira ) in single reaction ( figure 1 ) .
forty - six e. coli colonies isolated from the crab samples of the different mangroves ( 21 from meat and 25 colonies from hepatopancreas ) were confirmed by biochemical tests .
after biochemical characterization , the molecular test ( pcr ) revealed that 25 ( 54.3% ) were positive for the researched virulence genes , 9 presenting easta ( 36% ) , 13 presenting lt ( 52% ) , and 3 presenting stx ( 12% ) .
no colonies presenting vira , eaea , st , and agg genes were detected ( table 3 ) .
fourteen e. coli strains were isolated from itaca , with the presence of virulence genes , 2 presenting stx1 ( hepatopancreas ) , 7 presenting lt ( 4 in meat and 3 in hepatopancreas ) , and 5 presenting easta ( 4 in meat and 1 in hepatopancreas ) .
eight strains were detected in samples from the suru mangrove , where 4 strains showed the lt virulence gene ( 2 in meat and 2 in hepatopancreas ) and 4 strains showed the east virulence gene ( 2 in meat and 2 in hepatopancreas ) .
thirteen strains were detected in the samples from the piedade mangrove by means of the biochemical test , but only one showed the presence of the stx1 virulence gene ( meat ) , while 2 showed the presence of the lt virulence gene ( hepatopancreas ) .
suru mangrove samples showed the highest incidence of isolated vibrio ( 46 ) , followed by piedade ( 40 ) and itaca ( 33 ) .
one hundred and nineteen vibrio strains were confirmed by pcr in 90 samples ( meat : 68 , and hepatopancreas : 51 ) .
( 2010 )   using multiplex pcr ( identifying the gyrb and pnta genes ) to differentiate v. parahaemolyticus , v. cholerae , v. vulnificus , and other vibrio spp . from fish . among the researched vibrio genus , 61.3% ( 73/119 ) of the samples
pathogenic strains v. cholerae and v. parahaemolyticus were found with a frequency of 1.7% ( 02/119 ) and 37% ( 44/119 ) , respectively ( table 4 ) .
the highest incidence of v. parahaemolyticus was observed in samples from the suru mangrove ( 21 ) , followed by itaca ( 12 ) and piedade ( 11 ) .
no v. mimicus and v. vulnificus were detected in the present study ( table 4 ) .
twenty - six e. coli strains showed some resistance to the tested antimicrobials , with a high index of resistance .
e. coli strains isolated from the itaca samples showed high resistance ( 63% ) against gentamicin ( cn ) and tobramycin ( tob ) . 

e. coli strains found at piedade , itaca , and suru showed resistance to gentamicin ( 66% , 63% , and 22% , resp . ) .
the e. coli isolates from the piedade samples showed 33% resistance to chloramphenicol ( c ) . only strains found in crabs from the piedade mangrove showed resistance ( 16% ) to ampicillin ( amp ) .
no resistance to amoxicillin + clavulanic acid ( amc ) , levofloxacin ( lev ) , cefoxitin ( ctx ) , ofloxacin ( ofx ) , and ciprofloxacin ( cip ) was observed . among the 26 resistant e. coli strains , 12 were resistant to two or more antibiotics ( table 6 ) .
this pattern is mainly due to the indiscriminate use of antimicrobials and may cause serious impacts on human health [ 31 , 32 ] .
the e. coli strains showed multiresistance to several antimicrobial agents , with mar indices ranging from 0.12 to 0.31 , whereas 3 strains showed mar indexes from 0.12 to 0.25 and 3 strains presented mar indexes of 0.18 ( table 6 ) .
the resistance of the 26 e. coli strains was distributed as follows : 12 strains were resistant to gentamicin and tobramycin , 4 were resistant to amikacin and cephalothin , 3 were resistant to ciprofloxacin , tetracycline , ceftazidime , and cefoxitin , and one strain was resistant to ampicillin .
when 119 vibrio strains were analyzed only 72 isolates ( 60.5% ) showed resistance to some of the tested antimicrobials , with higher rates in those isolated from crabs samples from piedade ( 29 ) , followed by itaca ( 23 ) and surui ( 20 ) ( table 5 ) .
the vibrio strains from piedade , itaca , and suru showed resistance to ampicillin ( 86% , 78% , and 85% , resp . ) .
the strains from surui showed 5% resistance to amoxicillin + clavulanic acid ( amc ) , ampicillin / sulbactam ( sam ) , and chloramphenicol ( c ) .
the piedade strains showed resistance to levofloxacin ( lev ) and ciprofloxacin ( cip ) . no resistance to cefoxitin ( ctx ) , ceftazidime ( caz ) , tobramycin ( tob ) , and tetracycline ( te ) was observed . 

vibrio strains isolated from crabs showed multiresistance to several antimicrobial agents , presenting a mar index ranging from 0.12 to 0.25 ; 24 strains presented mar indices of 0.12 ( table 6 ) ; 5 strains showed mar indices of 0.18 ; and two strains showed mar indices of 0.25 mar indexes .
the resistance of the 72 strains was distributed as follows : 60 strains were resistant to ampicillin , 14 were resistant to amikacin ( ak ) , 10 were resistant to cephalothin ( kf ) , 8 were resistant to cefoxitin ( ctx ) , 5 were resistant to gentamicin ( cn ) , 3 were resistant to ciprofloxacin ( cip ) , and 1 strain was resistant to amoxicillin + clavulanic ( amc ) , ampicillin + sulbactam ( sam ) , levofloxacin ( lev ) , ofloxacin ( ofx ) , and chloramphenicol ( c ) .
the thermotolerant coliforms found in the present study are above the maximum permissible limit ( maximum tolerance of 5  10  nmp  g for coliforms at 45c ) in bivalve mollusks , crab meat , and similar samples , according to laws from the brazilian sanitary vigilance agency ( agncia nacional de vigilncia sanitria ( anvisa ) ) .
similar results were found with regard to the microbiological quality of a crab meat in 3 different points at praia do futuro , located in fortaleza , ce , brazil , where thermotolerant coliforms were detected ranging from 3.0 to 1,100  nmp  g in 90 analyzed crabs . according to de lima grisi and gorlach - lira ( 2010 )
, the presence of this group of bacteria is associated to the dumping of fecal material in the environment .
guanabara bay receives effluents without treatment daily and has become bacteria reservoir , which in turn has caused the contamination of fish and other biota in this region .
the expression of e. coli virulence genes is a public health risk , since these genes characterize the presence of toxins able to cause disease .
e. coli cells are the main pathogens associated to gastroenteritis of food origin in humans , provoking diarrhea , hemorrhagic colitis , and hemolytic - uremic syndrome
. however , some studies reporting human infection by e. coli due to crab consumption are available . despite the absence of the vira , eae ,
st , and agg genes in the present study , the confirmation of e. coli strains indicates recent fecal contamination in crabs , and this indicates that major care in the preparation of this type of food is required
. the results regarding the presence of vibrio can be explained by the salinity and temperature of the studied mangroves .
many studies show the presence of vibrio in aquatic animals such as fish , shrimp , and mussels , but , despite the importance of crabs , only some studies have been conducted on crab contamination by vibrio . however , its occurrence in marine food is pointed as a major cause of gastroenteritis in the united states and europe   and associated with cases in brazil and chile .
these results suggest a probable health risk for people that consume raw and undercooked seafood . according to alam et al .
( 2012 ) , v. vulnificus and v. mimicus are most commonly found in coccoid viable but not culturable form , while another study confirmed the presence of vibrio in crabs marketed in fortaleza , brazil , where only 10 strains were identified up to the species level : 2 v. alginolyticus and 8 v. parahaemolyticus but not any v. vulnificus and v. mimicus .
abd - elghany and sallam ( 2013 )   detected 10 v. parahaemolyticus isolates in crab by molecular identification in egypt and highlighted that reliable molecular detection methods should be included in routine seafood examinations , in addition to the conventional bacteriological methods .
these findings of antimicrobial susceptibility are in agreement with data from previous studies , which found that resistance to aminoglycosides , -lactamase , and penicillin is common among e. coli isolates from food of animal origin [ 4749 ] .
however , the resistance frequency in e. coli isolated in the present study was low when compared to other studies , where a resistance of 58% and 42% in raw fish samples from kenya and vietnam , respectively , was observed [ 48 , 50 ] .
mussel samples from niteri ( brazilian southeastern oceanic region ) showed 29% resistance to at least one antimicrobial , and strains isolated from mussels from the guanabara bay , rio de janeiro , showed 40% to 85% resistance to tested antimicrobials , indicating the intense presence of domestic and industrial effluents .
the percentage of high sensitivity to these antibiotics was also observed by rebouas et al .
( 2011 )   in strains isolated from shellfish and is associated with various resistance mechanisms found in gram - negative organisms . over time , vibrio strains exposed to antibiotics through the environment can acquire antimicrobial resistance transferable by mobile genetic elements and horizontal gene transfer .
thus , due to the presence of r - factors in the population , resistance developed through gene regulation of plasmids and chromosomes may be transferred vertically ( by heredity ) or horizontally . in the present study ,
this data was confirmed in another study , where , from 169 vibrio strains isolated from shrimp , only 3 were sensitive to ampicillin .
the high percentage of pathogenic vibrio with reduced susceptibility to ampicillin suggests a potential for the low efficiency of ampicillin in the treatment of vibrio infections .
many cases of multiple antimicrobial resistance have been reported from shellfish farms in countries where the activity is well developed , such as china , korea , and chile . according to the world health organization
, changes in the microbiota can induce the evolution of new pathogenic microorganisms and the development of new virulence factors in ancient pathogens , such as the development of resistance to antimicrobials or changes in their survival ability in adverse environmental conditions .
several e. coli and vibrio isolates were found in crabs ( ucides cordatus ) from different mangroves in the state of rio de janeiro , brazil . considering the current legislation , the presence of these pathogens in crab indicates contamination influenced by mangrove pollution , by using newer molecular methods and thus contributing to seafood safety .
the analyzed samples presented unsuitable hygienic - sanitary conditions , which can be considered a warning to the municipal health surveillance agency , since seafood is many times consumed without any subsequent thermal treatment or even sufficient thermal treatment able to eliminate pathogenic microorganisms , causing disorders to consumer health .
 the bacteriological quality of crabs from three different mangroves ( itaca , suru , and piedade ) from rio de janeiro state , brazil , was investigated using conventional and molecular methods . 
 the results revealed high counts for total coliforms in meat and hepatopancreas samples . 
 pcr analyses identified 25 escherichia coli colonies in the itaca , piedade , and suru samples , detecting 13 enterotoxigenic colonies and 9 enteroaggregative colonies . respectively , 12 , 11 , and 21 vibrio parahaemolyticus strains were detected in the itaca , piedade , and suru samples . 
 two v. cholerae strains were detected in the piedade samples . 
 the e. coli strains isolated in the present study showed resistance to gentamicin . 
 e. coli strains from the piedade samples showed 33% resistance to chloramphenicol and the strains also showed multiresistance to several antimicrobial agents with a mar index ranging from 0.12 to 0.31 . 
 vibrio strains from piedade , itaca , and suru showed 86% , 78% , and 85% resistance , respectively , to ampicillin . 
 the isolated vibrio strains showed multiresistance to several antimicrobial agents , with a mar index ranging from 0.12 to 0.25 . 
 the presence of these organisms in crab meat is an indication of microbial contamination , which may pose health risks to consumers when improperly cooked . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions