Inter-species competition of surface bacterial flora of pomegranate and their role in spoilage.

The surface of fruits is heterogenous in term of its microenvironment hence dictate the kind of microflora that develops during storage. A better understanding of spoilage organisms would lead to better preservation methods. The pomegranate was chosen, since its sturdy and spoils slow at room temperature and is ideal for studying fruit spoilage in-situ. In the current study we isolated organisms from fruit surface and study the spoilage and competition amongst microbial species. Total 17 unique bacterial isolates from pomegranate were identified. The 16S rRNA gene identification placed them in 8 major genera (Acinetobacter, Micrococcus, Pantoea, Microbacterium, Strenotrophomonas, Bacillus, Staphylococcus and Exiguobacterium). Competition assay among isolate suggested that Exiguobacterium is dominant species followed by Micrococcus, Pantoea and Bacillus. The consortium of 3 different combinations (5 bacteria each) of isolated bacteria showed the spoilage phenotype on pomegranate. Except for 3 bacterial isolates, the rest of the isolates produced any one or multiple enzymes associated with the food spoilage (cellulase, amylase, lactase, pectinase and protease). The isolates were checked for the presence of genes associated with antibiotic resistance and 78.9% of the tested micro-organisms were blaTEM positive. Aminoglycoside resistance genes were present in 10% of the tested microbes. This study demonstrated interspecies competition amongst spoilage organisms. This understanding of surface flora of fruit would give better insights to preserve fruits.

The cAMP receptor protein (CRP) enhances the competitive nature of Salmonella Typhimurium.

The cAMP receptor protein (CRP) is a global regulatory protein. We evaluated the role of CRP in starvation physiology in Salmonella Typhimurium. The Δcrp mutant survived 10 days of starvation. However, in a co-culture with the wild type in nutrient-rich medium, Δcrp died within 48 h. Similar co-culture results were observed with Escherichia coli and Staphylococcus aureus. Our study showed that the Δcrp mutant was not killed by toxins and the Type IV secretion system of the WT. The possibility of viable but non-culturable cells (VBNC) was also ruled out. However, when the overall metabolism of the co-culture was slowed down (anaerobic condition, inhibition by antibiotics and low temperature) that improved the survival of Δcrp in co-culture. But one more significant observation was that the Δcrp mutant survived in nutrient-free co-culture conditions. These two observations suggest that CRP protein is essential for efficient nutrient assimilation in a competitive environment. The cells without CRP protein are unable to evaluate the energy balance within the cell, and the cell spends energy to absorb nutrients. But the wild type cell absorbs nutrients at a faster rate than Δcrp mutant. This leads to a situation wherein the Δcrp is spending energy to absorb the nutrients but is unable to compete with the wild type. This futile metabolism leads to death. Hence, this study shows that CRP is a metabolism modulator in a complex nutrient environment. This study also highlights the need for innovative growth conditions to understand the unique function of a gene.

An improved, simple and field-deployable CRISPR-Cas12a assay for the detection of SARS-CoV-2.

AIMS: The RT-PCR is the most popular confirmatory test for SARS-CoV-2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR-based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high-quality lateral flow strips. The main goal of this study was to improve the Cas12a-based SARS-CoV-2 DETECTR method and develop a portable and field-deployable system to reduce the recurring consumable cost. METHODS AND RESULTS: Specific regions of N and E genes from SARS-CoV-2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop-mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a-based trans-cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT-LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans-cleavage reaction into a single ready-to-use mix. A portable, cost-effective battery-operated instrument, CRISPR-CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples. CONCLUSIONS: The simplified CRISPR-based SARS-CoV-2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR-CUBE. SIGNIFICANCE AND IMPACT OF STUDY: Significant improvement in Cas12 method, coupled with simple visualization of end point makes the method and instrument deployable at the point-of-care (POC) for SARS-CoV-2 detection, without any recurrent cost for the lateral flow strips which is used in other POC methods.

Ciprofloxacin induced antibiotic resistance in Salmonella Typhimurium mutants and genome analysis.

Antibiotic resistance of Salmonella species is well reported. Ciprofloxacin is the frontline antibiotic for salmonellosis. The repeated exposure to ciprofloxacin leads to resistant strains. After 20 cycles of antibiotic exposure, resistant bacterial clones were evaluated. The colony size of the mutants was small and had an extended lag phase compared to parent strain. The whole genome sequencing showed 40,513 mutations across the genome. Small percentage (5.2%) of mutations was non-synonymous. Four-fold more transitions were observed than transversions. Ratio of < 1 transition vs transversion showed a positive selection for antibiotic resistant trait. Mutation distribution across the genome was uniform. The native plasmid was an exception and 2 mutations were observed on 90 kb plasmid. The important genes like dnaE, gyrA, iroC, metH and rpoB involved in antibiotic resistance had point mutations. The genome analysis revealed most of the metabolic pathways were affected.

Role of cAMP receptor protein in phenotype and stress tolerance in Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is exposed to biotic and abiotic stresses. The survival of Salmonella in nature depends on the global regulators like cAMP receptor protein (CRP). The role of CRP in the phenotypic characteristics and stress tolerance was elucidated in S. Typhimurium using a crp gene null mutant (Δcrp). A 1.6-fold decrease in the cell size, a two-fold reduction in the colony size, and a 3.5-fold decrease in motility were observed in the Δcrp compared with the S. Typhimurium wild-type (WT). H2 S production on selective media was affected in the Δcrp. The utilisation of d-mannose, d-glycerol and d-mannitol was completely affected, whereas that of d-galactose and d-fructose was partially affected. The utilisation of d-arabinose was induced in the Δcrp. The growth rate of the Δcrp in Luria Bertani medium was unaffected. However, in the glucose-containing minimal medium, the growth rate of the Δcrp was reduced by 1.5-fold compared with the WT. The Δcrp was able to utilise ethanolamine as the sole carbon source similar to the WT. The Δcrp was more tolerant to heat and oxidative stress. Overexpression of heat and oxidative stress-related genes was observed in the Δcrp in the stationary phase. The Δcrp was less tolerant to radiation stress compared with the WT. The current findings decisively establish the CRP protein as a global regulator. The CRP affects multiple phenotypes, carbon metabolism and stress physiology of S. Typhimurium.

Understanding the influence of heavy water stress on the physiology of Salmonella Typhimurium.

The heavy isotope of water is used in understanding the physiology of bacteria. Deuterium (D2O) reduces chemical reaction kinetics. In the present study, the survivability of the food-borne pathogen Salmonella Typhimurium grown in D2O supplemented medium is studied under various stress conditions. The growth of S, Typhimurium was studied in rich (Luria Broth-LB) and minimal medium (M9) prepared in D2O. The reduced growth rate of S. tTyphimurium in M9 (2.4 fold) as compared to that in LB (1.6 fold) was observed. S. tTyphimurium grown in D2O supplemented medium was significantly more tolerant to heat and gamma radiation (1.2 fold), but was sensitive to extreme pH (both alkaline and acidic) and osmotic stress (10 fold). These results suggest that the change in the biological reaction kinetics in the cell due to D2O may modify the stress tolerance of S. tTyphimurium. This is the first study carried out to understand how a bacterial system (S. Typhimurium) in D2O responds to different stresses. This study suggests that investigations on bacterial physiology in D2O supplemented medium helps in understanding the underlying mechanisms of stress tolerance.

Glucose sensitizes the stationary and persistent population of Vibrio cholerae to ciprofloxacin.

The subject of analysis in this report was the antibiotic susceptibility of V. cholerae under glucose supplementation since the metabolites can significantly alter the antibiotic sensitivity of bacteria. Glucose could change the antibiotic susceptibility in a growth phase-dependent manner, however, the antibiotic susceptibility of exponentially growing cells was not affected in the presence of glucose. What has been shown is that the stationary phase cells which show higher antibiotic tolerance, could be sensitized to ciprofloxacin and ampicillin by glucose supplementation (tenfold sensitive). The glucose increases the respiration which in turn increases the metabolism and cell division rate. Furthermore, the addition of glucose could increase the susceptibility of persister cells to ciprofloxacin only. In general, the bacterial susceptibility can be increased by combining the antibiotics with glucose.

Inhibition of protein synthesis eradicates persister cells of V. cholerae.

In the present work, we studied the antibiotic-induced persister formation in Vibrio cholerae. Persisters vary with the bacterial growth phase with minimum persisters in log phase and maximum in stationary phase. Only 10% of the stationary phase cells of V. cholerae were tolerant of ampicillin and ciprofloxacin. In comparison, more than 90% of the stationary phase cells of E. coli were tolerant of ampicillin and ciprofloxacin. Frequency of ciprofloxacin-induced persisters of V. cholerae would vary with the bacteriological media used for the growth of the cells. In tryptone soy broth (TSB) and in buffered peptone water (BPW), V. cholerae could form more than 10% persisters, whereas in Luria-Bertani broth (LB) and alkaline peptone water (APW) persister fraction was less than 1%. When exposed to protein synthesis inhibitors (kanamycin, chloramphenicol, tetracycline, erythromycin and gentamicin), V. cholerae did not form persisters. Persister recovery assay, LIVE/DEAD analysis and QRDR sequence analysis showed that persister population neither included resistant mutants nor VBNC population. Starvation, anaerobic conditions and inhibition of ATP synthesis also induced persisters, but not when protein synthesis is inhibited. These observations suggest that the protein synthesis is critical for persister formation, persister maintenance, and also for dormancy maintenance in V. cholerae. Contrary to these observations, E. coli can form persisters when protein synthesis is inhibited, suggesting fundamental mechanistic differences between the two species.

Comparison of Starvation-Induced Persister Cells with Antibiotic-Induced Persister Cells.

The phenotypic heterogeneity in a large population arises because of fluctuation in microenvironments and stochastic gene expressions. In this report, we isolated two types of persistent sub-populations of Vibrio cholerae, one triggered by starvation and another by antibiotics. We characterised starvation-induced (E-cells) and antibiotic-induced (P-cell) persister cells for stress tolerance, colony morphology and toxin gene expressions. Both the sub-populations differ with respect to morphology, temperature tolerance and oxidative stress tolerance. The E-cells were smaller than the P-cells and formed tiny colonies (1-2 mm). The E-cells were more sensitive to heat and oxidative stress compared with P-cells. The up-regulated genes of P-cells include, genes of antioxidant enzymes (>5 fold), cholera toxin (>26 fold) and toxin: antitoxin protein hipA (>100 fold). Upon nutrient up-shift, the E-cells recovered after lag time of 6 h. However, such lag extension was not visible during P-cell recovery, suggesting that P-cell physiology is more akin to normal cells than E-cells. This is the first comparative report on the two different persister sub-populations of V. cholerae. The E-cells and P-cells are similar regarding antibiotic tolerance. However, the sub-populations differ significantly in stress tolerance and other phenotypes studied.

Development and evaluation of taxon-specific primers for the selected Caudovirales taxa.

The phage taxonomy is primarily based on the morphology derived from Transmission Electron Microscopic (TEM) studies. TEM based characterization is authentic and accepted by scientific community. However, TEM based identification is expensive and time consuming. After the phage isolation, before analysis TEM, a DNA based rapid method could be introduced. The DNA based method could dramatically reduce the number of samples analyzed by TEM and thereby increase the speed and reduce the cost of identification. In the present work, four environmental phage isolates were identified based on TEM studies and genome size. The identification of these four phages was validated using DNA based method. The taxon-specific DNA markers were identified through multiple sequence alignments. The primers were designed at conserved genes (DNA polymerase or integrase) of 4 different phage taxa viz. family Ackermannviridae, genus Jerseyvirus, genus T4virus, and genus P22virus. These primers were evaluated using both in vitro and in silico approach for the amplification of the target taxons. Majority of the primer sets were found to amplify member species of the targeted taxa in vitro. In In silico analysis, six primer sets intended for identification of family Ackermannviridae showed positive amplification of ≥86.7% classified species. Further, the primers targeting the genus Jerseyvirus and T4virus showed the amplification of 53.8% and ≥84.6% species, respectively. The present work is a case study performed to explore the possibility of use of taxon-specific primers for identification and taxonomic studies of newly isolated phages to supplement the TEM.

Isolation and Genome Sequence Characterization of Bacteriophage vB_SalM_PM10, a Cba120virus, Concurrently Infecting Salmonella enterica Serovars Typhimurium, Typhi, and Enteritidis.

The prevalence of multidrug-resistant Salmonella is ever increasing and calls for alternatives to antibiotics. The use of phages has been anticipated to reduce the multidrug-resistant human pathogens in food environment. Salmonella phage vB_SalM_PM10 (PM10) was isolated from sewage-polluted river in India. It shows an icosahedral head (94 ± 4 nm) along with a long contractile tail (106 ± 7 × 18 ± 2 nm), a morphotype of family Ackermannviridae. Additionally, the phage displayed the features resembling to existing Cba120viruses. Phage PM10 could infect S. enterica serovars Typhimurium, Typhi, and Enteritidis. The genome sequencing analysis of phage PM10 revealed circular 158.08 kb double-stranded DNA, with the GC content of 44.6%. Two hundred and nine ORFs, 171 putative promoters, 122 rho-independent terminators, and 5 transfer RNA encoding genes were found in the genome. The genome-wide comparisons and phylogenetic analyses showed that phage PM10 is closely related to Salmonella phage PhiSH19. Comparison of the tail-spike protein sequences encoded in PM10 and PhiSH19 genome showed the variation, which might have facilitated PM10's simultaneous infectivity to aforementioned S. enterica serovars. This is a varied host range than that of PhiSH19 or any other Cba120viruses.

Characterization of multiple antibiotic resistance of culturable microorganisms and metagenomic analysis of total microbial diversity of marine fish sold in retail shops in Mumbai, India.

Marine fish species were analyzed for culturable and total metagenomic microbial diversity, antibiotic resistance (AR) pattern, and horizontal gene transfer in culturable microorganisms. We observed a high AR microbial load of 3 to 4 log CFU g-1. Many fish pathogens like Providencia, Staphylococcus, Klebsiella pneumoniae, Enterobacter, Vagococcus, and Aeromonas veronii were isolated. Photobacterium and Vibrio were two major fish and human pathogens which were identified in the fish metagenome. Other pathogens that were identified were Shewanella, Acinetobacter, Psychrobacter, and Flavobacterium. Most of these pathogens were resistant to multiple antibiotics such as erythromycin, kanamycin, neomycin, streptomycin, penicillin, cefotaxime, bacitracin, rifampicin, trimethoprim, ciprofloxacin, and doxycycline with a high multiple antibiotic resistance index of 0.54-0.77. The fish microflora showed high prevalence of AR genes like bla TEM, Class I integron, tetA, aph(3')-IIIa, ermB, aadA, and sul1. Nineteen of 26 AR isolates harbored Class I integrons showing high co-resistance to trimethoprim, kanamycin, doxycycline, and cefotaxime. Mobile R-plasmids from 6 of the 12 AR pathogens were transferred to recipient E. coli after conjugation. The transconjugants harbored the same R-plasmid carrying bla CTX-M, dfr1, tetA, bla TEM, and cat genes. This study confirms that fish is a potential carrier of AR pathogens which can enter the human gut via food chain. To the best of our knowledge, this is the first study in the Indian subcontinent reporting a direct evidence of spread of AR pathogens to humans from specific marine fish consumption.

The ploidy of Vibrio cholerae is variable and is influenced by growth phase and nutrient levels.

The ploidy of Vibrio cholerae was quantified under different growth conditions. The V. cholerae was found to be (mero-) oligoploid or polyploid. The ploidy levels per cell were found to be growth phase regulated. The ploidy is highest during the early stationary phase (56-72 per cell) and lowest in the long-term starved state. In addition to growth phase, an external parameter such as nutrient level influences the ploidy, i.e. ploidy reduces rapidly at the onset of the starvation. The reduction is significant with P-value < 0.05 within 2 h of starvation. Even after prolonged starvation of 10 days, the ploidy number remained above 2 per cell. Failure to obtain a monoploid V. cholerae indicates that during starvation the genome is not distributed equally to daughter cells. The activity of DNase enzyme increased during starvation that decreased the ploidy. The ploidy was restored to the pre-starvation levels with nutrient supplementation.

Species specific PCR based detection of Escherichia coli from Indian foods.

Escherichia coli is a faecal indicator and certain virotypes are known as pathogens. Therefore, detection and prevention of E. coli in food is very important. The existing rapid methods concentrate on detecting the pathogenic E. coli instead of total E. coli population. Present study evaluates the use of two molecular markers (uidA and flanking region of uspA) specific for the E. coli in combination with microbiological method for confirmation. Majority of the isolates (77%) were positive for both the genes tested. However, 22% of the E. coli isolates were positive for any one of the two primer sets [uidA (9%) and flanking region of uspA (13%)]. High levels of E. coli incidences (92% samples) were observed in beef while low occurrence (19% samples) was found in sprouts. Low percentage (7.3%) of E. coli isolates was positive for virulence genes tested (lt, ipaH, aggR, eaeA, stx1 and stx2). Two isolates were positive for stx genes. However, none of the isolates including stx positive isolates were E. coli 0157:H7. Maximum number of the E. coli (44%) isolates was characterized under phylogenetic group B2. This phylogenetic group comprises of extra intestinal and virulent E. coli strains.

Expression of virulence and stress response genes in Aeromonas hydrophila under various stress conditions.

Aeromonas hydrophila has emerged as an important human pathogen as it causes gastroenteritis and extra-intestinal infections. Information regarding the influence of environmental stresses on gene expression profile of A. hydrophila is lacking. The impact of nutrient replenishment, nutrient deprivation, acid stress, and cold shock on housekeeping, general stress-response, and virulence genes was studied using quantitative real-time PCR in two A. hydrophila strains, CECT 839T , and A331. These sub-lethal stresses invoked significant changes in the expression of these genes in a strain-dependent manner. Overall, nutrient replenishment and deprivation significantly induced the expression of housekeeping (rpoD), general stress regulators (uspA and rpoS), and virulence (aer) genes, indicating their importance in regulating the survival and virulence of A. hydrophila under these stress conditions. rpoS gene was significantly induced under cold shock; whereas, acid stress significantly induced the expression of uspA gene. This is the first study to investigate the effect of environmental parameters on the expression of stress-response and virulence genes in A. hydrophila strains.

Radiation-resistant Macrococcus caseolyticus (A) isolated from radiation-processed semidried prawns.

A radiation-resistant bacterial isolate from gamma-radiation-processed (5 kGy) semidried prawns was identified as a new strain of Macrococcus caseolyticus and was designated as M. caseolyticus (A) on the basis of morphological and biochemical characterization and 16S rRNA sequencing. DNA-DNA hybridization studies with M. caseolyticus DSM 20597(T) further confirmed the isolate as M. caseolyticus. Major fatty acids present in M. caseolyticus (A) were C14:0, C16:1ω11c, and C18:1ω9c, whereas C15:0anteiso, C16:0iso, and C18:0iso were absent. The closest match for the isolate, as per fatty acid methyl ester analysis, was M. caseolyticus DSM 20597(T). However, the similarity index was significantly low (0.112), which indicates that the isolate could be a new strain of M. caseolyticus. The decimal reduction dose (D10) for M. caseolyticus (A), M. caseolyticus JCSC5402, and Staphylococcus aureus MTCC96 was 1.18, 0.607, and 0.19 kGy, respectively. This is the first report on radiation resistance of M. caseolyticus. Macrococcus caseolyticus (A) is more resistant to gamma and UV radiation stress than are M. caseolyticus JCSC5402 and S. aureus MTCC96; however, it is sensitive to heat as well as desiccation stress.

Induction, resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio vulnificus in artificial sea water.

Vibrio vulnificus, an important food-borne pathogen, is known to enter viable but nonculturable (VBNC) state under low temperature and low nutrition stress conditions. Present study examined the time required for induction of VBNC state and temperature which induces resuscitation of V. vulnificus YJ016. The change in cell morphology and gene expression during VBNC state and in resuscitated cells was also examined. V. vulnificus incubated in artificial sea water at 4 °C entered VBNC state after considerably extended time (70 days). An increase in temperature by 6 °C from the VBNC induction temperature (4 °C) resulted in resuscitation of VBNC cells; however, maximum resuscitation was observed when VBNC cells were held at 23 °C for 24 h. VBNC cells changed their morphology from comma shape to coccoid shape. Two rounds of induction of VBNC and resuscitation were possible with V. vulnificus cells; however, there was progressive reduction in number of resuscitated cells and after 190 days cells failed to resuscitate. Significant up-regulation of genes related to membrane proteins [porinH (10.4-fold), ompU (2.9-fold)], regulatory proteins [envZ (5.6-fold), toxR (4.5-fold), toxS (4.8-fold)], oxidative stress related protein katG (2.3-fold), cell division/maintenance proteins [ftsZ (4.3), mreB (6.5-fold)] and resuscitating promoter factor yeaZ (fourfold) was observed during resuscitation with respect to VBNC state indicating that these genes play a role during resuscitation. Gene expression data presented here would enhance our understanding of resuscitation of V. vulnificus from VBNC state. The results also highlight the importance of maintenance of low temperature during storage of seafood.

Comparative analysis of induction of osmotic-stress-dependent genes in Vibrio vulnificus exposed to hyper- and hypo-osmotic stress.

Vibrio vulnificus, a halophilic pathogenic bacterium of marine environments, encounters changes in salinity in its natural habitat and in the food-processing environment. The comparative response of V. vulnificus to hyperosmotic and hypoosmotic stress in terms of gene expression was investigated. Genes belonging to the proU operon for transport of compatible solutes and compatible solute synthesis were significantly upregulated (3- to 4.7-fold) under hyperosmotic stress. Under hypoosmotic stress, upregulation of genes coding for mechanosensitive channels of small conductance (mscS) was not observed. In hyperosmotic conditions a 2.3-fold decrease in the expression of aqpZ was observed. A 2-fold induction in gyrA was observed in V. vulnificus cells on exposure to hyperosmotic stress. groEL genes, VVA1659 (1.6-fold), and VV3106 (1-fold) were induced in hypoosmotic condition. Results of this study indicate that to manage hyperosmotic stress, V. vulnificus accumulated osmoprotectants through uptake or through endogenous synthesis of compatible solutes. Expression of mscS may not be necessary for immediate protection in cells exposed to hyper- and hypo-osmotic stress. Comparative analysis of important osmotic-stress-related genes showed up- or down-regulation of 14 genes in hyperosmotic stress as compared with up- or down-regulation of only 7 genes in hypoosmotic stress, indicating that the cells respond asymmetrically to hyper- and hypo-osmotic stress.

Characterization of Aeromonas strains isolated from Indian foods using rpoD gene sequencing and whole cell protein analysis.

Aeromonas are responsible for causing gastroenteritis and extra-intestinal infections in humans. Twenty-two Aeromonas strains isolated from different food sources were re-identified up to species level using rpoD gene sequence analysis. Biochemical tests and 16S rRNA gene sequencing were insufficient to identify Aeromonas till species level. However, incorporation of additional biochemical tests lead to correct identification of 95.5 % strains up to species level. The 16S rRNA gene sequencing was useful to identify Aeromonas isolates at the genus level only. Sequences of the rpoD gene showed greater discriminatory power than 16S rRNA gene and provided conclusive discrimination of the strains for which the phenotypic species identification was uncertain. All these 22 strains were accurately identified up to species level by rpoD gene as A. salmonicida (6), A. veronii bv. veronii (4), A. caviae (3), A. hydrophila (2), A. veronii bv. sobria (2), A. jandaei (1), A. trota (1), A. sobria (1), A. allosaccharophila (1) and A. bivalvium (1). All these strains were also characterized using whole cell protein (WCP) analysis by gradient SDS-PAGE and showed different whole cell protein (WCP) profile [22-28 polypeptide bands (~10 to >97 kDa)], indicating high genetic diversity. The present work emphasizes the use of molecular methods such as rpoD gene sequencing along with comprehensive biochemical tests for the rapid and accurate identification of Aeromonas isolates till species level. The WCP profile can be subsequently used to characterize Aeromonas isolates below species level.

Prevalence, characterization, and antimicrobial resistance of Aeromonas strains from various retail food products in Mumbai, India.

A total of 154 food samples (chicken, fish, and ready-to-eat sprouts) from various retail outlets in Mumbai, India, were analyzed for the presence of Aeromonas spp. over a period of 2 y (January 2006 to March 2008). Twenty-two Aeromonas isolates belonging to 7 different species were isolated from 18 (11.7%) food samples. The highest percentages of isolation were from chicken (28.6%) followed by fish (20%) and sprout (2.5%) samples. Aeromonas caviae, A. veronii bv. sobria, and A. salmonicida were the most frequently isolated species from sprouts, chicken, and fish samples, respectively. The genes encoding for putative virulence factors, cytotoxic enterotoxin (act), hemolysin (hly), aerolysin (aer), elastase (ahyB), and lipase (lip) were detected using polymerase chain reaction method in 59.1%, 40.9%, 22.7%, 54.5%, and 31.8% of the strains, respectively. The isolated Aeromonas strains were found to be positive for virulence factors, that is, amylase, DNase, gelatinase, protease, and lipase production. More than 60% isolates were also positive for ß-hemolytic activity. All these food isolates were found to be resistant to ampicillin and bacitracin, and sensitive to gentamicin, 3rd-generation cephalosporins (ceftazidime, cephotaxime, ceftriaxone), and chloramphenicol. Seventeen (77.2%) isolates harbored single and/or multiple plasmids (approximately 5 to >16 kb). The XbaI digestion patterns of chromosomal DNA of these isolates, using pulsed field gel electrophoresis, showed high genetic diversity among these isolates. Our results demonstrate the presence of various Aeromonas spp. with virulence potential and antimicrobial resistance in different food products marketed in Mumbai, India. The potential health risks posed by consumption of these raw or undercooked food products should not be underestimated.