Putative virulence factors of the Aeromonas spp. isolated from food and environment in Abu Dhabi, United Arab Emirates

Thirty randomly selected Aeromonas isolates from food and the environment in Abu Dhabi, United Arab Emirates, were characterized for putative virulence determinants, such as production of cytotoxin, cytotonic toxin, and hemolysin and their capacity to adhere to and invade Henle 407 cells in vitro. Seventy percent of the tested isolates were cytotoxin producers, and 80% were hemolytic. Cytotoxin was produced by 6 of 7 A. hydrophila strains, 6 of 13 A. caviae strains, and 6 of 7 A. veronii bv. sobria strains, mostly from food sources. A. schubertii, A. jandaei, and A. trota also produced both cytotoxin and hemolysin. All of the 30 isolates tested adhered to Henle 407 cells, but none were able to invade the cells, as determined with the in vitro assay. However, no significant correlation of the presence of these putative virulence factors was found among these aeromonad food isolates.     

Prevalence and antimicrobial susceptibilities of Vibrio, salmonella, and Aeromonas isolates from various uncooked seafoods in Thailand

Uncooked seafood samples were collected from open markets and supermarkets in Bangkok, Thailand, and were examined for the presence of Vibrio, Salmonella, and Aeromonas species from January to February 2008. From 120 samples, 272 bacterial isolates were identified through biochemical testing. Of all sea bass, shrimp, oyster, and blood cockle samples (30 of each) that were processed for culture, 114 (95%) samples had at least one detectable isolate of Vibrio, Salmonella, or Aeromonas, leaving only 6 (5%) samples free of them. All oyster sample (100%) had at least one pathogen, followed by sea bass (97%), blood cockles (97%), and shrimp (90%). Overall, 111 (92%) of all samples had detectable Vibrio spp., 32 (27%) had detectable Aeromonas spp., and 25 (21%) had detectable Salmonella enterica. There was no overall difference between positive samples collected from fresh markets versus supermarkets (relative risk, 0.97; 95% CI, 0.89 to 1.05). Resistance to ampicillin among isolated pathogens was relatively high (56%), while resistance to 12 other antibiotics, including azithromycin, ciprofloxacin, and trimethoprim-sulfamethoxazole, was relatively low (0, 0, and 3%, respectively). Study results indicate that uncooked seafood in Bangkok, Thailand, commonly harbors enteric pathogens and that consumption of uncooked seafood should be avoided to reduce foodborne illnesses.     

Comparison of methods for recovering Vibrio cholerae O1 from ice

Alkaline peptone water (1% peptone, 1% NaCl, pH 8.5) and Trypticase soy yeast extract broth (TSYB) supplemented with 2.5% NaCl (pH 8.5) or 1% NaCl (pH 7.5) were evaluated as enrichment broths for the isolation of Vibrio cholerae O1 from ice. Thirty samples of sterile and nonsterile mineral water were inoculated with cell suspensions of this bacterium, quickly frozen, and stored for 3 days at--18 degrees C. After thawing, samples were analyzed by a three-tube most-probable-number technique. Incubation in TSYB with 2.5% NaCl (pH 8.5) for 18 h at 37 degrees C yielded the highest recovery of V. cholerae O1 cells (P < 0.05), a result that might be attributable to the nutrients and to the NaCl concentration of the TSYB, both of which would promote V. cholerae O1 growth and prevent the growth of competitive microbiota.     

A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.     

Occurrence of the tdh and trh genes in Vibrio parahaemolyticus isolates from waters and raw shellfish collected in two French coastal areas and from seafood imported into France

The occurrence of the hemolysin genes, tdh and trh, in Vibrio parahaemolyticus strains isolated from environmental samples collected in two French coastal areas, clinical samples, and seafood products imported into France was studied. Polymerase chain reaction (PCR) with two sets of primers was used to detect the hemolysin genes. Most of the clinical isolates (91%) and 1.5% of the isolates from seafood possessed the hemolysin genes. Three and fifteen percent, respectively, of the two groups of environmental strains carried the hemolysin genes depending on the geographic site. The tdh and trh genes play important roles in virulence. Thus, our results indicate that pathogenic V. parahaemolyticus isolates are present in French coastal areas and in seafood imported into France. Furthermore, they may also be present in French seafood products.     

A New Procedure for the Differentiation of 01 Vibrio cholerae and Non-01 V. cholerae Recovered From Oysters

Live oysters were inoculated with 01 V. cholerae or non-01 V. cholerae and the liquor from the oysters was spread onto thiosulfate citrate bile salts agar plates. After incubation the colonies with yellow centers were picked and streaked onto sheep blood agar plates. Biochemical tests were performed on all colonies. Isolates exhibiting characteristic V. cholerae reactions were checked for serological identity and inoculated into a vibrio medium. After incubation, the isolates were further analyzed for lactic and succinic acids by gas-liquid chromatography. Peak height ratios of lactic/succinic acid provided a testing parameter to distinguish all isolates of 01 V. cholerae from non-01 V. cholerae isolates recovered from the oysters.     

副溶血性弧菌ERIC-PCR分型及毒力基因检测研究

目的:建立副溶血性弧菌ERIC-PCR分子分型技术,分析副溶血性弧菌标准菌株及分离株基因组DNA ERIC-PCR指纹图谱,并对副溶血性弧菌毒力基因进行检测,以了解不同来源副溶血性弧菌毒力基因携带情况.方法:提取副溶血性弧菌基因组DNA,以肠杆菌基因间共有重复序列(ERIC)为引物进行PCR扩增,PCR产物经琼脂糖凝胶电泳后用凝胶成像分析仪对图谱进行观察分析,并以相似性系数构建聚类图;通过PCR方法对直接耐热溶血素(TDH)和耐热直接相关溶血素(TRH)进行检测.结果:26株副溶血性弧菌均可扩增产生可重复的DNA指纹图谱,ERIC-PCR可将26株菌分为12个型,分辨力指数为0.926;只在临床分离株中检测到TDH基因,而除一株标准菌株外,所有菌株都未检测到TRH基因.结论:研究显示ERIC-PCR可从分子水平对副溶血性弧菌基因组DNA进行快速指纹图谱分析,同时结合毒力基因检测,能够为副溶血性弧菌食物中毒疾病的预防和流行病学调查提供科学依据.     

Foodborne disease outbreaks caused by sucrose-nonfermenting and beta-galactosidase-deficient variants of Vibrio cholerae

We reported four foodborne disease outbreaks in Taiwan caused by sucrose-nonfermenting and by beta-galactosidase-deficient variants of non-O1, non-O139 Vibrio cholerae. The sucrose-nonfermenting vibrios collected from three outbreaks were biochemically identified to be V. mimicus and the beta-galactosidase-deficient vibrios from an outbreak to be V. alginolyticus. However, molecular methods including DNA-DNA hybridization, fatty acid profile analysis, and sequence analysis of 16S rRNA, oriC, pyrH, recA, and rpoA indicated that these vibrios should be V. cholerae. These V. cholerae variants carried two hemolysin genes, hlyA and hlx, but contained neither cholera toxin gene, ctx, V. mimicus hemolysin gene, vmh, nor thermo-directed hemolysin, tdh. The sucrose-nonfermenting variants of V. cholerae shared a high level of genetic relatedness; they could derive from a common clone. In our record from 1995 to date, this was the first time that V. cholerae variants were discovered as etiologic agents for foodborne disease outbreaks in Taiwan.     

Adhesion and colonization of Vibrio cholerae O1 on shrimp and crab carapaces

The potential of Vibrio cholerae O1 to attach to and colonize the carapaces of shrimp and crabs was evaluated. One million cells of V. cholerae O1 were spread within a circle on the external surfaces of separated carapaces and stored at 22 +/- 0.2 degrees C in a moist environment to permit adherence. Attached vibrios were counted directly by an immunofluorescence technique and by the pour plate technique after detachment of the cells. To study the colonization process, rifampicin-resistant strains of V. cholerae O1 were used. V. cholerae O1 strains, including those resistant to rifampicin, were able to attach to shrimp and crab carapaces. Dorsal crab carapaces showed higher levels of attachment than ventral carapaces. Colonization of V. cholerae O1 on these carapaces was also demonstrated. Both attachment and colonization on the shrimp exoskeleton were optimal at a salinity of 1.0 to 1.5%, a pH of 6.0 to 7.0, and a temperature of 37 degrees C. Less than 2% attachment at 3 degrees C contrasted with >20% attachment at 37 degrees C. Even at 3% NaCl, some attachment was observed. Although attachment percentages may appear low (2 to 20%), they represent significant numbers, about 3.7 to 5.6 log10 CFU per carapace. A rugose V. cholerae O1 strain attached to and colonized the shrimp carapace in a fashion very similar to that of the smooth strain from which it was derived. The ability of V. cholerae O1 to attach to and colonize exoskeletons of edible crustaceans provides a potential means of survival in aquatic environments. Concentrations of vibrios that may be reached on a single crab or shrimp carapace are clearly of concern with regard to public health.     

Detection and molecular characterization of Vibrio parahaemolyticus isolated from seafood harvested along the southwest coast of India

The levels of total and tdh⁺ Vibrio parahaemolyticus were estimated in 83 seafood samples from southwest coast of India by colony hybridization. Conventional enrichment and isolation technique was also used to study the prevalence. Polymerase chain reaction (PCR) was performed on bacterial cell lyates for detection of total and pathogenic V. parahaemolyticus by amplification of specific genes. Of 83 samples tested, V. parahaemolyticus could be detected in 74 (89.2%) samples and tdh⁺ V. parahaemolyticus in 5 (6.0%) samples by colony hybridization. V. parahaemolyticus was detected in 68 (81.9%) of 83 samples after 18 h of enrichment by PCR, and isolated from 63 (75.9%) of 83 samples by conventional isolation. The virulence genes tdh and trh could be detected in 8.4% and 25.3%, respectively, in the sample enrichment broths by PCR. Use of colony hybridization following enrichment to achieve sensitive detection of tdh⁺ V. parahaemolyticus in seafood was evaluated using another set of 58 seafood samples. Thirty pathogenic V. parahaemolyticus strains isolated during the study were screened by PCR for genetic markers to be specific for the detection of the pandemic clone. Results of this study suggest that the GS-PCR may serve as a reliable genetic marker for the pandemic clone of V. parahaemolyticus.     

Biochemical and virulence characterization of viable but nonculturable cells of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a common foodborne pathogen frequently causing outbreaks in summer. Maintenance of virulence by the viable but nonculturable (VBNC) state of this pathogen would allow its threat to human health to persist. This study reports on the change in virulence and concomitant changes in activity of two enzymes and fatty acid profiles when V. parahaemolyticus ST550 entered the VBNC state in the modified Morita mineral salt-0.5% NaCl medium incubated at 4 degrees C. The major change in fatty acid composition occurred in the first week, with a rapid increase in C15:0 fatty acid and saturated/unsaturated ratio while a rapid decrease in C16:1 was observed. The activity level of the inducible protective enzyme superoxide dismutase became undetectable in the VBNC state, whereas that of constitutive glucose-6-phosphate dehydrogenase did not change in either the exponential phase or the VBNC state. Cytotoxicity against HEp-2 cells and a suckling mouse assay showed that virulence was lowered in the VBNC state compared with exponential-phase cells. Longer incubation times were required by the VBNC cells to achieve the same level of virulence as seen in exponential-phase cells. Culturable cells were recovered on selective agar medium from the VBNC cultures injected into suckling mice, probably as the result of in vivo resuscitation. Results of this study add to our understanding of the biochemical and physiological changes that have not been reported when V. parahaemolyticus enters into the VBNC state.     

Putative virulence properties of Aeromonas strains isolated from food, environmental and clinical sources in Italy: a comparative study

The distribution of virulence properties in 142 strains of Aeromonas isolated from diarrhoeic patients, food and surface water in Italy and identified by biochemical and molecular methods was investigated. The virulence properties studied were the presence of genes for the aerolysin (aerA), heat-stable cytotonic enterotoxin (ast), heat-labile cytotonic enterotoxin (alt), cytotoxic enterotoxin (act); and cytotoxicity for Vero cells and adhesion on Hep-2 cells. A. hydrophila and A. caviae were the species most commonly isolated from clinical and environmental samples (9/30; 30.0% and 5/27; 18.5%, respectively) while mesophilic A. salmonicida was most common in food samples (19/80; 23.7%). Out of 142 strains, 86 (60.6%) were positive for at least one of the virulence properties. All the toxin genes were present in 4/18 (22.3%) of clinical strains. Most of the food isolates (54/55; 98.2%) were cytotoxic and most of the environmental strains (12/13; 92.3%) were adhesive. The aerA gene was present in most toxigenic strains (72/86; 83.7%), irrespective of their origin. The growth temperature affected the expression of cytotoxicity and adhesivity. Aeromonas strains from food and surface water frequently had toxin gene patterns similar to those of clinical strains and expressed virulence properties at human body temperature. These findings indicate that aeromonads have the potential to cause human illness and confirm the role of food and water as vehicles for Aeromonas diseases.     

Preliminary examinations on the enterotoxigenicity of isolates of Klebsiella pneumoniae from seafoods.

One hundred and eighty-five seafood samples, consisting of 96 freshwater fish, 37 marine fish, 13 freshwater prawn, 13 marine prawn and 26 molluscs were screened for presence of Klebsiella. Out of these, 12 isolates of Klebsiella were identified, Four K. pneumoniae var. ozaenae were isolated from marine fish samples and eight K. pneumoniae var. pneumoniae, six from freshwater fish and two from freshwater prawns. All 12 isolates were tested for enterotoxigenicity by the vasopermeability factor test in rabbits, the mouse foot pad test, the latex agglutination test and the coagglutination test. One isolate of K. pneumoniae var pneumoniae, isolated from fresh water prawn was found enterotoxigenic.     

Relationship between the effects of stress induced by human bile juice and acid treatment in Vibrio cholerae

The effects of low pH and human bile juice on Vibrio cholerae were investigated. A mild stress condition (exposure to acid shock at pH 5.5 or exposure to 3 mg of bile per ml for 20 min) slightly decreased (by < or = 1 log unit) V. cholerae cell viability. However, these treatments induced tolerance to subsequent exposures to more severe stress. In the O1 strain, four proteins were induced in response to acid shock (ca. 101, 94, 90, and 75 kDa), whereas only one protein (ca. 101 kDa) was induced in response to acid shock in the O139 strain. Eleven proteins were induced in response to bile shock in the O1 strain (ca. 106, 103, 101, 96, 88, 86, 84, 80, 66, 56, and 46 kDa), whereas only one protein was induced in response to bile shock in the O139 strain (ca. 88 kDa). V. cholerae O1 and O139 cells that had been preexposed to mild acid shock were twofold more resistant to pH 4.5 (with times required to inactivate 90% of the cell population [D-values] of 59 to 73 min) than were control cells (with D-values of 24 to 27 min). Likewise, cells that were preexposed to mild bile shock (3 mg/ml) were almost twofold more tolerant of severe bile shock (30 mg/ml; D-values, 68 to 87 min) than were control cells (with D-values of 37 to 43 min). These protective effects persisted for at least 1 h after the initial shock but were abolished when chloramphenicol was added to the culture during the shock. Cells preexposed to acid shock exhibited cross-protection against subsequent bile shock. However, cells preexposed to bile shock exhibited no changes in acid tolerance. Bile shock induced a modest reduction (0 to 20%) in enterotoxin production in V. cholerae, whereas acid shock had no effect on enterotoxin levels. Adaptation to acid and bile juice and protection against bile shock in response to preexposure to acid shock would be predicted to enhance the survival of V. cholerae in hosts and in foods. Thus, these adaptations may play an important role in the development of cholera disease.     

Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish

The presence of virulence genes and integrons was determined in 81 strains of Aeromonas veronii isolated from farm-raised catfish. Polymerase chain reaction (PCR) protocols were used to determine the presence of genes for cytotoxic enterotoxin (act), aerolysin (aerA), two cytotonic enterotoxins (ast, alt), lipase (lip), glycerophospholipid:cholesterol acyltransferase (gcaT), serine protease (ser), DNases (exu), elastase (ahyB) and the structural gene flagellin (fla) in the template DNA. Oligonucleotide primers amplified a 231-bp region of the act gene from the template DNA of 97.0% of the isolates. Primers specific for the amplification of the aerA gene amplified a 431-bp region of the aerA gene from the template DNA of 96.0% of the isolates. None of the isolates contained ast or alt genes. Oligonucleotide primers specific for the amplification of lip, gcaT, ser and fla genes, amplified their respective amplicons from 85.0, 78.0, 82.0 and 80.0% of the isolates. None of the isolates contained exu or the elastase genes. Several of the isolates (48.0%) contained class I integrons that confer resistance to multiple antibiotics; various sizes between 0.6 and 3.1 kb were found. None of the isolates contained Class II integrons. Our results indicate that farm-raised catfish may be a source of pathogenic A. veronii and that the potential health risks posed by virulent strains of A. veronii should not be underestimated.     

The impact of the postharvest environment on the viability and virulence of decay fungi

Postharvest decay of fruits, vegetables, and grains by fungal pathogens causes significant economic losses. Infected produce presents a potential health risk since some decay fungi produce mycotoxins that are hazardous to human health. Infections are the result of the interplay between host resistance and pathogen virulence. Both of these processes, however, are significantly impacted by environmental factors, such as temperature, UV, oxidative stress, and water activity. In the present review, the impact of various physical postharvest treatments (e.g., heat and UV) on the viability and virulence of postharvest pathogens is reviewed and discussed. Oxidative injury, protein impairment, and cell wall degradation have all been proposed as the mechanisms by which these abiotic stresses reduce fungal viability and pathogenicity. The response of decay fungi to pH and the ability of pathogens to modulate the pH of the host environment also affect pathogenicity. The effects of the manipulation of the postharvest environment by ethylene, natural edible coatings, and controlled atmosphere storage on fungal viability are also discussed. Lastly, avenues of future research are proposed.     

Plant-derived antimicrobials reduce Listeria monocytogenes virulence factors in vitro, and down-regulate expression of virulence genes

Listeria monocytogenes (LM) is a major foodborne pathogen causing septicemia, meningitis and death in humans. LM infection is preceded by its attachment to and invasion of human intestinal epithelium followed by systemic spread. The major virulence factors in LM include motility, hemolysin and lecithinase production. Reducing LM attachment to and invasion of host tissue and production of virulence factors could potentially control listeriosis in humans. This study investigated the efficacy of sub-inhibitory concentrations (SICs, concentrations not inhibiting bacterial growth) of three, generally regarded as safe (GRAS)-status, plant-derived antimicrobial compounds in reducing LM attachment to and invasion of human colon adenocarcinoma (Caco-2) and human brain microvascular endothelial cells (HBMEC). Additionally, the effect of these compounds on the aforementioned LM virulence factors was studied. The compounds and their respective SICs used relative to their MICs were trans-cinnamaldehyde (TC 0.50mM, 0.75mM with the MIC of 0.90mM), carvacrol (CR 0.50mM, 0.65mM with the MIC of 0.75mM), and thymol (TY 0.33mM, 0.50mM with the MIC of 0.60mM). All three-plant antimicrobials reduced LM adhesion to and invasion of Caco-2 and HBMEC (p<0.05). The compounds also decreased LM motility, hemolysin production and lecithinase activity (p<0.05). Real-time PCR data revealed that TC, CR, and TY down-regulated the expression of LM virulence genes by >3.0 folds compared to controls (p<0.05). Results suggest that TC, CR, and TY could potentially be used to control LM infection; however, in vivo studies are necessary to validate these results.     

Control of the transmission of Vibrio cholerae and other enteropathogens by foods originating from endemic areas in South America and elsewhere as a model situation.

The cholera-pandemic raging in South and Middle America and endemic cholera in other countries call for measures of health protection of the local population, but particularly with respect to the young, old, pregnant and immunocompromised citizens of countries importing food from the areas where the disease has struck. Instead of harshly barring importation, a more humanitarian policy is recommended, relying on assistance of areas presenting risks, with the introduction of and adherence to rigorous measures of longitudinally integrated microbiological safety assurance. This model is equally applicable to other enteric diseases transmitted by food. Examples are given of how canned foods of neutral pH, fishery products, vegetables and certain fruits should be processed for safety. Importation monitoring strategies, linked to this proactive approach to consumer protection, are briefly summarized.     

Influence of iron-chelated growth conditions on outer membrane protein production and virulence of Vibrio tubiashii

Growth of two Vibrio tubiashii strains under iron-chelated conditions resulted in the production of a hydroxymate-like siderophore, and expression of outer membrane proteins with homologies to proteins in Vibrio cholerae and Vibrio vulnificus which were not seen in cells grown under non-chelated growth conditions. PCR analysis using primers based on Listonella anguillarum’s ferric uptake Repressor protein (fur) gene detected a 316 bp fur gene homolog which also had sequence homology to the fur genes of V. cholerae and V. vulnificus. V. tubiashii cultured under iron-chelated growth conditions induced a greater fluid accumulation (FA) response in suckling mice than cells which were cultured under iron non-chelated growth conditions. Our observations that V. tubiashii possesses a fur-like gene homolog and expresses unique OMPs, a hydroxymate-like siderophore, and produces an increased fluid accumulation response in suckling mice when grown under iron-chelated condition support previous findings that V. tubiashii may have the essential components for the survival and establishment of infections and this report represents the first observations of competent iron acquisition system in V. tubiashii which is similar to those produced by other marine vibrios, many of which are pathogenic for humans.     

The phylogenetic group,antimicrobial susceptibility,and virulence genes of Escherichia coli from clinical bovine mastitis

Bovine mastitis is still a central problem on dairy farms despite control programs, and Escherichia coli is a crucial pathogen during the development of bovine mastitis. The virulence genes, antimicrobial susceptibility, and mortality of mice infected with different E. coli isolates from bovine mastitis were determined in this study. According to the presence of the specific genes chuA, yjaA, and TspE4.C2, these isolates mainly belonged to 2 different groups: group A (47/79) and group B1 (22/79). The ompC gene was detected in all the isolates, followed by fimH (89.9%), ECs3703 (88.6%), and ompF (73.4%), whereas most of the virulence genes were not detected in these isolates. The results of the antimicrobial susceptibility tests indicated that the isolates were susceptible to the fluoroquinolones and aminoglycosides. An inverse relationship was shown between the expression level of ompF and antimicrobial resistance; additionally, the isolates that were nonsusceptible to at least 4 classes of antimicrobial agents showed a lower mortality to mice in comparison with the susceptible isolates. This study indicated that antibiotic resistance had emerged in E. coli from bovine mastitis in this area, and appropriate measures should be taken to avoid potential threats to humans and other animals.