Diverse Profiles of N‐acyl Homoserine l‐Lactones,Biofilm, Virulence Genes and Integrons in Food‐Borne Aeromonas Isolates

Aeromonas are regarded as opportunistic as well as primary pathogens of humans and fish, and are associated with gastroenteritis and septicemia in humans. Production of N‐acyl‐homoserine lactone (AHL) signal molecules and biofilm was determined in 22 Aeromonas isolates, from different food products in India, using thin‐layer chromatography (TLC) analysis and microtiter‐plate assay, respectively. Overall, highly heterogeneous patterns of AHL production were observed, with the production of N‐butanoyl homoserine lactone (C4‐HSL) and N‐hexanoyl homoserine lactone (C6‐HSL) by the majority (81.8%) of Aeromonas food isolates. Moreover, putative N‐pentanoyl homoserine lactone (C5‐HSL), N‐heptanoyl homoserine lactone (C7‐HSL), and N‐octanoyl homoserine lactone (C8‐HSL) were produced by 72.7%, 27.3%, and 9.1% of isolates, respectively. This is the 1st report of production of C7‐HSL by Aeromonas species. Aeromonas food isolates were highly variable in their biofilm forming abilities with majority of them as weak biofilm producers in 2 different media, TSB and M9 minimal medium supplemented with 0.4% glucose. The genes encoding for putative virulence factors, glycerophospholipid cholesterol acyltransferase (gcat), heat‐labile cytotonic enterotoxin (alt), heat‐stable cytotonic enterotoxin (ast), serine protease (ser), polar flagella (fla), and lateral flagella (lafA) were present in 95.5%, 59.1%, 22.7%, 81.8%, 77.3%, and 22.7% of the strains, respectively. Class 1 integrons (100 to 3000 bp) were found in 68.2% of food isolates; whereas, 50% isolates contained class 2 integrons (150 to 1600 bp). This study provides a baseline data on the diversity of AHLs, biofilm forming ability and presence of virulence genes and integrons in Aeromonas food isolates from India.     

Detection of potentially pathogenic Aeromonas isolates from ready‐to‐eat seafood products by PCR analysis

This study provides data on the prevalence of potentially pathogenic Aeromonas spp. in ready‐to‐eat (RTE) seafood products by evaluating the occurrence of Aeromonas spp. and the presence of virulence‐associated genes. Aeromonas spp. was detected in 57/81 (70.3%) RTE seafood samples. Specifically, Aeromonas spp. was highlighted in 19/21 (90.5%) sushi, in 18/21 (85.7%) sea salad, 11/12 (91.7%) surimi and 9/12 (75%) peeled shrimp samples. Aeromonas spp. was not observed in marinated anchovies and octopus salad samples. Then, PCRs aimed at the hlyA, aerA, alt and ast genes, encoding, respectively, haemolysin A, aerolysin, aeromonas labile temperature cytotonic enterotoxin and aeromonas stable temperature cytotonic enterotoxin, demonstrated a widespread distribution of these genes among Aeromonas isolates. The results underline the need to implement an adequate control plan performing an intensive and continuous monitoring to guarantee the human health.     

Emetic toxin producing Bacillus cereus Korean isolates contain genes encoding diarrheal-related enterotoxins

Bacillus cereus can cause the diarrheal and emetic type of food poisoning but the symptoms of emetic food poisoning caused by B. cereus occasionally include emesis and diarrhea. The enterotoxin characteristics of emetic toxin (cereulide) producing B. cereus were needed to be determined. Therefore, forty B. cereus strains isolated from various sources in Korea were investigated for the presence of enterotoxin genes. All strains were confirmed to produce the emetic toxin using HPLC-MS methods. The rates of the nheABC, hblCDA, entFM and cytK genes amongst emetic toxin producing B. cereus strains were 82.5, 7.5, 50.0 and 27.5%, respectively. Pattern III harbored nheABC and entFM genes and pattern V processed entFM gene and were shown to be the major patterns, being present in 55.0% (21 of 40) of the emetic toxin producing B. cereus strains. Our findings revealed that 34 (85.0%) of 40 emetic toxin producing B. cereus strains isolated in Korea have the potential to cause diarrheal and emetic type of food poisoning, simultaneously. Thus, emetic toxin and enterotoxin genes should be constantly screened to provide insight into B. cereus food poisoning.     

Toxin profile, antibiotic resistance, and phenotypic and molecular characterization of Bacillus cereus in Sunsik

Sunsik, a ready-to-eat food in Korea, is comprised of various agricultural and marine products, and has been an important concern in Bacillus cereus food poisoning. The aim of this study was to investigate the toxin profiles, genotypic and phenotypic patterns as well as antibiotic resistance of B. cereus strains isolated from Sunsik. A subtyping method known as automated repetitive sequence-based PCR system (DiversiLab™) was used to assess the intraspecific biodiversity of these isolates. Thirty-five B. cereus strains were isolated from 100 commercial Sunsik samples, all of which harbored at least 1 enterotoxin gene. The detection rates of nheABC, hblCDA, cytK, and entFM enterotoxin gene among all isolates were 97%, 86%, 77%, and 100%, respectively. Most strains also produced corresponding enterotoxins such as HBL (83%) and NHE (94%). One strain (2.9%) carried the emetic toxin genes, including ces and EM1, and was positive for the HEp-2 cell emetic toxin assay. Most strains were positive for various biochemical tests such as salicin hydrolysis (86%), starch fermentation (89%), hemolysis (89%), motility test (100%) and lecithinase hydrolysis (89%). All isolates were susceptible to most antibiotics although they were highly resistant to β-lactam antibiotics. By using the automated rep-PCR system, all isolates were successfully differentiated, indicating the diversity of B. cereus strains present in Sunsik.     

Biochemical and toxic diversity of Bacillus cereus in a pasta and meat dish associated with a food-poisoning case

A dish of pasta and minced meat caused severe food-poisoning involving both emesis and diarrhoea in two adult persons. Emetic toxin producing strains of Bacillus cereus formed the majority (68% of 122) of strains identified in this food. Haemolytic diarrhoeal toxin was produced by 26% of the strains studied and 6% of the strains produced neither emetic nor haemolytic diarrhoeal toxin. The B. cereus strains isolated from this dish could be divided into four biochemically distinct groups and two different colony morphologies. All emetic toxin producing strains (n=83) were negative for both haemolytic enterotoxin and starch hydrolysis in contrast to haemolytic enterotoxin producers (n=32). Colonies of emetic toxin producing strains were poorly haemolytic, ⩽2 mm zones, in contrast to the diarrhoeal colonies, 4–5 mm zones. This disparity persisted after extended incubation using blood agar supplemented with lithium chloride. Despite the wide diversity of B. cereus biotypes in this single food all emetic toxin producers exhibited narrow haemolysis with negative starch hydrolysis. The findings emphasize that colonies with different properties should be isolated when food-poisoning cases are studied.     

RE-PCR variability and toxigenic profile of food poisoning, foodborne and soil-associated Bacillus cereus isolates from Brazil

Twenty-three Bacillus cereus isolates from food poisoning outbreaks associated with a diarrheal-type syndrome, fourteen foodborne isolates not associated with food poisoning and fifteen isolates from Brazilian soil samples were analyzed for the presence and genetic diversity (by RE-PCR) of the virulence genes ces (emetic toxin, cereulide), plcR-papR (pleiotropic regulator PlcR and peptide PapR), nheA (a component of the NHE complex), bceT (diarrheal enterotoxin bc-D-ENT), gyrB (B subunit of DNA gyrase), cytK-2 (necrotic enterotoxin cytotoxin K-2), and plcA (phosphatidylcholine-specific phospholipase C). Additionally, these isolates were phenotypically characterized for motility, hemolytic and lecithinase activities, as well as HBL enterotoxin production. The group of isolates associated with food poisoning had the highest occurrence of the phenotypically analyzed factors and the most frequent occurrence and highest genetic diversity of the plcR-papR, nheA, bceT, cytK-2, plcA, and gyrB genes. An analysis of molecular variance (AMOVA), in which all loci were analyzed, demonstrated that the genetic variation intragroup of isolates (92%) was significantly higher than that intergroup (8%) (P < 0.05). These results were corroborated by an analysis of the genetic differentiation between the groups, which was low/moderate, the result of a high degree of allele sharing. Our results suggest that B. cereus isolates with the potential to cause food poisoning outbreaks do not have a specific genetic profile characterized by the presence of a particular gene or allele among the genes assessed. On the contrary, different combinations of genes encoding virulence factors may be present in different isolates of B. cereus that potentially cause food poisoning outbreaks.     

Detection of genes for enterotoxins (ent) and toxic shock syndrome toxin-1 (tst) in mammary isolates of Staphylococcus aureus by polymerase-chain-reaction

During a two-year-period (1997–1998) 94 field strains of Staphylococcus aureus isolated from cases of bovine mastitis were investigated for the presence of genes for staphylococcal enterotoxin (ent) and toxic shock syndrome toxin-1 (TSST-1; tst). Polymerase-chain reaction (PCR) was performed using six pairs of relevant oligonucleotide primers. Thirty-four isolates were positive for one (18 strains) or two (16 strains) toxin genes. Three field strains were positive for enterotoxin A gene (sea), two for enterotoxin B gene (seb), 22 for enterotoxin C gene (sec), four for enterotoxin D gene (sed) and 19 for TSST1 gene (tst). The enterotoxin E gene (see) sequence was not found. The combination of sec and tst showed the highest incidence. A high correlation between PCR results and toxin protein detection by a commercial enzyme linked immunosorbent assay (ELISA) system and a reversed passive latex-agglutination (RPLA) test was observed. In these immunoassays, only one strain of S. aureus produced equivocal results for production of enterotoxin A, giving an overall concordance between genotypic and phenotypic identification of 98.9%.     

Molecular characterization and clonal diversity of methicillin-susceptible Staphylococcus aureus in milk of cows with mastitis in Brazil

Mastitis is an important disease for the dairy industry worldwide, causing economic losses and reducing milk quality and production. Staphylococcus aureus is a worldwide agent of this intramammary infection, which also causes foodborne diseases. The objective of this study was to determine the frequency of methicillin-susceptible Staphylococcus aureus (MSSA) isolates in milk of mastitis cows in Brazil and to analyze the genetic lineages and the content of antimicrobial resistance genes and virulence factors among these isolates. Fifty-six MSSA isolates were recovered from 1,484 milk samples (positive for the California mastitis test) of 518 cows from 11 different farms in Brazil (representing 51% of total Staph. aureus obtained), and they were further characterized. Methicillin-susceptible Staphylococcus aureus were isolated from 3.7% of California mastitis test-positive tested milk samples and from 6.2% of tested mastitic cows. Methicillin-susceptible Staphylococcus aureus isolates were characterized by spa typing, agr typing, and multilocus sequence typing, and resistance and virulence traits were investigated by PCR. Seven spa types were identified among MSSA (% of isolates): t127 (44.6), t605 (37.5), t002, t1784, t2066 (1.8), and 2 new ones: t10856 (10.7) and t10852 (1.8). Five distinct sequence types (ST) were detected (% of isolates): ST1 (46.4), ST126 (37.5), ST133 (10.7), ST5 (3.6), and a novel ST registered as ST2493 (1.8). Resistances were detected for streptomycin, chloramphenicol, and tetracycline. One strain contained the chloramphenicol resistance gene (fexA; included within transposon Tn558) and 3 strains contained the tetracycline resistance gene [tet(K)]. Methicillin-susceptible Staphylococcus aureus strains were susceptible to most of the antibiotics studied and lacked the virulence genes of Panton-Valentine leukocidin (lukF/S-PV), toxic shock syndrome toxin 1 (tst), exfoliative toxin A (eta), and exfoliative toxin B (etb), as well as the genes of the immune evasion cluster. Methicillin-susceptible Staphylococcus aureus isolates were detected in a relatively low proportion of cows with mastitis (6.2%) and recovered isolates presented high diversity of genetic lineages, with CC1 and CC126 the predominant clonal complexes, and CC133 also being detected. Larger epidemiological studies with molecular characterization of isolates are required to deepen the knowledge on the circulating genetic lineages among the cow population with mastitis.     

Multiplex PCR detection of enterotoxin genes in Aeromonas spp. from suspect food samples in northern Taiwan

Aeromonads possess an array of virulence factors and are causative agents of a number of human infections. Among them, genes of one cytotoxic (Act) and two cytotonic (Alt, Ast) enterotoxins are implicated in a human diarrheal disease. A rapid, specific, simultaneous detection of these enterotoxin genes in suspected food poisoning samples is not yet reported. Hence, a multiplex PCR assay was designed to amplify the cytotoxic (act), heat-labile cytotonic (alt), and heat-stable cytotonic (ast) enterotoxin genes of aeromonads. The PCR assay was tested with 133 Aeromonas spp. isolated from suspect food poisoning samples and retail samples of poultry and fish from wet markets in and around Taipei, Northern Taiwan. The Aeromonas spp. isolates were divided into six genotypes based on absence or presence of one or more enterotoxin genes. Of these 133 isolates, Aeromonas caviae (52.5%) and Aeromonas hydrophila (43.4%) were the most frequently isolated species from food poisoning samples and retail samples, respectively. Among the species, A. hydrophila had a significantly higher proportion for harboring three enterotoxin genes than had the others, whereas Aeromonas encheleia, considered a nonpathogen, was found harboring three enterotoxin genes. The multiplex PCR assays are rapid and specific, and provide a useful tool for the detection and genotyping of enterotoxin genes of aeromonads.     

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.     

Comparison of the enterotoxigenic types,toxic shock syndrome toxin I (TSST-1) strains and antibiotic susceptibilities for enterotoxigenicStaphylococcus aureusstrains isolated from food and clinical samples

The distribution of enterotoxin types and toxic shock syndrome toxin I (TSST-1) strains for 176Staphylococcus aureusstrains obtained from food samples and 62S. aureusstrains isolated from clinical samples were compared. It was found that for both the food and clinical isolates, staphylococcal enterotoxin A (SEA) strains accounted for the major part (75% and 45% of the total enterotoxigenic strains for food and clinical isolates, respectively) followed by SEB or SEAB, SEC and SED strains. For food isolates, none of theS. aureusstrains was TSST-1 strain while for clinical isolates, three strains (1 SEC, 1 SED and 1 SEAB strain) were found to be TSST-1 strains. When susceptibilities for these enterotoxigenicS. aureusstrains to antibiotics, such as penicillin, oxacillin, vancomycin, methicillin, streptomycin, tetracycline, gentamycin and kanamycin were compared, results showed that 51.6% of the food isolates were resistant to penicillin G only but sensitive to the other antibiotics tested. Also, 8 of the 64 enterotoxigenic strains isolated from food samples were all sensitive to antibiotics while none of the enterotoxigenic strains from clinical samples showed this antibiotic susceptibility pattern. No methicillin resistantS. aureus(MRSA) strains could be found among the food isolates. On the other hand, the majority (42.4%) of the enterotoxigenicS. aureusstrains from clinical samples were penicillin and/or other antibiotics resistant MRSA strains. Since MRSA strains often posed the therapeutic problem, the MRSA strains were further confirmed by PCR assay using the primers specific formecA gene. It was found that results obtained from the disc agar diffusion method and the PCR method were the same.     

Genetic diversity, antimicrobial resistance and toxigenic profiles of Bacillus cereus isolated from food in Brazil over three decades

Bacillus cereus is an ever-present problem. It is widely distributed in several environments such as soil and plants and is commonly isolated from food and additives. In this study we analyzed 97 foodborne B. cereus sensu stricto strains isolated in Brazil in the 1980's, 1990's and 2000's in order to investigate the genetic diversity (assessed by Rep-PCR), antimicrobial resistance and toxigenic profiles (presence of hblA, hblC and hblD; nheA, nheB and nheC as well as cytK, ces and entFM genes) of such strains. The majority of the strains (79, 81.4%) were β-hemolytic. The NHE complex was found in 82 strains (84.5%) and HBL complex was found in 61 (62.9%) strains. All strains were negative to ces. The cytK-2 gene was found in 44 (45.4%) strains. The predominant toxigenic pattern was type I (32, 33%) which included strains positive for all toxin genes but ces. Computer assisted cluster analysis of Rep-PCR profiles showed a high genetic diversity. Seven major clusters comprising two or more strains were found and cluster 1 was predominant (ten strains, nine of them showing 100% similarity). This cluster included strains isolated in the 1980's and the 1990's. Cluster analysis of Rep-PCR profiles based on decade of isolation, source, hemolytic pattern, toxigenic and antibiotic resistance patterns revealed a similar clustering pattern as found in the analysis including all strains. The inability to observe a predominant band pattern when Rep-PCR cluster analysis was based on decade of isolation suggests that this diversity has been maintained over time. All strains were susceptible to gentamicin. We detected resistance to tetracycline (11 strains showing intermediate resistance and nine completely resistant strains), clindamycin (ten intermediate strains) and vancomycin (one strain). Clindamycin resistance showed statistical association with strains isolated in 2000's. The predominant resistance pattern was type A (72, 72.2%) which included strains susceptible to all drugs tested. Our results suggest that the majority of the strains present in several types of food in Brazil pose a potential risk to cause food poisoning due to the high prevalence of toxin genes found in these strains. However, additional studies involving cytotoxicity tests and affiliation of these strains to phylogenetic groups based on molecular data would be useful to better evaluate this potential and could provide a more accurate indication of the risk.     

Enterotoxigenic Profiling of Emetic Toxin‐ and Enterotoxin‐Producing Bacillus cereus,Isolated from Food,Environmental, and Clinical Samples by Multiplex PCR

Bacillus cereus comprises the largest group of endospore‐forming bacteria and can cause emetic and diarrheal food poisoning. A total of 496 B. cereus strains isolated from various sources (food, environmental, clinical) were assessed by a multiplex PCR for the presence of enterotoxin genes. The detection rate of nheA, entFM, hblC, and cytK enterotoxin genes among all B. cereus strains was 92.33%, 77.21%, 59.47%, and 47.58%, respectively. Enterotoxigenic profiles were determined in emetic toxin‐ (8 patterns) and enterotoxin‐producing strains (12 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. Our findings revealed that B. cereus must be considered a serious health hazard and Bacillus thuringiensis should be considered of a greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.     

The occurrence of virulence traits among high-level aminoglycosides resistant Enterococcus isolates obtained from feces of humans, animals, and birds in South Korea

Enterococcus isolates (1500) obtained from the feces of 48 humans, 209 domesticated food animals, and 155 wild geese in South Korea were characterized with respect to species status by PCR analyses and resistance to antibiotics. Of the 1500 strains examined, the majority (n = 577) were Enterococcus faecalis from 224 (54.4%) of the samples feces, while 299 were of E. faecium from 125 of the samples (30.3%), 224 were E. hirae from 101 (24.5%) of the samples, 94 were E. casseliflavus from 43 (10.4%) of the samples, and one was E. gallinarum. While 305 isolated from 125 (30.3%) of the samples were unidentified species. Approximately 15, 60, 50, 55, 3, and 40% of samples obtained from beef cattle, chickens, ducks, swine, wild geese, and humans, respectively, yielded Enterococcus isolates that were resistant to high-levels of aminoglycosides (i.e., of gentamicin, kanamycin, and streptomycin, minimum inhibitory concentrations were > 1000 mg/l). The 180 Enterococcus isolates that showed high levels of resistance to aminoglycoside antibiotics (HLAR) were screened for virulence genes encoding for aggregation substance (agg), cytolysin activator (cylA), gelatinase (gelE) and surface protein (esp). Of those, the gelE gene was found most frequently in chickens and ducks of the HLAR isolates, while 56 E. faecalis and 13 E. faecium HLAR were gelatinase positive and showed hemolysin activity. Multiple antibiotic resistant Enterococcus isolates carrying virulence genes were most frequently isolated from poultry and swine, and were mostly E. faecalis or E. faecium. These findings suggest that restriction of the use of antibiotics in food animal operations in South Korea, especially those involved in poultry and swine production would be desirable.     

Prevalence of staphylococcal enterotoxins, toxin genes and genetic-relatedness of foodborne Staphylococcus aureus strains isolated in the Marmara Region of Turkey

Staphylococcus aureus is a major foodborne pathogen and it has the ability to produce a number of extracellular toxins. We analyzed 1070 food samples obtained from retail markets and dairy farms in the Marmara Region of Turkey for the presence of S. aureus. Out of 147 isolates, 92 (62.6%) were enterotoxigenic. PCR was used to investigate the presence of staphylococcal enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu), exfoliative toxin genes (eta and etb) and the toxic − shock syndrome toxin gene (tst). The PCR results showed that 53.3% of the isolates contained staphylococcal enterotoxin-like (SEl) toxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq and seu) which were more frequent than classical enterotoxin genes (sea to see). Furthermore, seo, sei, sem, seg, seu and sec were found in 37.0, 32.7, 30.4, 29.3, 29.3 and 27.2% of the isolates, respectively. The tst gene was detected and confirmed by DNA sequencing in 9 isolates. The presence of eta and etb were not found in the isolates. Enterotoxigenic capabilities of isolates with SEA-SEE were investigated by ELISA. Enterotoxigenic S. aureus isolates produced one to three enterotoxins, with the most frequently produced types being enterotoxin A and C. There was a correlation of 72.1% between production of a specific toxin and the presence of the respective genes. PFGE analysis was used to identify genetic-relatedness of enterotoxigenic S. aureus isolates and the results revealed that 13 groups of isolates from different or the same origin that contained the same genes showed 100% homology with indistinguishable band patterns. The other enterotoxigenic isolates showed related band patterns with 72-86% homology in sea-, 61-90% homology in sec-, 80-96% homology in seh-, and 69-96% homology in sep-positive isolates. To our knowledge, this is the first study to examine enterotoxins and related gene contents of S. aureus food isolates in the Marmara Region of Turkey.     

Pulsed field, PCR ribotyping and multiplex PCR analysis of Yersinia enterocolitica strains isolated from meat food in San Luis Argentina

The characterization of phenotypic and genotypic virulence markers of Yersinia enterocolitica strains belonging to biotypes (B) 1A, 2 and 3, mostly isolated from food in San Luis, Argentina, and the assessment of their genotypic diversity using PFGE and PCR ribotyping, were performed in our laboratory for the first time. Thirty five Y. enterocolitica strains, two reference strains and 33 strains isolated in our laboratory were studied. The presence of virF, ail, ystA, and myfA genes was investigated by multiplex PCR. The pathogenic potential of B1A strains, the most predominant biotype of Y. enterocolitica strains isolated from meat in our region, was investigated by simple PCR. Four B1A strains were positive for ystB gene. Four Y. enterocolitica 2/O:9 (bio/serotype) and two 3/O:5 strains isolated in our laboratory showed virulence-related results in the phenotypic tests and multiplex PCR. A good correlation between the expression of virulence markers and their corresponding genotypes was observed for most strains. Sixteen genomic types (GT) and 9 different intergenic spacer region (SR) groups were generated by PFGE and PCR ribotyping, respectively. In both cases the Y. enterocolitica 2/O:9 strains were separately clustered from 1A and 3/O:5 strains. Meat foods might be vehicles of transmission of pathogenic Y. enterocolitica strains in our region.     

Incidence, diversity and toxin gene characteristics of Bacillus cereus group strains isolated from food products marketed in Belgium

The major objectives of this study were to determine the incidence, diversity and characteristics of Bacillus cereus group spp. isolated from food products marketed in Belgium. The food products investigated in this study included cooked pasta, lasagna, béchamel sauce, bolognaise sauce, fresh minced beef, fresh-cut vegetables and raw basmati rice. B. cereus group spp. were detected in 56.3% (324 of 575) of the samples giving rise to 380 strains. The highest incidence (100%) occurred in the raw basmati rice. Although only 10 (2.6%) of the 380 isolates were determined to be psychrotolerant (able to grow at ≤ 7 °C), 25 (6.2%), 189 (49.7%) and 334 (87.9%) isolates were able to grow at mild temperature abuse conditions of 8 °C, 9 °C and 10 °C, respectively. The large diversity of the isolates obtained (overall and between isolates obtained from the same product type) was highlighted by the results of the (GTG)₅ PCR fingerprinting of 80 selected isolates. Sixty-one of these 80 isolates belonged to 15 distinct clusters (≥ 85% Pearson correlation) whereas the remaining 19 were each clustered separately. Further diversity was also found in the distribution of toxin genes as 16 different profiles were observed in the 80 selected isolates. Whilst none of 80 selected strains harboured the ces gene required for the production of the emetic toxin cereulide, 42 strains (52.5%) carried all seven genes required for the production of the diarrhoeal enterotoxins: haemolytic BL, non-haemolytic enterotoxin and cytotoxin K. The results of this study highlight not only the omnipresence but also the highly diverse ecology of B. cereus spp. within and across several food product types available on the retail market in Belgium. They should also provide the impetus for more studies to enable detailed risk assessment studies to be performed.     

Phylogenetic identification of Aeromonas from pigs slaughtered for consumption in slaughterhouses at the North of Portugal

In the present study, 710 isolates of Aeromonas spp. have been collected from pig carcasses, diaphragm muscle, faeces, dehairing equipment and water in slaughterhouses at the North of Portugal. The isolates were obtained from a total of 154 samples. All presumptive Aeromonas isolates were subjected to ERIC-PCR analysis and those which presented a different pattern were taken and the species classified by gyrB gene sequencing. We have found the species A. hydrophila, A. salmonicida, A. bestiarum, A. caviae, A. media, A. veronii, A. allosaccharophila, A. simiae and A. aquariorum. To our knowledge, this extent of Aeromonas species diversity has not been previously described from meat or from the slaughter environment, perhaps due to the unreliability of available identification methods. A noticeable level of isolate redundancy (strains with identical gyrB sequence) from different samples collected in different dates was also obtained, indicating that only a few predominant strains of these species persist at the slaughter system. It is also important to emphasise the presence of Aeromonas species previously associated with illness in man.     

Improved multiplex PCR assay for simultaneous detection of Bacillus cereus emetic and enterotoxic strains

Toxin producing Bacillus cereus can cause enterotoxic and/or emetic food poisoning. In the present study, a multiplex PCR assay was developed to detect all toxin genes known to be involved in food poisoning of B. cereus in a single reaction. Specific primers for the detection of enterotoxic (entFM, hblC, nheA, and cytK) genes and emetic toxin production (2 primer pairs: ces, CER) were designed based on the GeneBank sequences. The developed multiplex PCR assay was evaluated in pure culture and artificially inoculated milk, using 43 B. cereus strains and non-target strains. In brief, sensitivity in pure culture was 10-fold or more higher than artificially inoculated milk in multiplex PCR detection limit assay. The presented PCR assay is a developed molecular tool for the rapid simultaneous detection of emetic and enterotoxin producing B. cereus strains.