Retail Food Samples Research Articles

Antimicrobial Susceptibility and Molecular Typing of Methicillin-ResistantStaphylococcus aureusin Retail Foods in Shaanxi, China

The aims of this study were to evaluate the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in retail foods in Shaanxi, China and to investigate antimicrobial resistance and molecular characteristics of these strains. A total of 1979 retail food samples were randomly collected during 2008-2012 from supermarkets and farmers markets and screened for S. aureus, and then S. aureus isolates were further examined to determine whether they were MRSA. MRSA isolates were further characterized by antimicrobial susceptibility test, pulsed-field gel electrophoresis, spa typing, multilocus sequence typing, and SCCmec typing, and were examined for genes encoding enterotoxins, exfoliative toxins, Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin 1. Among all the samples examined, four (1.4%) raw milk samples, six (2.3%) chicken samples, one (0.6%) pork sample, three (0.6%) ready-to-eat food samples, and three (2.5%) dumpling samples were positive for MRSA. No MRSA isolates were recovered from infant foods. A total of 23 MRSA isolates were recovered from the 17 MRSA-positive samples. Antimicrobial susceptibility tests showed that, among these MRSA isolates, resistance was most frequently observed to penicillin, ampicillin, oxacillin, cefoxitin, and clindamycin (each 100%), followed by erythromycin (95.7%) and clarithromycin (87.0%). The commonly detected toxin genes were pvl, seg, seb, sed, followed by see, sec, and sei. Seven spa types (t189, t377, t437, t899, t10793, t5762, and a new spa type) and three SCCmec types (II, IVb, and V) were identified. More than half (52.2%) of the MRSA isolates belonged to ST9, followed by ST88, ST59, ST188, ST72, and ST630. Our findings indicate that MRSA in food could be from both animal and human origin. Although the prevalence is low, the presence of multidrug resistant and enterotoxigenic MRSA strains in foods poses a potential threat to consumers and emphasizes the need for better control of sources of contamination.

Implementation of classical, molecular biological and immunoenzymatic methods in isolation and detection of Listeria monocytogenes in food

Food borne pathogens and spoilage bacteria are influencing the safety and quality of food and animal feed and can result in serious adverse effects to human and animal health as well as to the food quality. Consequently, microbiological quality control in the food industry has become the priority of the food producers and it aims towards minimizing the risks related to food pathogens and spoilage bacteria. One of the most important pathogens in the food industry is Listeria monocytogenes. Listeriosis has a significant public health and economic impact because of its high hospitalization and mortality rate. Most people infected with Listeria are hospitalized and mortality is approximately 30 %. Therefore it is necessary to undertake efficient control measures, especially concerning the necessity of a rapid and accurate detection of this pathogen in the food industry as well as in retail food samples. Recently used conventional methods are often time consuming and require intensive work. However, obtained results can often be false considering the presence of viable but not cultivable microorganisms. Advances in biotechnology and bioinformatics have resulted in the development of novel testing technologies that enable tracking, more reliable and faster detection of food pathogens. Furthermore, molecular-biology methods, although still not applied routinely in everyday practice, are the promising alternative which can replace current reference methods in this area.

Prevalence and characterization of Listeria monocytogenes isolated from retail food in Henan, China

Listeria monocytogenes, an important foodborne pathogen, is the causal agent of listeriosis. In this study, a total of 954 food samples originating from raw meat, cooked meat products, seafood, and vegetables purchased from supermarkets and open-air markets in Henan province, China, were analyzed for the presence of L. monocytogenes. All L. monocytogenes isolates were subjected to serotyping, pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance. The overall percentage of L. monocytogenes prevalence was 6.2% (n = 59) with the highest rate of 7.4% for cooked meat products followed by raw meat (6.7%). The isolates belonged to five serotypes (1/2a, 1/2b, 1/2c, 4b, and 4c), with serotype 1/2a being predominant (55.9%). PFGE revealed a low genetic diversity among the isolates, irrespective of their sources, suggesting that dominant clones are widespread in different food products in Henan. Resistance to cefotaxime (30.5%) and ciprofloxacin (13.5%) was most often, whereas resistance to tetracycline, trimethoprim/sulfamethoxazole, and erythromycin was observed less frequently. The presence of L. monocytogenes in food products and antimicrobial resistance among the isolates represents a potential public health risk. Our results indicate that effective hygienic measures and bacteriological controls are necessary in China to reduce the contamination of retail food samples by L. monocytogenes.

Development and validation of an LC-MS/MS method for the detection of phomopsin A in lupin and lupin-containing retail food samples from the Netherlands

Phomopsins (PHO) are mycotoxins produced by the fungus Diaporthe toxica (also referred to as Phomopsis leptostromiformis). Lupin is the most important host crop for this fungus and PHO are suspected as cause of lupinosis, a deadly liver disease, in sheep. Lupin is currently in use to replace genetically modified soy in many food products available on the European market. However, a validated method for analysis of PHO is not available until now. In this work, a dilute-and-shoot LC-MS/MS-based method was developed for the quantitative determination and identification of phomopsin A (PHO-A) in lupin and lupin-containing food. The method involved extraction by a mixture of acetonitrile/water/acetic acid (80/20/1 v/v), dilution of the sample in water, and direct injection of the crude extract after centrifugation. The method was validated at 5 and 25 µg PHO-A kg−1 product. The average recovery and RSD obtained were 79% and 9%, respectively. The LOQ (the lowest level for which adequate recovery and RSD were demonstrated) was 5 µg PHO-A kg−1. Identification of PHO-A was based on retention time and two transitions (789 > 226 and 789 > 323). Using the average of solvent standards from the sequence as a reference, retention times were all within ± 0.03 min and ion ratios were within ± 12%, which is compliant with European Union requirements. The LOD (S/N = 3 for the least sensitive transition) was 1 µg PHO-A kg−1 product. Forty-two samples of lupin and lupin-containing food products were collected in 2011–2012 from grocery stores and internet shops in the Netherlands and analysed. In none of the samples was PHO-A detected.

Surveillance and characterisation of Cronobacter spp. in Czech retail food and environmental samples

Cronobacter spp. (formerly Enterobacter sakazakii) are emerging, opportunistic pathogens that are linked with food-borne infections in neonates and infants. In the present study, 291 samples of food, 36 samples from a dairy farm and 140 samples of dust from vacuum cleaners were examined for the presence of Cronobacter spp. using chromogenic media and biochemical tests. Altogether, 72 Cronobacter spp. strains were isolated in accordance with the reference standard ČSN P ISO/TS 22964 (2006). No Cronobacter spp. strains were detected in 10 samples of infant milk formula or in samples from a dairy farm. Twelve out of 20 positive food samples were dry products. The incidence of Cronobacter spp. in instant and powdered products and spices (12 positive isolates out of 82 samples) was significantly higher than that in other foods (P = 0.002), but lower than that in samples of dust (52 isolates; P < 0.001). The incidence of Cronobacter spp. in dust from restaurants, bars and hotels (13 positive isolates in 20 samples) was significantly higher than that in dust from households (P = 0.010). The polymerase chain reaction assay for the species-specific detection of the rpoB gene was performed in 49 isolates. Thirty-four Cronobacter spp. isolates were identified as Cronobacter sakazakii, nine isolates as Cronobacter malonaticus and one isolate as Cronobacter turicensis.

Isolation and Characterization of Listeria monocytogenes Isolates from Retail Foods in Shaanxi Province, China

Listeria monocytogenes is an important foodborne pathogen of public health concern. A total of 902 retail food samples, including 342 ready-to-eat (RTE) foods, 366 infant foods, and 194 raw chickens were collected randomly in supermarkets and farmers' markets in 12 geographic areas in Shaanxi Province, China and screened for L. monocytogenes. All L. monocytogenes isolates were characterized by antimicrobial susceptibility testing, serotyping, and pulsed-field gel electrophoresis (PFGE). Twenty-seven (3.0%) samples were positive for L. monocytogenes, and 39 L. monocytogenes isolates were recovered from positive samples. Of these L. monocytogenes isolates, 21 isolates (53.8%) showed resistance to at least one antimicrobial. The isolates displayed resistance most frequently to oxacillin (18 isolates, 46.2%), followed by tetracycline (five isolates, 12.8%), erythromycin (four isolates, 10.3%), trimethoprim/sulfamethoxazole (three isolates, 7.7%), chloramphenicol (two isolates, 5.1%), and vancomycin (one isolate, 2.6%). All isolates were sensitive or displayed intermediate resistance to gentamicin, ampicillin, ciprofloxacin, and amikacin. Four serotypes including serotype 1/2b, 4b, 4e, and 1/2a were identified in those foodborne isolates. PFGE analysis demonstrated that some isolates with the same PFGE patterns came from different food sources, and isolates from the same food source tend to cluster closely. Presence of L. monocytogenes of clinically important serotypes in retail foods and their antimicrobial resistance constitute a potential risk for the public. Appropriate measures should be taken by government, industry, and consumers to reduce the risk posed by this ubiquitous pathogen.

Sorbate and benzoate in Turkish retail foodstuffs

From 2008 to 2011, surveys were conducted to determine the levels of benzoic and sorbic acids and their respective salts in 983 retail food samples which included sauces, vegetable and fruit preparations, flavoured syrups, food supplements, cereals, bakery products, jelly, synthetic cream, sprays, mustards, jam and preserves, molasses, chewing gum, confectionery, non-alcoholic beverages, tea, wine, vinegar, brine and beers. The analysis involved methanol extraction of the foodstuff and direct determination by HPLC with UV detection. Quality assurance was employed with each batch of samples. Accuracy was ensured through regular participation in proficiency tests. Over this four-year period, a total of 23 samples (2.3%), some syrups, tomato sauces and fruit contained individual or combined levels of sorbic and benzoic acids above regulatory limits. Unauthorised use of benzoic acid was also detected in a syrup sample, bakery products and fruit preserves.

Expression of efflux pump gene lde in ciprofloxacin‐resistant foodborne isolates of Listeria monocytogenes

The expression of efflux pump gene lde in ciprofloxacin resistant (Cip(R) ) and susceptible strains of Listeria monocytogenes collected from retail food samples was investigated. For two Cip(R) strains, the MICs of ciprofloxacin decreased four- to eightfold in the presence of reserpine; however, no significant alterations were observed with naturally sensitive isolates. Overexpression of the lde gene induced by ciprofloxacin was observed in two resistant isolates. The present findings indicate that expression of lde and the MICs of ciprofloxacin are well correlated with the presence and absence of reserpine, suggesting that Lde might be involved in ciprofloxacin resistance of L. monocytogenes.

Rapid detection of food-borne Listeria monocytogenes by real-time quantitative loop-mediated isothermal amplification

The purpose of this study was to develop a real-time quantitative loop-mediated isothermal amplication (LAMP) method for the rapid, sensitive, and convenient detection of Listeria monocytogenes in food. The LAMP method could amplify the hlyA gene of L. monocytogenes successfully at 63°C with a loopamp real-time turbidimeter. The detection limits of the LAMP for hlyA gene were 6 colony forming units (CFU)/tube. A standard curve was generated for L. monocytogenes LAMP by plotting the graph based different log CFU values of L. monocytogenes and time of positivity through real-time monitoring of the amplication. Then, the LAMP method was employed to test 94 retail food samples effectively. Sensitivity in detection of L. monocytogenes by the LAMP was higher than that of PCR and none of the conventional methodpositive samples was missed by the LAMP method.

The Prevalence and Concentration of Bacillus cereus in Retail Food Products in Brisbane, Australia

Illness associated with Bacillus cereus may be underreported as very few of those affected seek medical attention owing to the mild nature and short duration of symptoms. For this reason there is little information on the prevalence and concentration of this pathogen in retail food products. A total of 1263 retail food samples were examined for B. cereus using the Australian Standard 1766.2.6 (1991): spread plate technique on polymyxin pyruvate egg yolk mannitol bromothymol blue agar, of which the limit of detection was log(10) 2.0 cfu/g. Bacillus cereus was not detected in samples of skim milk powder, sandwiches, sushi, fresh beef mince, tortillas, or shelf stable stir-fry sauces. Bacillus cereus was detected in the following food samples: uncooked pizza bases (1 of 63 samples, log(10) count of 2.0 cfu/g), cooked pizzas (8 of 175, mean log(10) 3.4 cfu/g), cooked meat pies (7 of 157, mean log(10) 2.2 cfu/g), cooked sausage rolls (5 of 153, mean log(10) 2.6 cfu/g), processed meats (1 of 350, log(10) 3.3 cfu/g), and raw diced chicken (3 of 55, mean log(10) 4.3 cfu/g). It appears that composite food products have more positive detection samples because the numerous ingredients may introduce spores into the foods. This study provides valuable data on the distribution, prevalence, and concentration of B. cereus in selected retail products.

Benzo (a) pyrene residue surveillance in retail food in the city of Xiamen, China: report from 121 samples

To investigate Benzo (a) pyrene (BaP) residue in retail food of Xiamen. BaP residue in 121 retail food samples collected from Xiamen were determined by a rapid BaP detector based on derivative constant-energy synchronous fluorescence technique. BaP was detected in 84.3% samples and the concentration were ranged from 0.17 to 59.0 microg/kg. There were 49.6% samples exceeding 5.00 microg/kg, and most of them were roasting food (1.44 - 54.10 microg/kg), processed meat products (0.17 - 59.00 microg/kg) and aquatic products (2.79 - 36.80 microg/kg). The BaP concentration in 34 samples collected from roadside stands were 1.78 - 49.60 microg/kg, of which the rate of the samples exceeding 5.00 microg/kg was 88.2%. The BaP contamination in retail food samples from Xiamen is serious.

Structural Elucidation of T-2 Toxin Thermal Degradation Products and Investigations toward Their Occurrence in Retail Food

The stability of T-2 toxin under the conditions of baking or cooking was investigated using heating experiments with the model substances alpha-d-glucose, alpha-d-methyl-glucopyranosid, N-alpha-acetyl-l-lysine methyl ester, and N-alpha-acetyl-cysteine methyl ester. The reaction residue was screened for degradation products using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD). Although T-2 toxin was degraded under all conditions, only heating of T-2 toxin with alpha-d-glucose produced a mixture of three degradation products, which were isolated and identified by MS and nuclear magnetic resonance (NMR) experiments. The reaction mechanism for the formation of the T-2 degradation products was elucidated by quantum chemical calculations. The relevance of these degradation products was investigated by baking experiments as well as the analysis of retail food samples. In cell-culture studies using immortalized human kidney epithelial (IHKE) cells, the T-2 degradation products were less cytotoxic (formazan dye cytotoxicity assay) compared to T-2 toxin.

Prevalence of Escherichia coli O157 from Cattle and Foods in the Mekong Delta, Vietnam

From July 2001 to September 2004, 571 fecal samples from cattle and 363 retail food samples including 150 of beef, 50 of chicken meat, 51 of duck meat and 112 of shrimp were examined for the prevalence of Escherichia coli O157 in the Mekong Delta, Southern Vietnam. E. coli O157 was isolated from 2.1% (12/571) of the cattle and from 15% (6/40) of the farms, but was not isolated from foods. E. coli O157 was isolated from 4% (3/75) of calves (≤6 month) and 1.8% (9/496) of cattle (>6 month). Of 15 isolates, 12 (80%) possessed the eae and EHEC-hlyA genes and 13 (86.7%) carried the stx2 gene. No stx1gene was detected from any O157 isolates. Of 15 isolates, 12 (80%) were O157 : H7 and 3 (20%) were O157 : H-. Three of 15 isolates showed antibiotic resistance to one or more antibiotics.These results indicate that this pathogen is distributed in cattle in the Mekong Delta, Vietnam. This is the first isolation of E. coli O157 in the Mekong Delta, Southern Vietnam.

Occurrence of Campylobacter, Salmonella, Yersinia enterocolitica and Listeria monocytogenes in Some Retail Food Products in Novi Sad

The official reporting system in the Province of Vojvodina (PV) indicates that cases of human salmonellosis were partly covered by complete epidemiological investigation including laboratory analysis of the suspected food. Intestinal campylobacteriosis and yersiniosis and four cases of septicemias caused by Listeria monocytogenes were not fully epidemiologically investigated. Actual country legislation on food safety does not include provisions for a routine control of the above mentioned pathogens except for Salmonella. In the PV, there are no other sources of data that contribute to risk assessment of the above food-borne diseases. A pilot investigation, performed in Novi Sad, indicated that 8.17% out of the total number of 257 retail food samples (90 of fresh meat and 167 of ready-to-eat food) had been contaminated with one of the tested bacteria Campylobacter or Salmonella or Listeria monocytogenes. Yersinia enterocolitica was not detected in any of the tested samples. Fresh poultry meat and other fresh meats were the dominant sources of the detected pathogens compared to samples of ready-to-eat food (p < 0.05). Campylobacter was detected in 18.8% and 10.0% samples of fresh poultry and other fresh meat respectively, which was not statistically significant (p > 0.05). Salmonella was detected in 3.3% samples of fresh poultry meat. Listeria monocytogenes was detected in 5.0% samples of fresh poultry and in 3.3% samples of other fresh meat, the difference was not statistically significant (p > 0.05). One sample (0.6%) of ready to eat food was contaminated with Campylobacter and one (0.6%) with Salmonella.

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1–10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1–5 CFU L. monocytogenes per 25 g/ml of food sample in 30 h. The performance of the assay was compared with the Roche Diagnostics ‘LightCycler foodproof Listeria monocytogenes Detection Kit’. Both methods detected L. monocytogenes in all artificially contaminated retail samples ( n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.

Prevalence of Foodborne Microorganisms in Retail Foods in Thailand

Worldwide, foodborne illness is often associated with consumption of meats and poultry products sold at retail markets. A cross-sectional retail food study was conducted in Bangkok, Thailand to assess the prevalence of bacterial pathogens on retail food samples. Raw chicken, beef, pork, and chicken eggs were purchased from fresh markets and supermarkets and tested for Salmonella spp., Campylobacter spp., Arcobacter spp., and Enterococcus spp. Suspect bacterial pathogens were isolated by differential culture and Salmonella species were serotyped. A total of 200 samples were collected from 50 markets between May and August 2003. Of the 200 samples tested, 121 (61%) were positive for at least one Salmonella spp. serogroup. A total of 175 Salmonella spp. were isolated. The most common serotype was Salmonella Anatum, followed by S. Corvallis and S. Derby. Campylobacter spp. were found in 31 (15.5%) of 200 samples. C. jejuni was isolated from 15% of fresh market chicken samples and 35% of supermarket chicken samples. Arcobacter spp. were isolated from 42 (21%) samples; fresh market chicken had significantly higher A. butzleri contamination than supermarket chicken. The presence of Enterococcus spp., an indication of fecal contamination, was detected in 188 (94%) samples, including 100% of the beef and pork sources. Few studies have examined retail food contamination in Thailand. In particular, the high prevalence of samples with Arcobacter spp. warrants further study to determine pathogenicity.

Antibiotic resistance of retail food and human Campylobacter isolates on the island of Ireland from 2001–2002

The antimicrobial resistance profiles of Campylobacter isolates recovered from a range of retail food samples (n=374) and humans (n=314) to eight antimicrobial compounds were investigated. High levels of resistance in food C. jejuni isolates were observed for ceftiofur (58%), ampicillin (25%) and nalidixic acid (17%) with lower levels observed for streptomycin (7.9%) and chloramphenicol (8.3%). A total of 80% of human C. jejuni isolates were resistant to ceftiofur, while 17% showed resistance to ampicillin and nalidixic acid, 8.6% to streptomycin and 4.1% to chloramphenicol. Resistance to clinically relevant antimicrobials such as erythromycin, ciprofloxacin and tetracycline was 6.7, 12, and 15% respectively for all food isolates and was similar to corresponding resistance prevalences observed for human isolates, where 6.4, 12 and 13% respectively were found to be resistant. Comparisons of C. jejuni isolates in each location showed a high degree of similarity although some regional variations did exist. Comparison of total C. jejuni and C. coli populations showed minor differences, with C. jejuni isolates more resistant to ampicillin and ceftiofur. Multidrug resistance patterns showed some profiles common to human and clinical isolates.

Incidence of Enterohemorrhagic Escherichia coli, Escherichia coli O157, Salmonella, and Listeria monocytogenes in Retail Fresh Ground Beef, Sprouts, and Mushrooms

The objective of this study was to determine the prevalence of enterohemorrhagic Escherichia coli (EHEC), E. coli O157, Salmonella, and Listeria monocytogenes in retail food samples from Seattle, Wash. A total of 2,050 samples of ground beef (1,750 samples), mushrooms (100 samples), and sprouts (200 samples) were collected over a 12-month period and analyzed for the presence of these pathogens. PCR assays, followed by culture confirmation were used to determine the presence or absence of each organism. Of the 1,750 ground beef samples analyzed, 61 (3.5%) were positive for EHEC, and 20 (1.1%) of these were positive for E. coli O157. Salmonella was present in 67 (3.8%) of the 1,750 ground beef samples. Of 512 ground beef samples analyzed, 18 (3.5%) were positive for L. monocytogenes. EHEC was found in 12 (6.0%) of the 200 sprout samples, and 3 (1.5%) of these yielded E. coli O157. Of the 200 total sprout samples, 14 (7.0%) were positive for Salmonella and none were positive for L. monocytogenes. Among the 100 mushroom samples, 4 (4.0%) were positive for EHEC but none of these 4 samples were positive for E. coli O157. Salmonella was detected in 5 (5.0%) of the mushroom samples, and L. monocytogenes was found in 1 (1.0%) of the samples.

Most Campylobacter subtypes from sporadic infections can be found in retail poultry products and food animals

The subtypes of Campylobacter isolates from human infections in two Danish counties were compared to isolates from retail food samples and faecal samples from chickens, pigs and cattle. During a 1-year period, 1285 Campylobacter isolates from these sources were typed by two methods: 'Penner' heat-stable serotyping and automated ribotyping (RiboPrinting). C. jejuni was the dominating species, but C. coli was more prevalent among food and chicken isolates (16%) compared to human isolates (4%). In total, 356 different combined sero-ribotypes (subtypes) were found. A large subtype overlap was seen between human isolates and isolates from food (66%), chickens (59%) and cattle (83%). This was verified by PFGE typing of 212 isolates representing selected subtypes. All frequent (n>3) subtypes found in food were also present in humans. Sixty-one per cent of the isolates from domestically acquired infections had subtypes that were also found in food as opposed to 31% of travel-associated infections. The results showed differences in the various Campylobacter populations, e.g. the Danish population as reflected in the domestically acquired infections and the Danish-produced food was more uniform than the isolates originating from outside the country. The study shows that most C. jejuni subtypes found in poultry food samples, broiler chickens, and cattle were represented in the domestically acquired cases, indicating that C. jejuni from these reservoirs are likely sources of human infections in Denmark.

The usefulness of molecular techniques to assess the presence of Aeromonas spp. harboring virulence markers in foods

A total of 78 raw and 123 processed and ready-to-eat retail food samples were used to assess the presence of motile Aeromonas spp. harboring virulence genes (cytotoxic enterotoxin and hemolysin genes) using a recently described PCR method in comparison with the conventional cultivation method based on the use of Ampicillin-Dextrin Agar (ADA) medium. With the ADA-based method, 65/201 (32.3%) samples showed presumptive Aeromonas spp. colonies whereas the PCR method revealed the presence of Aeromonas spp. harboring the targeted virulence genes in 51/201 (25.4%) of the tested samples. The rate of contaminated samples and the presence of pathogenic Aeromonas were significantly lower with both methods for processed than in case of raw samples. A polyphasic identification approach including biochemical and molecular techniques was applied to a selection of 34 PCR-positive presumptive Aeromonas isolates. Following fatty acid methyl ester (FAME) analysis and amplified fragment length polymorphism (AFLP) fingerprinting, a total of 33 isolates (97%) could be identified to the DNA hybridization group (HG) level. The majority of these isolates belonged to the species Aeromonas hydrophila HG3 (50%) and Aeromonas veronii biovar sobria (HG8/10) (38%). Molecular characterization of PCR amplicons obtained from these strains by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) fingerprinting and PCR-Amplicon Sequence Analysis (PCR-ASA) allowed classification of all strains in a known PCR-RFLP and PCR-ASA type. In conclusion, the current findings demonstrate that the combined use of PCR-based virulence marker detection, PCR-RFLP and PCR-ASA offers a rapid, sensitive, and specific system to assess the presence and prevalence of Aeromonas spp. harboring virulence markers in food samples.