Virulence markers of Escherichia coli strains isolated from traditional cheeses made from unpasteurised sheep milk in Slovakia

Numerous virulence factors including adhesins, host cell surface-modifying factors, invasins, toxins, and secretion systems are involved in Escherichia coli pathogenicity mechanisms. Strains of the same pathotype are genetically similar and carry the same virulence determinants which are ideal targets for the determination of the pathogenic potential of any given E. coli isolate.We examined 95 strains of E. coli isolated from two kinds of traditional Slovak cheeses made from raw sheep milk in course of one year. We tested those strains with the goal to determine some of 11 selected genes of virulence factors which at least partially represent all frequent pathotypes of E. coli. Markers of VTEC (VT1, VT2, VT2e), ETEC (ST I, ST II, LT I), EPEC (EaeA), EIEC (Einv), EAEC (Eagg), and UPEC (CNF1, CNF2) were examined using PCR assays. The result of our testing was the determination of nine potentially pathogenic strains from 95 isolates (9.72%). We did not prove VT1 and VT2 factors as well as Einv and Eagg in any strains. We detected ST I in two strains, ST II and LT I in one strain, and VT2e in one strain. In this study most frequent virulence markers were CNF 1 (three strains) and EaeA (three strains).     

Virulence factors among enterococci isolated from traditional fermented meat products produced in the North of Portugal

The aim of this work was to characterize Enterococcus spp. isolated from Alheira, Chouriça de Vinhais and Salpicão de Vinhais, fermented meat products produced in the North of Portugal, concerning their potential pathogenicity. One hundred and eighty two isolates (76 identified as Enterococcus faecalis, 44 as Enterococcus faecium, 1 as Enterococcus casseliflavus and 61 as Enterococcus spp.) were studied. Twenty six percent of isolates were gelatinase producers. None of the isolates produced lipase nor DNase activities. Hemolytic activity using sheep and human blood from two types (A and O) was assessed. One isolate was β-hemolytic in human blood. Results obtained in sheep blood were quite different from those obtained in human blood. Biofilm production in batch and in fed-batch mode was evaluated. In batch mode, only 28.0% and 3.9% of isolates were classified as moderate and strong biofilm producers, respectively, and in fed-batch mode, 35.7% and 63.2% of isolates were classified as moderate and strong biofilm producers, respectively. The presence of 13 virulence genes (efaAfs, efaAfm, esp, agg, cylM, cylB, cylA, cylL_L cylL_s and gelE) were investigated by PCR. The majority of enterococcal isolates showed the presence of one or more virulence factors, the most frequent genotype being efaAfs⁺ gelE⁺ agg⁺ (41.5%). E. faecalis isolates harbored multiple virulence traits, while E. faecium isolates were generally free of virulence determinants. Phenotypic and genotypic evidence of potential virulence factors were identified in Enterococcus spp. isolates, which is a reason of concern.     

Technological properties of Lactobacillus plantarum strains isolated from Chinese traditional low salt fermented whole fish

A total of 21 Lactobacillus plantarum were isolated from a Chinese traditional low salt fermented whole fish product “Suan yu” and identified by biochemical and molecular methods. The isolates were screened for acidifying and amino-biogenic ability for proteolysis, lipolysis, antimicrobial activity, and sensory in order to select the most suitable candidates as the starter cultures. All the strains had high acidifying activities and were able to reduce the pH to lower than 4.5 in 36, 24 and 16 h at 15, 25 and 37 °C respectively. Major strains showed antimicrobial activities against Listeria monocytogenes, Staphylococcus aureus and Escherichia coli, and ten L. plantarum strains could produce bacteriocins. Five L. plantarum strains exhibited weak proteolytic activities against myofibrillar proteins, as evidenced by the agar plate assay and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). None of the strains showed lipolytic activity. Major L. plantarum strains displayed different enzymatic profiles. Besides, the isolates mostly showed negative-decarboxylase activities, but Lp-10 had a decarboxylase activity against l-tyrosine. Furthermore, the fermented fish surimi inoculated with Lp-15 and Lp-21 strains respectively scored high for overall acceptability. Therefore, Lp-15 and Lp-21 presented the best technological properties, which should be given first priority as the starter culture for manufacturing fermented fish products.     

Occurrence of coagulase-positive Staphylococcus in various food products commercialized in Botucatu,SP, Brazil and detection of toxins from food and isolated strains

The aim of this work was to assess the microbiological quality of commercialized desserts, sandwiches and finger food in Botucatu, SP, for human consumption. A total of 172 food samples were analyzed for fecal coliforms and coagulase-positive Staphylococcus and 69 (40.1%) were in disagreement with the standards established by Decree No. 12 (Brazilian Food Sanitation Standard, 2001). Coagulase-positive Staphylococcus was isolated from 26 (15.1%) samples. Toxins were not isolated directly from foods but 27 (54%) coagulase-positive Staphylococcus strains were enterotoxigenic, and toxin type C was the most frequently detected. These results suggest that these products may act as an important vehicle of transmission for well-established pathogens.     

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shellfish in Shanghai

Vibrio parahaemolyticus is a marine and estuarine bacterium that poses the greatest threat to human health worldwide. It has been the leading bacterial cause of seafood-borne illness. This study investigated the prevalence and drug resistance of V. parahaemolyticus isolated from retail shellfish in Shanghai. A total of 140 shellfish samples were collected from February 2014 to February 2015. The occurrence of V. parahaemolyticus in shellfish was 34.3%, which has increased compared to previous reports. In addition, discernible differences of total presumptive V. parahaemolyticus counts (TPVPC) were also observed in shellfish between market A and B. The results from PCR assays indicated that thermostable direct hemolysin (tdh) gene was positive in two isolates (2.1%), and the thermostable direct hemolysin-related hemolysin (trh) gene was not detected in all isolates. Antibiotic resistance profiles of those isolates were as follows: ampicillin (87.5%), cephazolin (31.3%), cephalothin (6.3%), amoxicillin/clavulanic acid (6.3%), piperacillin (6.3%), and amikacin (3.2%). Thirty-three out of 96 isolates were resistant to two or more antimicrobial agents. It is suggested that V. parahaemolyticus in retail shellfish could be a potential risk to consumers in Shanghai.     

Molecular characterizations of pathogenic Vibrio parahaemolyticus isolated from Southern Taiwan oyster-growing environment

Ninety-four virulent strains of Vibrio parahaemolyticus containing tdh and/or trh genes isolated from seawater, sediment, and oysters collected in Penghu, Tainan, and Changhua Lukang coastal water were characterized by analyses of O-group antigens, pulsed-field gel electrophoresis (PFGE) after digestion with SfiI enzyme, and randomly amplified polymorphic DNAs (RAPD). Analysis of virulence markers (tdh and trh) revealed that 60.6% of the isolates possessed only tdh, 13.8% of them possessed only trh, and 25.5% of them possessed both tdh and trh. Ten O-group serotypes (O1, O2, O3, O4, O5, O6, O7, O8, O10, and O11) were identified among the isolates with O1 group (28 isolates) being the most prevalent, followed by the O3 group (16 isolates) and O5 group (15 isolates). PFGE typing of the isolates revealed 94 patterns while RAPD analyses categorized the isolates into five major groups. Both PFGE and RAPD analyses showed high index of discrimination (DI) values. This study identified diverse serotypes and genotypes of virulent V. parahaemolyticus distributed in the oyster-growing environments, which can be used for risk assessment of V. parahaemolyticus infection associated with oyster consumption in Taiwan.     

Inhibitor activities of two Lactobacillus strains,isolated from sourdough,against rope-forming Bacillus strains

The effect of two different sourdoughs, produced with Lactobacillus plantarum LMO25 and Lactobacillus alimentarius LMO7, with antimicrobial activities on inhibition of rope-forming Bacillus strains in wheat bread was studied. Addition of 15% or 20% low pH (pH 3.5–4.0) sourdough to bread dough, which were produced by using two strains (Lb. plantarum LMO25 and Lb. alimentarius LMO7) separately, prevented the generation of visual rope caused by Bacillus subtilis and Bacillus licheniformis. However, adding 10% sourdough was not enough to prevent the generation of visual rope. When repeated with sourdoughs with a higher pH (pH > 4), additives at 10% or 15% did not prevent the generation of rope, whereas additives at 20% prevented the generation of visual rope caused by both B. subtilis and B. licheniformis.     

Determination of inorganic trace elements in edible marine fish from Rio de Janeiro State, Brazil

Fish and seafood may represent risk for human health since they can accumulate contaminants from aquatic environment and magnify them up the food chain. The purpose of this study was to analyze and evaluate the levels of aluminum, zinc, iron, manganese, cobalt, copper, arsenics, selenium, cadmium, barium, lead and bismuth in 11 fish species (Salmo salar, Sardinella brasiliensis, Pomatomus saltatrix, Micropogonias furnieri, Cynoscion leiarchus, Caranx crysos, Priacanthus arenatus, Mugil cephalus, Genypterus brasiliensis, Lopholatilus villarii and Pseudopercis numida) captured at Rio de Janeiro State Coast, Brazil. Concentration ranges (mg kg⁻¹ of wet weight) of the selected elements were compared with those reported in other studies. In some cases, comparison of certain elements in the same fish species was difficult due to the lack of data. Aluminum concentration was significantly high in all samples and only M. cephalus, C. leiarchus e C. crysos presented arsenic concentrations below 1 mg kg⁻¹, limit recommended by Brazilian legislation.     

Mismatch between antimicrobial resistance phenotype and genotype of pathogenic Vibrio parahaemolyticus isolated from seafood

Antimicrobial resistance phenotypes (18 antimicrobials; disk diffusion method) and genotypes (38 antimicrobial resistance genes; PCR) of 20 pathogenic Vibrio parahaemolyticus strains isolated from seafood in Shanghai wholesale markets between 2009 and 2013 were evaluated. Seventeen isolates (85%) were resistant to one or more antimicrobials, and highest resistance was observed to ampicillin (85%) and cephazolin (30%). And the isolates with tdh displayed higher resistant rates than isolates with trh. Eight antimicrobial resistance genes (strB, aadA2, strA, tetA, floR, sulI, sulII, and sulIII) were detected in these isolates. Surprisingly, the antimicrobial resistance phenotypes and genotypes of these isolates were not consistent: some isolates were resistant to β-lactam or aminoglycoside, whereas the corresponding genes were negative. Comparatively, aminoglycoside, tetracycline and chloramphenicol resistance genes occurred in susceptibility isolates. This research reveals the mismatch phenomenon between the antimicrobial resistance phenotype and genotype of seafood-derived pathogenic V. parahaemolyticus, and that susceptibility isolates might be a potential risk source for storage and transmission of resistance genes.     

A survey on bacteria isolated as hydrogen sulfide-producers from marine fish

Aim of the work was to identify sulfide-producers isolated from tuna and swordfish and to evaluate some physiological characteristics, particularly those that could be related to spoiling potential.16S rDNA sequencing revealed Shewanella baltica as the main species, followed by Serratia spp. and other Shewanella species, while RAPD- and rep-PCR analyses indicated the presence of several biotypes. Shewanella baltica and Shewanella putrefaciens showed rapid growth at 4 and 8 °C, production of TMA and H₂S, amino acids decarboxylation and proteolytic activity also at refrigeration temperatures, therefore being potentially able to modify texture and sensory characteristics of finfish. Extracellular DNAse activity and growth in presence of high salt concentrations can provide a competitive advantage in unfavourable environments.Our data provide new insights into specific metabolic features of Shewanella spp., rarely studied before, such as extracellular DNAse activity and amino acid decarboxylating activity. Moreover, our results highlight the clear necessity of more specific media and research methods to count H₂S-producing bacteria.     

Virulence factors,antibiotic resistance,and bacteriocins in enterococci from artisan foods of animal origin

A collection of 25 isolates from foods of animal origin (including mainly milk and cheese, together with meat and ham) was studied. Enterococci were identified at species levels as E. faecalis (9 isolates) and E. faecium (16 isolates). Investigation of virulence factors by PCR amplification revealed incomplete sets of cytolysin genes both in E. faecalis and E. faecium isolates. Among E. faecalis, PCR amplification revealed a high incidence of genes encoding for enterococcal surface protein esp (7/9 isolates), enterococcal antigen efaA_fs (6/9), aggregation substance agg (2/9) and sex-pheromone encoding genes ccf, cob, cpd (which were detected in 9, 5 and 3 out of 9 isolates, respectively). By contrast, only esp (7/16 isolates) and efaA_fm (10/16) were detected among E. faecium. Antibiotic resistance detected at higher frequencies included rifampicin, nitrofurantoin, ciprofloxacin and levofloxacin. Vancomycin resistance was also detected among E. faecalis and E. faecium. E. faecalis isolates showed decarboxylating activity mostly for tyrosine (5/9 isolates), while E. faecium isolates showed a broader decarboxylating capacity, involving tyrosine (11/16 isolates) ornithine (6/16), lysine (4/16) and histidine (3/16). Six isolates produced bacteriocins, and genes encoding for enterocins A, B, P, L50, and 1071 were detected. Many isolates tested positive for several of the traits investigated, which raises concerns about their possible role as reservoirs for dissemination of antibiotic resistance and virulence traits in foods.     

Production of histamine and tyramine by bacteria isolated from Portuguese vacuum-packed cold-smoked fish

An agar medium containing histidine or tyrosine incubated anaerobically was used for detecting the decarboxylating activity of bacteria isolated from Portuguese vacuum-packed cold-smoked fish during chilled storage. The capacity of each bacterial isolate to produce histamine and tyramine was studied at 25 and 5 °C incubated for 48 h and 10 days, respectively. More strains produced histamine and tyramine at 25 °C compared with 5 °C although lactic acid bacteria (LAB) strains exhibited similar results at the two different temperatures. Tyramine was produced by majority of the isolates tested although very low concentrations were produced at 5 °C as confirmed by high-pressure liquid chromatography (HPLC). Tyrosine-agar was shown to be a good indicator medium for detection of bacteria that produced high levels of tyramine, since typical colonies surrounded by a translucent halo were easily recognised. LAB identified as Lc. Lactis lactis 1 and Carnobacterium divergens were detected as tyramine-producing bacteria. Acinetobacter spp. and Pseudomonas spp., isolated from all Portuguese smoked fish products, were negative on histidine-agar, but HPLC identified considerable quantities of histamine produced in a broth medium.     

Occurrence and traceability of Listeria monocytogenes strains isolated from sheep's milk cheese-making plants environment

The aim of the study was to conduct an extensive survey on Listeria monocytogenes and Listeria spp. environmental contamination in 13 cheese-making plants. A total of 409 environmental and food samples were collected during years 2011–2013. Listeria spp. contamination was observed in all the facilities, while L. monocytogenes was recovered from 12 facilities with a prevalence ranging between 3.0% and 22.6%. Floor drains were the most contaminated sampling sites (48.8% of positive samples), serving as harbourage site for subsequent contamination. Out of 616 isolates, 277 (45.0%) were Listeria innocua, 274 (44.5%) L. monocytogenes, 41 (6.6%) Listeria ivanovii, 14 (2.3%) Listeria welshimeri and 10 (1.6%) Listeria gravyi. Serotyping carried out by PCR and agglutination method for L. monocytogenes revealed that 169 strains (61.7%) were serotype 1/2a, 65 (23.7%) 4b, 20 (7.3%) 1/2b, 10 (3.6%) 3a, 7 (2.5%) 1/2c and 3 (1.1%) 3b. PFGE conducted on L. monocytogenes isolates using AscI and ApaI restriction enzymes, yielded 6 clusters. Two predominant PFGE clusters were observed including respectively 36 and 32 strains. Within cheese-making plants, L. monocytogenes showed wide variability with strains distributed up to 4 different clusters. Pulsotypes isolated from raw milk filter were never detected in the processing environment, indicating that the contamination originated from sources other than raw milk. The isolation of strains with similar profile from different sampling sites, within and among cheese-making plants, indicated the possible transfer of L. monocytogenes contamination along production lines and from one facility to another. Strains recovered from food were confirmed as originating from the processing environment.     

Prevalence,strain identification and antimicrobial resistance of Campylobacter spp. isolated from slaughtered pig carcasses in Brazil

The aim of this study was to investigate the rate of contamination, species identification and antimicrobial resistance of thermophilic Campylobacter spp. in pig carcasses during the slaughter process in a slaughterhouse in Brazil. Two hundred and fifty-nine samples were collected at 7 different stages of the slaughter process for Campylobacter determination by both qualitative and quantitative methods. Typical colonies were subjected to API Campy, real-time polymerase chain reaction (PCR) and antimicrobial resistance testing. Campylobacter was found in 18.9% of the carcasses and 3.5% of the samples. Dehairing was the slaughter stage with the highest Campylobacter contamination (55.6%). All Campylobacter strains were confirmed by real-time PCR and showed multi-drug resistance to cephalothin, nalidixic acid, norfloxacin, tetracycline and trimethoprim. None of the strains were resistant to amoxicillin/clavulanic acid, ampicillin and chloramphenicol. Despite the low occurrence of Campylobacter spp. in pig samples, the antimicrobial resistance of Campylobacter strains represents a considerable risk for the consumption of pork meat and confirms the need for continuous monitoring of Campylobacter in the pig production chain.     

Methylmercury in fish species used in preparing sashimi: A case study in Brazil

The objective of this work was to determine the organic and total mercury contents in twelve fish species used in preparing sashimi in Japanese restaurants and estimate the exposure to organic mercury due to the consumption of this food. The mercury species were quantified by atomic absorption with thermal decomposition and amalgamation. Total mercury was analyzed directly, whereas organic mercury was quantified after extraction with toluene in an acid medium using a microwave assisted system. Needlefish and tuna showed the highest levels of mercury species and salmon and mullet the lowest levels. The mean ratios between MeHg⁺/total Hg were 93% and 87% for sandperch and octopus, respectively, indicating that the most toxic form (organic Hg) predominates in these species. Exposure to methylmercury was estimated based on the PTWI and the results showed that the ingestion of 2 portions of needlefish and tuna exceeded the values established by 100%.     

Heterogeneity in genetic and phenotypic characteristics of Saccharomyces cerevisiae strains isolated from red and white wine fermentations

The genetic and phenotypic variability of 27 Saccharomyces cerevisiae isolates from naturally fermented Greek wines was studied. RAPD-PCR allowed species identification of the isolates, while mtDNA restriction analysis enabled a considerably high discrimination at strain level. Moreover results on hydrolase profiles obtained by the API-ZYM system and some technological characteristics of the test strains, suggested a high heterogeneity and allowed the selection of strains with interesting vinification properties.     

Prevalence and antimicrobial susceptibility of Vibrio species related to food safety isolated from shrimp cultured at inland ponds in Thailand

Inland farming of marine shrimp species is a relatively recent development, in response to increasing demand for the products. This study aimed to investigate the prevalence and antimicrobial resistance of potentially pathogenic Vibrio species in shrimps cultured at inland ponds with low salinity in Thailand. Of 16 shrimp samples (white-leg shrimps and black-tiger shrimps) from ponds with water temperatures from 29 to 32 °C, and salinities from 1 to 5 parts per trillion, 15 contained Vibrio cholerae (densities: 62–252,000 MPN/g). Vibrio parahaemolyticus was present in six samples (370–6,300,000 MPN/g), and Vibrio vulnificus was present in two samples (16–1300 MPN/g). V. parahaemolyticus has higher affinity for non-native white-leg shrimp than for native black-tiger shrimp. The minimum inhibitory concentrations were then measured for 12 common antimicrobial agents. Resistance to ampicillin and oxytetracycline (8% and 2% of the isolates) was documented in V. cholerae isolates, resistance to ampicillin and oxytetracycline (72% and 3%) was found in V. parahaemolyticus isolates, and resistance to nalidixic acid (20%) was detected in V. vulnificus isolates. β-lactamase and tetracycline resistance genes were detected in the resistant Vibrio isolates. The oxytetracycline-resistance phenotype was eliminated by plasmid curing, suggesting that the resistance was related to R-plasmids. Our results show that shrimps cultured even in low salinity ponds are potentially contaminated with pathogenic Vibrio species, and that shrimp are a vehicle for transfer of species with antimicrobial resistance genes to the public.     

Behavior of Vibrio parahemolyticus cocktail including pathogenic and nonpathogenic strains on cooked shrimp

Pathogenic Vibrio parahaemolyticus, an important foodborne bacterium, coexists with nonpathogenic strains of V. parahaemolyticus in the environment. However, current predictive models for V. parahaemolyticus usually focused on single strain without considering the pathogenic and nonpathogenic bacteria cocktail coexist situation. In this study, the behavior of four V. parahaemolyticus strains (F18: tlh⁺/trh⁺/tdh⁻, ATCC 17802: tlh⁺/trh⁺/tdh⁻, F36: tlh⁺/trh⁻/tdh⁻, ATCC 33847: tlh⁺/trh⁻/tdh⁺) and their cocktails on cooked shrimp were investigated at different temperatures (4, 7, 10, 15, 20, 25 and 30 °C). Total bacteria counts were enumerated by traditional plate count method, periodically. Results showed both V. parahaemolyticus cocktails and four single strains grew in a temperature range of 15–30 °C and died at 4–7 °C. At 10 °C, for the cocktails, it inactivated at initial 100 h and then grew rapidly; but for single strain, only one strain F18 displayed the similar growth tendency, while the others grew all the time. Compared with single strains, the primary and secondary model analysis showed that the cocktails displayed a better growth activity with higher maximum growth rate and shorter lag time. The above results indicated that modeling bacterial growth by using a cocktail of V. parahaemolyticus strains may be a better option for simulating growth in the real environment. This study will fill in the gap of predictive microbiology and improve significantly the accuracy of microbial risk assessment.     

Prevalence,genetic characterization and antimicrobial resistance of Salmonella isolated from fresh pork sausages in Porto Alegre,Brazil

A total of 336 samples of fresh pork sausage randomly obtained from supermarkets and butcher shops in Porto Alegre, Brazil, were examined for the presence of Salmonella serovars. Salmonella enterica was detected in 82 (24.4%) of the samples, with a most probable number count ranging from 0.03 MPN g^?1 to 460 MPN g^?1. Strains belonging to the most isolated S. enterica serovars (Brandenburg, Panama, Derby and Typhimurium) were further characterized by XbaI-macrorestriction, resulting in a total of 17 profiles. Resistance to tetracycline was the most prevalent among the Salmonella isolates. S. panama and S. typhimurium presented the greatest number of resistance phenotypes.     

Biofilm formation of Campylobacter strains isolated from raw chickens and its reduction with DNase I treatment

Campylobacter is a well-known bacterial agent that causes foodborne gastroenteritis. Its biofilm-forming ability is known to be important for its survival in harsh conditions. Poultry is a main reservoir of Campylobacter. In this study, we evaluated the biofilm-forming ability, motility, and antibiotic susceptibility of Campylobacter strains isolated from commercially purchased chickens of various sources in South Korea. From 37 (30%) out of 124 chickens, a total of 78 Campylobacter isolates were obtained, and the ability of these strains to form biofilms was studied on polystyrene surfaces. We identified seven biofilm-forming strains of Campylobacter jejuni and Campylobacter coli, respectively, out of 60 C. jejuni and 18 C. coli strains. Our study demonstrated that motility is inconsistent with biofilm-forming ability, suggesting that motility is not a single factor affecting biofilm formation of Campylobacter. Moreover, there was no clear relationship between antibiotic resistance and biofilm-forming ability. DNase I treatment significantly inhibited the biofilm formation or degraded the mature biofilms of 3 C. coli and 1 C. jejuni biofilm-forming strains (p < 0.05). It suggests that extracellular DNA plays a significant role in the biofilm formation of these strains. Collectively, our study demonstrated that biofilm-forming ability is not strongly correlated with motility and antibiotic resistance in Campylobacter and that extracellular DNA is required for biofilm formation of the isolated Campylobacter strains. It also suggests that DNase I is a potential method for the control of Campylobacter biofilms.