Attachment of shiga toxigenic Escherichia coli to beef muscle and adipose tissue

Shiga toxigenic Escherichia coli (STEC) serotypes are important foodborne pathogens that cause gastrointestinal disease worldwide. An understanding of how STEC strains attach to surfaces may provide insight into the potential persistence of and contamination with STEC in food environments. The initial attachment of a selection of STEC serotypes to beef muscle and adipose tissue was evaluated for isolates grown in planktonic and sessile culture. Initial experiments were performed to determine whether attachment differed among STEC strains and between the two modes of growth. Viable counts were obtained for loosely and strongly attached cells, and the strength of attachment (Sr) was calculated. All bacterial isolates grown in sessile culture attached in higher numbers to muscle and adipose tissue than did bacteria in planktonic cultures. For all attachment assays performed, mean concentrations for loosely attached cells were consistently higher than concentrations for strongly attached cells. The mean concentrations for strongly attached bacteria for planktonic and sessile cultures were significantly higher (P < 0.05) on adipose than on muscle tissue. However, some strains of STEC, particularly those from sessile culture, did not differ in their attachment to muscle or adipose tissue. Sr values were not significantly different (P > 0.05) among STEC isolates for all assays. No correlation was found between bacterial hydrophobicity and surface charge values (previously determined) and production of surface structures, viable counts, and Sr values. STEC grown in planktonic and sessile culture seems to behave differently with respect to attachment to muscle and adipose tissue. Cells in sessile culture may have a greater potential to strongly attach to meat surfaces.     

Attachment of Shiga toxigenic Escherichia coli to stainless steel

Shiga toxigenic Escherichia coli (STEC) are important foodborne pathogens causing gastrointestinal disease worldwide. Bacterial attachment to food surfaces, such as stainless steel may lead to cross contamination of foods and subsequent foodborne disease. A variety of STEC isolates, including E. coli O157:H7/H- strains, were grown in planktonic (broth) and sessile (agar) culture, following which initial attachment to stainless steel was determined using epifluorescence microscopy. Experiments were performed to determine whether the number of bacteria attached to stainless steel differed between STEC strains and between the two modes of growth. No relationship was found between STEC strains and the number of bacteria attached to stainless steel. Five STEC strains, including one non-toxigenic O157 isolate, attached in significantly greater (p<0.05) numbers to stainless steel following growth in planktonic culture compared to sessile culture. In contrast, two clinical strains of O157:H7 attached in significantly greater (p<0.05) numbers following growth in sessile culture compared to planktonic culture. Thirteen out of twenty E. coli strains showed no significant difference (p>0.05) in attachment when grown in planktonic or sessile culture. The change of interfacial free energy between the bacterial strains and stainless steel was calculated and the influence of free energy in attachment was determined. Although a significant variation (p<0.05) in free energy values was found between STEC strains, no correlation was found between free energy values and bacterial counts on stainless steel. In addition, no correlation was also found between bacterial hydrophobicity and surface charge values or production of surface structures (type I fimbriae or flagella) (previously determined) with the number of bacteria attached to stainless steel. The results of this study suggest that different growth conditions (planktonic and sessile) can influence the attachment of STEC to stainless steel. Factors other than bacterial physicochemical properties and these surface structures may also influence STEC attachment to stainless steel.     

Role of O-antigen on the Escherichia coli O157:H7 cells hydrophobicity, charge and ability to attach to lettuce

Environmental factors encountered during growing and harvesting may contribute to Escherichia coli O157:H7 contamination of lettuce. Limited nutrients and extended exposure to water may cause E. coli O157:H7 to shed its O antigen. Absence of the O157-polysaccharide antigen could affect the cell's physicochemical properties (hydrophobicity and cell charge) and ultimately influence its attachment to surfaces. The objectives of this study were to evaluate the effect of the E. coli O157:H7 O-antigen on the cell's overall hydrophobicity, charge and ability to attach to cut edge and whole leaf iceberg lettuce surfaces. Three strains of E. coli O157:H7 (86-24 wild type; F-12, mutant lacking the O-antigen and pRFBE, plasmid for O157 gene reintroduced) were examined for their hydrophobicity, overall charge and ability to attach to lettuce. Overall, E. coli O157:H7 attached at higher levels to cut surfaces over whole leaf surfaces (P = 0.008) for all strains and treatments. Additionally, the strain lacking the O-antigen (F12) — attached significantly less to lettuce (P = 0.015) than the strains expressing the antigen (WT and pRFBE). Cells lacking the O antigen (strain F-12) were also significantly more hydrophobic than strains 86-24 or pRFBE (P ≤ 0.05). Surface charge differed among the strains tested (P ≤ 0.05); however, it did not appear to influence bacterial attachment to lettuce surfaces. The charge was not fully restored in the pRFBE strain (expression of O-antigen reintroduced), therefore, no conclusions can be made pertaining to the effect of charge on attachment in this study. Results indicate that E. coli O157:H7 cells which lack the O-antigen have greater hydrophobicity and attach at lower concentrations than cells expressing the O-antigen, to iceberg lettuce surfaces.     

Survival of poultry-derived Campylobacter jejuni of multilocus sequence type clonal complexes 21 and 45 under freeze,chill, oxidative,acid and heat stresses

The application of multilocus sequence typing (MLST) for studying Campylobacter jejuni diversity reveals that MLST clonal complex (CC) 21 and CC-45 occupies significant proportion in the diverse population of C. jejuni. These two complexes are ecologically abundant and represent an interesting subpopulation for studying C. jejuni survival under different stress conditions. In the present study we characterize and compare 19 C. jejuni strains assigned to CC-21 and CC-45, isolated from chicken meat, based on laboratory stress models maintained in Muller-Hinton broth. Model conditions were mimicking freeze, chill, oxidative, acid and heat stresses. Results show that survival patterns varied between the strains. C. jejuni strains of CC-21 survived significantly better than C. jejuni strains of CC-45 under heat (P value = 0.022) and chill (P value = 0.001) stress models. On the other hand, C. jejuni strains of CC-45 showed significantly better survival compared to C. jejuni strains of CC-21 in response to oxidative (P value = 0.003) and freeze (P value = 0.021) stress models. C. jejuni strains assigned to the founder ST-45 showed significantly better survival (P value = 0.017) under heat stress model compared to their ancestral sequence types. However, an association between survival fitness and the diversification of a clonal group cannot be demonstrated directly from the obtained results. In conclusion, findings of the present study show that genotypic variations of C. jejuni might play a role in enabling certain lineages to be selected when encountering adverse and stressful environments. In future stress response studies, it is recommended to consider the effect of genotypic diversity among C. jejuni strains as that might bias the experimental findings.     

Molecular characterization and antibiotic resistance profiling of Campylobacter isolated from cattle in Polish slaughterhouses

A total of 812 samples from bovine hides and the corresponding carcasses collected at the slaughterhouse level in the eastern part of Poland were examined for the presence of Campylobacter jejuni and Campylobacter coli. Recovered isolates were confirmed using species-specific PCR, characterized by the presence of 11 putative virulence genes and antimicrobial susceptibility was determined using a microbroth dilution method. Furthermore, the genotypic relatedness of the isolates was determined by PFGE profiling and virulence pattern cluster analysis. The prevalence of Campylobacter was 25.6% and 2.7% in bovine hide and carcass samples, respectively. The presence of virulence markers varied between C. jejuni and C. coli species however, the majority of strains possessed the cadF, flhA, flaA genes, irrespective of the bacterial species and origin. The lower number of the strains was positive for the invasive associated markers – virB11 and wlaN. Antibiotic profiling showed that campylobacters were most frequently resistant to quinolones and fluoroquinolones (nalidixic acid and ciprofloxacin, 38.3% of each, respectively) followed by streptomycin (24.3%) and tetracycline (20.9%). Resistance to erythromycin and gentamicin was demonstrated in 4.3% and 2.6% of strains, respectively. Comparisons of the PFGE and virulence marker profiles of the isolates reflected the high genetic diversity of Campylobacter tested. Moreover, a poor correlation between the PFGE type, pathogenic gene marker and antimicrobial resistance patterns was observed.     

Attachment of Listeria monocytogenes on ready-to-eat meats

Five individual strains of Listeria monocytogenes and a mixed cocktail of all five were studied for attachment on frankfurters, ham, bologna, and roast beef relative to their cell surface characteristics. The ratio of strongly attached (sessile) L. monocytogenes cells compared with total (sessile and planktonic) attached cells on ready-to-eat meats was also determined. Because bacterial cell surfaces were characterized by net negative charge and hydrophobicity, electrostatic interaction chromatography and cationized ferritin methods were chosen to study net negative charge distribution on the bacterial cell surface, whereas hydrophobic interaction chromatography and contact angle measurement were used to examine the cell surface hydrophobicity. No differences (P > 0.05) were observed in cell surface charge or cell surface hydrophobicity among strains. Approximately 84 to 87% L. monocytogenes were found to attach strongly to ready-to-eat meats within 5 min. No differences (P > 0.05) were found among strains or among meats. Micrographs observed from scanning electron microscopy showed no differences among the strains but showed a difference in age of cells (mixed culture) in terms of surface negative charge distribution. More surface negatively charged sites were observed at 0 and 7 days and much fewer at 3 days during storage of washed, harvested cells in buffer at 4 degrees C (aged cells under cold and nutrient deprivation), indicating a possible change in cell surface properties. Because no difference in strains was observed, the contact angle measurement study was carried out with the five-strain mixed culture. The surface hydrophobicity increased in frankfurters, decreased in roast beef, and was unchanged in ham and bologna as a result of inoculation.     

PorA specific primers for the identification of Campylobacter species in food and clinical samples

Campylobacteriosis is a public health problem with considerable socio-economic impact. As the European Food Safety Authority has emphasized the importance of a surveillance programme for campylobacteriosis, the aim of the present study was the optimization of a specific and sensitive PCR protocol able to detect Campylobacter species responsible for gastrointestinal infections. Raw poultry meat samples were analysed for the presence of Campylobacter sp., by plating onto mCCD (Modified Charcoal-Cefoperazone-Deoxycholate) Agar and Campylobacter Selective Preston Agar and using four sets of species-specific primers for Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis and Campylobacter lari designed to bridge the porA gene. The resulting primers demonstrated a sensitivity of 0.01 ng/μl for the C. coli-specific, C. lari-specific, and C. upsaliensis-specific primer sets and 0.5 ng/μl for the C. jejuni-specific primer sets using DNA from pure cultures. Non-specific amplification of non-target DNA was not observed indicating excellent specificity. The primers were useful for the analyses of poultry meat samples both for direct plating onto mCCDA, and for DNA extracted directly from the cells grown for 48 h in Preston enrichment broth. The sets of primers were also useful when used for species identification of human isolates.     

Effects of physicochemical surface characteristics of Listeria monocytogenes strains on attachment to glass

Seven strains of Listeria monocytogenes frequently involved in foodborne disease (epidemic strains) and 14 sporadic strains were examined to compare the attachment and subsequent biofilm growth on glass slides at 37 degrees C. Epidemic strains at 3 h incubation had significantly higher attachment values than sporadic strains (P<0.001), but subsequent biofilm growth over 24 h was not dependent on initial attachment. To better understand this phenomenon, the surface hydrophobicity and charge, as well as the extracellular carbohydrate content of the 21 L. monocytogenes strains were studied to determine if these surface characteristics had an effect on bacterial attachment to glass. Hydrophobicity was measured by the bacterial adherence to hydrocarbon (BATH) and polystyrene adherence methods. Hydrophobicity values obtained with the BATH method were linearly correlated with those from the polystyrene adherence method (r=0.64, P<0.001), but no correlation was found between hydrophobicity and bacterial attachment to glass. Hydrophobicity and surface charge measured as electrophoretic mobility (EM) were correlated (r=0.77, P<0.001); however, there was no correlation between the degree of attachment and surface charge. Colorimetric measurements of the total extracellular carbohydrates revealed that attached cells produced significantly (P<0.05) higher levels than planktonic cells after a 3 h time period. Analysis of co-variance (Nested ANCOVA) furthermore demonstrated that total carbohydrates produced by planktonic cells had a significant positive effect on 24 h biofilm growth (P=0.006). This is the first report to indicate that the ability of a L. monocytogenes strain to produce high levels of extracellular carbohydrates may increase its ability to form a biofilm. Genetic studies targeting carbohydrate synthesis pathways of L. monocytogenes will be required to fully understand the importance of this observation.     

Survival of Campylobacter jejuni on beef and pork under vacuum packaged and retail storage conditions: examination of the role of natural meat microflora on C. jejuni survival

The ability of Campylobacter jejuni ATCC 11168 to survive on beef and pork stored under chilled, vacuum packaged and retail display conditions were examined. In addition, the effect of natural microflora on commercial beef and pork on the survival of C. jejuni under these storage conditions was examined. When sterile cores of beef and pork were inoculated with ∼10⁵ to 10⁶ cfu cm⁻²C. jejuni, and were stored under aerobic or vacuum packaged conditions at −1.5 or 4 °C, its numbers dropped significantly and C. jejuni could not be enumerated by direct plating after 21 d of the 6 wks study. In contrast, survival of C. jejuni on commercial vacuum packaged beef and pork was significantly enhanced, resulting in only 1 log cfu cm⁻² reduction at the end of 6 wks. During 7 d of display in a retail case, numbers of C. jejuni dropped quickly, but could be enumerated by direct plating even after the 7 d. The presence of high numbers of inoculated C. jejuni on beef and pork had no significant effect on the natural microflora numbers compared to uninoculated controls when the meat was stored either in vacuum or in a retail display case. These results show that natural microflora on vacuum packaged meat afford enhanced survival of C. jejuni present on the surfaces of both beef and pork when stored at refrigeration temperatures. Hence, strict hygienic practices or the implementation of decontamination technologies are recommended to ensure safety of meat with respect to this pathogen.     

Using of infrared spectroscopy to study the survival and injury of Escherichia coli O157:H7, Campylobacter jejuni and Pseudomonas aeruginosa under cold stress in low nutrient media

The inactivation and sublethal injury of Escherichia coli O157:H7, Campylobacter jejuni and Pseudomonas aeruginosa at three temperatures (22 °C, 4 °C and −18 °C) were studied using traditional microbiological tests and mid-infrared spectroscopy (4000-400 cm⁻¹). Bacteria were cultivated in diluted nutrient matrices with a high initial inoculation (∼10⁷ CFU/ml) levels. Both E. coli O157:H7 and P. aeruginosa survived and cell numbers increased at 22 °C for 5 days while C. jejuni numbers decreased one log₁₀ CFU/ml. A two log CFU/ml decrease was observed for the three pathogens held at 4 °C for 12 days. C. jejuni survived poorly following incubation at −18 °C for 20 days while levels of E. coli O157:H7 and P. aeruginosa remained high (10⁴ CFU/ml). Temperature stress response of microbes was observed by infrared spectroscopy in polysaccharide, protein, lipid, and nucleic acid regions and was strain specific. Level of cold injury could be predicted using cluster, discriminant function and class analog analysis models. Pathogens may produce oligosaccharides and potentially other components in response to stress as indicated by changes in spectral features at 1200-900 cm⁻¹ following freezing.     

Campylobacter contamination of broiler caeca and carcasses at the slaughterhouse and correlation with Salmonella contamination

In order to estimate the prevalence of Campylobacter spp. and Salmonella spp. on broiler chicken carcasses and the prevalence of Campylobacter spp. in caeca, 58 French slaughterhouses were investigated in 2008. Enumeration of Campylobacter spp. was also performed in order to study the relation between caeca and carcass contamination. A pool of 10 caeca and one carcass were collected from 425 different batches over a 12-month period in 2008. Salmonella was isolated on 32 carcasses leading to a prevalence of 7.5% ([5.0-10.0]_95%CI). The prevalence of Campylobacter was 77.2% ([73.2-81.2]_95%CI) in caeca and 87.5% ([84.4-90.7]_95%CI) on carcasses. No significant correlation was found between Campylobacter and Salmonella. Positive values of Campylobacter were normally distributed and the average level was 8.05 log₁₀ cfu/g ([7.94-8.16]_95%CI) in caeca and 2.39 cfu/g ([2.30-2.48]_95%CI) on carcasses. A positive correlation (r = 0.59) was found between the mean of Campylobacter in caeca and on carcasses (p < 0.001). Thus, carcasses from batches with Campylobacter-positive caeca had significantly (p < 0.001) higher numbers of Campylobacter per gram than batches with negative caeca. These results show that Campylobacter can be present in both matrices and reduction in caeca could be a possible way to reduce the amount of bacteria on carcasses. Of the 2504 identifications performed, 3 species of Campylobacter (Campylobacter jejuni, Campylobacter coli and Campylobacter lari) were identified. The main species recovered were C. jejuni and C. coli, which were isolated in 55.3% and 44.5% of positive samples, respectively. These two species were equally represented in caeca but C. jejuni was the most frequently isolated on carcasses with 57.1% and 42.5% of positive carcasses for C. jejuni and C. coli, respectively. This study underlines that target a reduction of Campylobacter on final products requires a decrease of contamination in caeca.     

Microbiological quality of fresh lettuce from organic and conventional production

Previously there was no available information on the levels of indicator bacteria and the prevalence of pathogens in fresh lettuce grown in organic and conventional farms in Spain. A total of 72 lettuce samples (18 farms for 4 repetitions each) for each type of the agriculture were examined in order to assess the bacteriological quality of the lettuces, in particular the prevalence of selected pathogens. The lettuce samples were analyzed for the presence of aerobic mesophilic, psychrotrophic microorganisms, yeasts and moulds, Enterobacteriaceae, mesophilic lactic acid bacteria, Pseudomonas spp. and presumptive Escherichia coli, Salmonella spp. and Listeria monocytogenes. The mean aerobic mesophilic counts (AM) were 6.35 ± 0.69 log₁₀ cfu g⁻¹ and 5.67 ± 0.80 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. The mean counts of psychrotrophic microorganisms were 5.82 ± 1.01 log₁₀ cfu g⁻¹ and 5.41 ± 0.92 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Yeasts and moulds (YM) mean counts were 4.74 ± 0.83 log₁₀ cfu g⁻¹ and 4.21 ± 0.96 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Lactic acid bacteria (LAB) were present in low numbers and the mean counts were 2.41 ± 1.10 log₁₀ cfu g⁻¹ and 1.99 ± 0.91 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Pseudomonas spp. mean counts were 5.49 ± 1.37 log₁₀ cfu g⁻¹ and 4.98 ± 1.26 log₁₀ cfu g⁻¹ in organic and conventional lettuce, respectively. The mean counts for Enterobacteriaceae were 5.16 ± 1.01 log₁₀ cfu g⁻¹ and 3.80 ± 1.53 log₁₀ cfu g⁻¹ in organic and conventional lettuce, respectively. E. coli was detected in 22.2% (16 samples) of organic lettuce and in 12.5% (9 samples) of conventional lettuce. None of the lettuce samples was positive for E. coli O157:H7, L. monocytogenes and Salmonella spp. From the samples analyzed by principal component analysis (PCA) a pattern with two different groups (conventional and organic) can be observed, being the highest difference between both kinds of samples the Enterobacteriaceae count.     

Quantitative analysis of viable,stressed and dead cells of Campylobacter jejuni strain 81-176

Campylobacter jejuni is an important foodborne gastrointestinal pathogen and highly sensitive to environmental stresses. Research has shown that changes in culturability, cell morphology, and viability occur when C. jejuni cells are subjected to stresses. In this study, real-time PCR, ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR), BacLight bacterial viability staining, and agar plate counting methods were used to quantitatively analyze viable, stressed, and dead C. jejuni strain 81-176. The real-time PCR assay provides highly sensitive and specific quantification of total genome copies of C. jejuni culture in different growth phases. Our results also reveal that real-time PCR can be used for direct quantification of Campylobacter genome release into Phosphate Buffered Saline (PBS) as an indicator of cell lysis. Using EMA-PCR, we obtained a dynamic range of greater than 3 logs for differentiating viable vs. dead cells. The viability and morphological characteristics of the stressed cells after one-week incubation at 25 °C, in air, and under nutrient-poor conditions were investigated. Our results indicated that, over 99% of the stressed cells were converted from the spiral to the coccoid form and became non-culturable. However, more than 96% of the coccoid cells retained their membrane integrity as suggested by both the BacLight staining and EMA-PCR analyses. Thus, to detect C. jejuni under stress conditions, conventional culturing method in conjunction with EMA-PCR or BacLight staining might be a more appropriate approach.     

The biofilm forming potential of bacterial species in the genus Campylobacter

The biofilm forming abilities of 16 strains representative of 14 of the 16 species comprising the genus Campylobacter were determined on glass, stainless steel, and polystyrene plastic. The formation of biofilms has been suggested as a means by which Campylobacter is able to persist within an inhospitable environment. Of the eight microaerophilic Campylobacter species, including two strains each of Campylobacter jejuni and Campylobacter fetus, only C. jejuni strain 81–176 reliably produced a visible biofilm on multiple surfaces. Alternately, all six strains of the anaerobic Campylobacter species reliably produced visible biofilms on multiple surfaces. Electron micrographs of the individual biofilms showed relatively homogeneous biofilms produced by the anaerobic strains, while the microaerophilic C. jejuni strain 81–176 produced a biofilm containing similar quantities of both the spiral and coccoid forms. This survey suggests a difference in the biofilm forming potentials and the morphologies of the bacteria comprising the biofilms between anaerobic and microaerophilic species of Campylobacter. Additionally, differences observed in the biofilm forming ability of two strains of C. jejuni suggest the need for a further investigation of the biofilm forming potential of this species using a larger number of strains.     

A “successful allele” at Campylobacter jejuni contingency locus Cj0170 regulates motility; “successful alleles” at locus Cj0045 are strongly associated with mouse colonization

Campylobacter jejuni is an important foodborne pathogen of humans and its primary reservoir is the gastrointestinal (GI) tract of chickens. Our previous studies demonstrated that phase variation to specific “successful alleles” at C. jejuni contingency loci Cj0045 (successful alleles carry 9G or 10G homopolymeric tracts) and Cj0170 (successful allele carries a 10G homopolymeric tract) in C. jejuni populations is strongly associated with colonization and enteritis in C57BL/6 IL-10 deficient mice. In the current study, we strengthened the association between locus Cj0170, Cj0045, and mouse colonization. We generated 8 independent strains derived from C. jejuni 11168 strain KanR4 that carried a Cj0170 gene disruption and these were all non motile. Two randomly chosen strains with the Cj0170 gene disruption (DM0170-2 and DM0170-6) were gavaged into mice. DM0170-2 and DM0170-6 failed to colonize mice while the control strain that carried a “successful” Cj0170 10G allele was motile and did colonize mice. In parallel studies, when we inoculated C. jejuni strain 33292 into mice, the “unsuccessful” Cj0045 11G allele experienced phase variation to “successful” 9G and 10G alleles in 2 independent experiments prior to d4 post inoculation in mice while the “successful” 9G allele in the control strain remained stable through d21 post inoculation or shifted to other successful alleles. These data confirm that locus Cj0170 regulates motility in C. jejuni strain KanR4 and is a virulence factor in the mouse model. The data also support a possible role of locus Cj0045 as a virulence factor in strain 33292 in infection of mice.     

Development and evaluation of aptamer magnetic capture assay in conjunction with real-time PCR for detection of Campylobacter jejuni

A prototype method for the concentration and detection of Campylobacter jejuni was developed using a previously reported biotinylated DNA aptamer in conjunction with qPCR. The so-called aptamer-based magnetic capture-qPCR (AMC-qPCR) assay was compared to a similar immunomagnetic separation (IMS)-qPCR assay. In small volume experiments (300 μl) applied to serially diluted C. jejuni suspended in buffer containing a mixed culture of other common food borne pathogens, the lower detection limit of the AMC-qPCR method was 1.1 log₁₀/300 μl C. jejuni cells, one log₁₀ better (lower) than that of IMS-qPCR (2.1 log₁₀ CFU/300 μl). AMC-qPCR capture efficiency was 10–13% at assay detection limit. In 10 ml scale-up experiments, the lower detection limit of AMC-qPCR was 2.0 log₁₀ CFU/10 ml with corresponding capture efficiency of 4–7%. Nucleic acid aptamers are promising alternatives to antibodies for magnetic bead-based capture followed by qPCR detection.     

Decontamination treatments can increase the prevalence of resistance to antibiotics of Escherichia coli naturally present on poultry

The main objective of this study was to determine the ability of various decontaminants to increase the prevalence of resistance to antibiotics in Escherichia coli populations on poultry. Chicken legs were dipped for 15 min into aqueous solutions (wt/vol) of trisodium phosphate (TSP; 12%), acidified sodium chlorite (ASC; 1200 ppm), ascorbic acid (AA; 2%) or citric acid (CA; 2%), or tap water (control). Samples were analyzed immediately after treatment (day 0) and after five days of storage at 7 ± 1 °C. A total of 250 E. coli isolates (50 from each group of samples; 25 on day 0 and 25 on day 5) were tested against twelve antibiotics of clinical significance by means of a standard disc-diffusion technique. A high prevalence of resistance to antibiotics was observed for E. coli strains from control samples, with three (6.0%) isolates sensitive, three (6.0%) resistant to one antibiotic and 44 (88.0%) isolates resistant to two or more antibiotics. Isolates from control samples had a lower prevalence of resistance than those from treated samples to ampicillin-sulbactam (P < 0.01, samples treated with TSP), amoxicillin-clavulanic acid (P < 0.001, ASC, AA and CA), cephotaxime (P < 0.05, TSP), trimethoprim-sulphamethoxazole (P < 0.05, AA; P < 0.01, CA), tetracycline (P < 0.01, CA), ciprofloxacin (P < 0.001, ASC; P < 0.05, AA; P < 0.01, CA) and nitrofurantoin (P < 0.01, TSP). These results suggest that the chemical decontaminants tested could favor the emergence, selection and/or proliferation of antibiotic-resistant strains in microbial populations on poultry meat.     

Cultured C2C12 cell lines as a model for assessment of bacterial attachment to bovine primary muscle cells

The mechanisms of bacterial attachment to meat tissues need to be understood to enhance meat safety interventions. However, little is known about attachment of foodborne pathogens to meat muscle cells. In this study, attachment of six Escherichia coli and two Salmonella strains to primary bovine muscle cells and a cultured muscle cell line, C₂C₁₂, was measured, including the effect of temperature. At 37 °C, all but one strain (EC623) attached to C₂C₁₂ cells, whereas only five of eight strains (M23Sr, H10407, EC473, Sal1729a and Sal691) attached to primary cells. At 10 °C, two strains (H10407 and EC473) attached to C₂C₁₂ cells, compared to four strains (M23Sr, EC614, H10407 and Sal1729a) of primary cells. Comparing all strains at both temperatures, EC614 displayed the highest CFU per C₂C₁₂ cell (4.60 ± 2.02 CFU/muscle cell at 37 °C), whereas greater numbers of M23Sr attached per primary cell (51.88 ± 39.43 CFU/muscle cell at 37 °C). This study indicates that primary bovine muscle cells may provide a more relevant model system to study bacterial attachment to beef carcasses compared to cell lines such as C₂C₁₂.     

Transcriptional and functional responses of Escherichia coli O157:H7 growing in the lettuce rhizoplane

Lettuce and spinach are increasingly implicated in foodborne illness outbreaks due to contamination by Escherichia coli O157:H7. While this bacterium has been shown to colonize and survive on lettuce leaf surfaces, little is known about its interaction with the roots of growing lettuce plants. In these studies, a microarray analyses, mutant construction and confocal microscopy were used to gain an understanding of structure and function of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots. After three days of interaction with lettuce roots, 94 and 109 E. coli O157:H7 genes were significantly up- and down-regulated at least 1.5 fold, respectively. While genes involved in biofilm modulation (ycfR and ybiM) were significantly up-regulated, 40 of 109 (37%) of genes involved in protein synthesis were significantly repressed. E. coli O157:H7 was 2 logs less efficient in lettuce root colonization than was E. coli K12. We also unambiguously showed that a ΔycfR mutant of E. coli O157:H7 was unable to attach to or colonize lettuce roots. Taken together these results indicate that bacterial genes involved in attachment and biofilm formation are likely important for contamination of lettuce plants with Shiga toxin-producing E. coli strains.     

Attachment strength and biofilm forming ability of Bacillus cereus on green-leafy vegetables: cabbage and lettuce

The present study was designed to investigate the ability of six Bacillus cereus strains to attach and form biofilm on cabbage and lettuce surfaces. These six strains were; a reference strain DSMZ 345 and five biofilm-producing strains (aquatic strains; TUB8, TUB30, TUB31, TUB32 and TUB33) isolated from drinking-water distribution network. Hydrophobicity, biofilm formation ability, attachment strength (S_R) of spores and vegetative cells of the six B. cereus strains were also determined. Due to their high hydrophobicity, spores of all strains had high ability to attach polystyrene and did not affect by dilution of tryptone soy broth (TSB, 1:20 v/v) in the in vitro experiment. Significant (p < 0.05) enhancement in vitro biofilm formation by vegetative cells of B. cereus was recorded in the diluted TSB. The highest biofilm formation on cabbage and lettuce surfaces was obtained by spores and vegetative cells of all tested strains on the 4^th hour of the incubation period. These populations were significantly (p < 0.05) increased by elongating incubation time from 4 h to 24 h except DSMZ 345 and TUB8. Biofilm formation behavior obtained by B. cereus spores and vegetative cells on the polystyrene surface was different compared with that recorded on produce surface. The S_R of both spores and vegetative cells of the studied strains to the lettuce surface was higher than that of the cabbage surface. The hydrophobicity, biofilm formation and S_R of spores and vegetative cells of the biofilm-producing strains were higher than that of the reference strain DSMZ 345. Scanning electron microscopy (SEM) exposed random distribution of cells either on the surface or cut edge, without clear obvious affinity for the surface structures. Increasing in the presence of large clusters of cells on leaf surfaces was demonstrated after 4 and 24 h.In conclusion, use of aquatic environmental isolates is more useful for studying biofilm formation than the reference strain. Lettuce surface supported the attachment of B. cereus spores and vegetative cells compared with the cabbage surface. Further investigations are required to improve our knowledge of biofilm formation mechanisms by the human pathogenic microorganisms, especially by using the environmental and clinical isolates. To ensure safety level of green-leafy vegetables, biofilm formation after harvest should be considered as critical control point during handling of these vegetables.