Comparison of two possible routes of pathogen contamination of spinach leaves in a hydroponic cultivation system

The route of pathogen contamination (from roots versus from leaves) of spinach leaves was investigated with a hydroponic cultivation system. Three major bacterial pathogens, Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes, were inoculated into the hydroponic solution, in which the spinach was grown to give concentrations of 10? and 103 CFU/ml. In parallel, the pathogens were inoculated onto the growing leaf surface by pipetting, to give concentrations of 10? and 103 CFU per leaf. Although contamination was observed at a high rate through the root system by the higher inoculum (10? CFU) for all the pathogens tested, the contamination was rare when the lower inoculum (103 CFU) was applied. In contrast, contamination through the leaf occurred at a very low rate, even when the inoculum level was high. For all the pathogens tested in the present study, the probability of contamination was promoted through the roots and with higher inoculum levels. The probability of contamination was analyzed with logistic regression. The logistic regression model showed that the odds ratio of contamination from the roots versus from the leaves was 6.93, which suggested that the risk of contamination from the roots was 6.93 times higher than the risk of contamination from the leaves. In addition, the risk of contamination by L. monocytogenes was about 0.3 times that of Salmonella enterica subsp. enterica serovars Typhimurium and Enteritidis and E. coli O157:H7. The results of the present study indicate that the principal route of pathogen contamination of growing spinach leaves in a hydroponic system is from the plant's roots, rather than from leaf contamination itself.     

Simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella in apple cider and produce by a multiplex PCR

With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli 0157:H7, Salmonella, and Shigella. Under the optimized conditions, the assay yielded a 252-bp product from E. coli O157:H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed. In the specificity test, 10 E. coli O157:H7 strains and one E. coli O157:NM strain showed the expected 252-bp amplicon. Seven other E. coli strains yielded no signal. Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species. No nonspecific amplification was observed with DNA from 48 other bacterial strains. Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 x 10(-1) CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 x 10(1) CFU/g in alfalfa sprouts. The whole procedure can be easily completed within 30 h. The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E. coli O157:H7, Salmonella, and/or Shigella.     

Antimicrobial activity of plant extracts against Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes on fresh lettuce

Plant extracts have been found to be effective in reducing microorganisms. This study evaluated antimicrobial activity of 12 plant extracts against Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes by using a disk diffusion assay, and Syzygium aromaticum (clove) showed the highest inhibitory effect. To investigate the efficacy of clove extract that inactivates pathogens on lettuce, inoculated lettuce with S. Typhimurium, E. coli O157:H7, and L. monocytogenes was treated with diluted clove extracts or distilled water for 0, 1, 3, 5, and 10 min. Clove extract treatment significantly reduced populations of the 3 tested pathogens from the surface of lettuce. Practical Application: This result indicated that clove extract is a useful antimicrobial agent to reduce the microbial level of foodborne pathogens on fresh lettuce. It also might be a natural antimicrobial for reducing or replacing chemical sanitizers in food preservation.     

Survival of Escherichia coli O157:H7 and Salmonella enterica serovars Typhimurium in iceberg lettuce and the antimicrobial effect of rice vinegar against E. coli O157:H7

The microbiological safety of fresh produce is a significant concern of consumers and industry. After applying at an inoculated level (about 10(6) CFUg(-1)) of E. coli O157:H7 and Salmonella enterica serovars Typhimurium on shredded iceberg lettuce and water samples individually, they were stored at 4 degrees C for 14 days and 22 degrees C for 7 days to monitor the growth and survival of pathogens. The results showed that at the end of 4 degrees C storage, populations of two pathogens in lettuce and water decreased approximately 1 log CFUg(-1). However, microbial levels on shredded lettuce increased 3 logs within 3 days at 22 degrees C. Vinegar (acetic acid) had been used to reduce populations of foodborne pathogens in foods; hence, the antimicrobial effect of rice vinegar on the survival of E. coli O157:H7 in inoculated lettuce (10(4) and 10(7) CFUg(-1)) is examined in this study. Results were observed that the treatment of inoculated lettuce (10(7) CFUg(-1)) with commercial vinegar containing 5% acetic acid (pH 3.0) for 5 min would reduce 3 logs population at 25 degrees C. Less than a 1-log decrease in bacterial numbers was recovered during 5 min exposure to 0.5% (pH 3.26) acetic acid.     

Association of Escherichia coli O157:H7 with preharvest leaf lettuce upon exposure to contaminated irrigation water.

Recent foodborne outbreaks have linked infection by enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 to the consumption of contaminated lettuce. Contamination via food handler error and on-the-farm contamination are thought to be responsible for several outbreaks. Though recent studies have examined the application of EHEC to store-bought lettuce, little is known about the attachment of EHEC to growing plants. We investigated the association of lettuce seedlings with EHEC O157:H7 strains implicated in lettuce or fruit outbreaks using hydroponic and soil model systems. EHEC strains that express the green fluorescent protein were observed by stereomicroscopy and confocal laser scanning microscopy to determine adherence patterns on growing lettuce seedlings. Bacteria adhered preferentially to plant roots in both model systems and to seed coats in the hydroponic system. Two of five nonpathogenic E. coli strains showed decreased adherence to seedling roots in the hydroponic system. EHEC was associated with plants in as few as 3 days in soil, and contamination levels were dose-dependent. EHEC levels associated with young plants inoculated with a low dose suggested that the bacteria had multiplied. These data suggest that preharvest crop contamination via contaminated irrigation water can occur through plant roots.     

Biofilm formation by multidrug-resistant Salmonella enterica serotype typhimurium phage type DT104 and other pathogens

The biofilm-forming capability of Salmonella enterica serotypes Typhimurium and Heidelberg, Pseudomonas aeruginosa, Listeria monocytogenes, Escherichia coli O157:H7, Klebsiella pneumoniae, and Acinetobacter baumannii isolated from humans, animal farms, and retail meat products was evaluated by using a microplate assay. The tested bacterial species showed interstrain variation in their capabilities to form biofilms. Strong biofilm-forming strains of S. enterica serotypes, E. coli O157: H7, P. aeruginosa, K. pneumoniae, and A. baumannii were resistant to at least four of the tested antibiotics. To understand their potential in forming biofilms in food-processing environments, the strong biofilm formers grown in beef, turkey, and lettuce broths were further investigated on stainless steel and glass surfaces. Among the tested strains, Salmonella Typhimurium phage type DT104 (Salmonella Typhimurium DT104) isolated from retail beef formed the strongest biofilm on stainless steel and glass in beef and turkey broths. K. pneumoniae, L. monocytogenes, and P. aeruginosa were also able to form strong biofilms on the tested surface materials. Salmonella Typhimurium DT104 developed a biofilm on stainless steel in beef and turkey broths through (i) initial attachment to the surface, (ii) formation of microcolonies, and (iii) biofilm maturation. These findings indicated that Salmonella Typhimurium DT104 alongwith other bacterial pathogens could be a source of cross-contamination during handling and processing of food.     

Inactivation kinetics of inoculated Escherichia coli O157:H7 and Salmonella enterica on lettuce by chlorine dioxide gas

The purpose of this investigation was to study inactivation kinetics of inoculated Escherichia coli O157:H7 and Salmonella enterica on lettuce leaves by ClO(2) gas at different concentrations (0.5, 1.0, 1.5, 3.0, and 5.0 mg l(-1)) for 10 min and to determine the effect of ClO(2) gas on the quality and shelf life of lettuce during storage at 4 degrees C for 7 days. One hundred microliters of each targeted organism was separately spot-inoculated onto the surface (5 cm(2)) of lettuce (approximately 8-9 log CFU ml(-1)), air-dried, and treated with ClO(2) gas at 22 degrees C and 90-95% relative humidity for 10 min. Surviving bacterial populations on lettuce were determined using a membrane transferring method, which included a non-selective medium followed by a selective medium. The inactivation kinetics of E. coli O157:H7 and S. enterica was determined using first-order kinetics to establish D-values and z-values. The D-values of E. coli and S. enterica were 2.9+/-0.1 and 3.8+/-0.5 min, respectively, at 5.0 mg l(-1) ClO(2) gas. The z-values of E. coli and S. enterica were 16.2+/-2.4 and 21.4+/-0.5 mg l(-1), respectively. A 5 log CFU reduction (recommended by the United States Food and Drug Administration) for E. coli and S. enterica could be achieved with 5.0 mg l(-1) ClO(2) gas for 14.5 and 19.0 min, respectively. Treatment with ClO(2) gas significantly reduced inherent microflora on lettuce and microbial counts remained significantly (p<0.05) lower than the uninoculated control during storage at 4 degrees C for 7 days. However, treatment with ClO(2) gas had a significantly (p<0.05) negative impact on visual leaf quality. These results showed that treatment with ClO(2) gas significantly reduced selected pathogens and inherent microorganisms on lettuce; however, the processing conditions would likely need to be altered for consumer acceptance.     

Efficiency of sodium hypochlorite, fumaric acid, and mild heat in killing native microflora and Escherichia coli O157:H7, Salmonella typhimurium DT104, and Staphylococcus aureus attached to fresh-cut lettuce

The effect of the disinfectant sodium hypochlorite (NaClO), with or without mild heat (50 degrees C) and fumaric acid, on native bacteria and the foodborne pathogens Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium DT104 attached to iceberg lettuce leaves was examined. The retail lettuce examined consistently harbored 6 to 7 log CFU/g of native bacteria throughout the study period. Inner leaves supported 1 to 2 log CFU/g fewer bacteria than outer leaves. About 70% of the native bacterial flora was removed by washing five times with 0.85% NaCl. S. aureus, E. coli, and Salmonella allowed to attach to lettuce leaves for 5 min were more easily removed by washing than when allowed to attach for 1 h or 2 days, with more S. aureus being removed than E. coli or Salmonella Typhimurium. An increase of time for attachment of pathogens from 5 min to 2 days leads to decreased efficiency of the washing and sanitizing treatment. Treatment with fumaric acid (50 mM for 10 min at room temperature) was the most effective, although it caused browning of the lettuce, with up to a 2-log reduction observed. The combination of 200 ppm of sodium hypochlorite and mild heat treatment at 50 degrees C for 1 min reduced the pathogen populations by 94 to 98% (1.2- to 1.7-log reduction) without increasing browning.     

Transfer and internalisation of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in cabbage cultivated on contaminated manure-amended soil under tropical field conditions in Sub-Saharan Africa

Surface contamination and internalisation of Escherichia coli O157:H7 and Salmonella Typhimurium in cabbage leaf tissues at harvest (120 days post-transplantation) following amendment of contaminated bovine manure to soil at different times during crop cultivation were investigated under tropical field conditions in the Central Agro-Ecological Zone of Uganda. Fresh bovine manure inoculated with rifampicin-resistant derivatives of non-virulent strains of E. coli O157:H7 and S. Typhimurium was incorporated into the soil to achieve inoculum concentrations of 4 and 7 log CFU/g at the point of transplantation, 56 or 105 days post-transplantation of cabbage seedlings. Frequent sampling of the soil enabled the accurate identification of the survival kinetics in soil, which could be described by the Double Weibull model in all but one of the cases. The persistence of 4 log CFU/g E. coli O157:H7 and S. Typhimurium in the soil was limited, i.e. only inocula applied 105 days post-transplantation were still present at harvest. Moreover, no internalisation in cabbage leaf tissues was observed. In contrast, at the 7 log CFU/g inoculum level, E. coli O157:H7 and S. Typhimurium survived in the soil throughout the cultivation period. All plants (18/18) examined for leaf contamination were positive for E. coli O157:H7 at harvest irrespective of the time of manure application. A similar incidence of leaf contamination was found for S. Typhimurium. On the other hand, only plants (18/18) cultivated on soil amended with contaminated manure at the point of transplantation showed internalised E. coli O157:H7 and S. Typhimurium at harvest. These results demonstrate that under tropical field conditions, the risk of surface contamination and internalisation of E. coli O157:H7 and S. Typhimurium in cabbage leaf tissues at harvest depend on the inoculum concentration and the time of manure application. Moreover, the internalisation of E. coli O157:H7 and S. Typhimurium in cabbage leaf tissues at harvest seems to be limited to the worst case situation, i.e., when highly contaminated manure is introduced into the soil at the time of transplantation of cabbage seedlings.     

Influence of inoculation method, spot inoculation site, and inoculation size on the efficacy of acidic electrolyzed water against pathogens on lettuce

The influence of bacterial inoculation methods on the efficacy of sanitizers against pathogens was examined. Dip and spot inoculation methods were employed in this study to evaluate the effectiveness of acidic electrolyzed water (AcEW) and chlorinated water (200 ppm free available chlorine) against Escherichia coli O157:H7 and Salmonella spp. Ten pieces of lettuce leaf (5 by 5 cm) were inoculated by each method then immersed in 1.5 liters of AcEW, chlorinated water, or sterile distilled water for 1 min with agitation (150 rpm) at room temperature. The outer (abaxial) and inner (adaxial) surfaces of the lettuce leaf were distinguished in the spot inoculation. Initial inoculated pathogen population was in the range 7.3 to 7.8 log CFU/g. Treatment with AcEW and chlorinated water resulted in a 1 log CFU/g or less reduction of E. coli O157:H7 and Salmonella populations inoculated with the dip method. Spot inoculation of the inner surface of the lettuce leaf with AcEW and chlorinated water reduced the number of E. coli O157:H7 and Salmonella by approximately 2.7 and 2.5 log CFU/g, respectively. Spot inoculation of the outer surface of the lettuce leaf with both sanitizers resulted in approximately 4.6 and 4.4 log CFU/g reductions of E. coli O157:H7 and Salmonella, respectively. The influence of inoculation population size was also examined. Each sanitizer could not completely eliminate the pathogens when E. coli O157:H7 and Salmonella cells inoculated on the lettuce were of low population size (10(3) to 10(4) CFU/g), regardless of the inoculation technique.     

Comparison of the attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Pseudomonas fluorescens to lettuce leaves

Attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens on iceberg lettuce was evaluated by plate count and confocal scanning laser microscopy (CSLM). Attachment of each microorganism (approximately 10(8) CFU/ml) on the surface and the cut edge of lettuce leaves was determined. E. coli O157:H7 and L. monocytogenes attached preferentially to cut edges, while P. fluorescens attached preferentially to the intact surfaces. Differences in attachment at the two sites were greatest with L. monocytogenes. Salmonella Typhimurium attached equally to the two sites. At the surface, P. fluorescens attached in greatest number, followed by E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium. Attached microorganisms on lettuce were stained with fluorescein isothiocyanate and visualized by CSLM. Images at the surface and the cut edge of lettuce confirmed the plate count data. In addition, microcolony formation by P. fluorescens was observed on the lettuce surface. Some cells of each microorganism at the cut edge were located within the lettuce tissues, indicating that penetration occurred from the cut edge surface. The results of this study indicate that different species of microorganisms attach differently to lettuce structures, and CSLM can be successfully used to detect these differences.     

Pathogenic potential of Salmonella Typhimurium DT104 following sequential passage through soil, packaged fresh-cut lettuce and a model gastrointestinal tract

From a quantitative microbial risk assessment perspective it is important to know whether certain food environments influence the pathogenic potential of pathogens and to what extent. The purpose of the present study was to examine the pathogenic potential of S. Typhimurium DT104, measured as the capability to survive a simulated gastrointestinal tract system and the capability of adhering to and invading differentiated Caco-2 cells, after sequential incubation (without intermediate culturing) into soil, lettuce and cut lettuce stored under modified atmosphere (MAP) conditions. Two S. Typhimurium DT104 strains were used, one isolated from a pig carcass and one isolated from lettuce. The most important result of the present study is that the sequential incubation of S. Typhimurium in soil and lettuce slightly increased the capability of surviving the simulated gastric fluid, increased the capability to grow in the simulated intestinal fluid but decreased the capability of epithelial attachment and invasion and decreased the overall survival probability of the gastrointestinal tract system. Some variation in responses between the strains was observed, with the lettuce strain maintaining higher epithelial attachment capability and the carcass strains maintaining higher epithelial invasion capability. This study provided quantitative data on the effect of environmental and food matrices on the pathogenic potential of S. Typhimurium DT104 using a realistic system of sequential incubations in environmental and food matrices, followed by simulated gastrointestinal tract passage without intermediate culturing. These results could aid the development of more realistic quantitative microbial risk assessments.     

Efficacy of aerosolized peroxyacetic acid as a sanitizer of lettuce leaves

Aerosolized sanitizer was investigated as a potential alternative to aqueous and gaseous sanitizers for produce. Peroxyacetic acid was aerosolized (5.42 to 11.42 microm particle diameter) by a commercially available nebulizer into a model cabinet. Iceberg lettuce leaves were inoculated with three strains each of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium and then treated with aerosolized peroxyacetic acid for 10, 30, or 60 min in a model aerosol cabinet at room temperature (22 +/- 2 degrees C). After treatment, surviving healthy and injured bacterial cells were enumerated on appropriate selective agars or using the overlay agar method. Inoculated iceberg lettuce leaves exposed to aerosolized peroxyacetic acid for 10 min exhibited a 0.8-log reduction in E. coli O157:H7, a 0.3-log reduction in Salmonella Typhimurium, and a 2.5-log reduction in L. monocytogenes when compared with the control. After 30 min of treatment, the three pathogens were reduced by 2.2, 3.3, and 2.7 log, and after 60 min, the reductions were 3.4, 4.5, and 3.8 log, respectively. Aerosolization may be a new and convenient method for sanitizing produce for storage or shipping.     

Growth and survival of foodborne pathogens in beer

This work aimed to assess the growth and survival of four foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus) in beer. The effects of ethanol, pH, and storage temperature were investigated for the gram-negative pathogens (E. coli O157:H7 and Salmonella Typhimurium), whereas the presence of hops ensured that the gram-positive pathogens (L. monocytogenes and S. aureus) were rapidly inactivated in alcohol-free beer. The pathogens E. coli O157:H7 and Salmonella Typhimurium could not grow in the mid-strength or full-strength beers, although they could survive for more than 30 days in the mid-strength beer when held at 4°C. These pathogens grew rapidly in the alcohol-free beer; however, growth was prevented when the pH of the alcohol-free beer was lowered from the "as received" value of 4.3 to 4.0. Pathogen survival in all beers was prolonged at lowered storage temperatures.     

Efficacy of chlorine dioxide gas as a sanitizer of lettuce leaves

Aqueous solutions of sodium hypochlorite or hypochlorous acid are typically used to sanitize fresh fruits and vegetables. However, pathogenic organisms occasionally survive aqueous sanitization in sufficient numbers to cause disease outbreaks. Chlorine dioxide (ClO2) gas generated by a dry chemical sachet was tested against foodborne pathogens on lettuce leaves. Lettuce leaves were inoculated with cocktail of three strains each of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium and treated with CLO2 gas for 30 min, 1 h, and 3 h in a model gas cabinet at room temperature (22 +/- 2 degrees C). After treatment, surviving cells, including injured cells, were enumerated on appropriate selective agar or using the overlay agar method, respectively. Total ClO2, generated by the gas packs was 4.3, 6.7, and 8.7 mg after 30 min, 1 h, and 3 h of treatment, respectively. Inoculated lettuce leaves exposed to ClO2 gas for 30 min experienced a 3.4-log reduction in E. coli, a 4.3-log reduction in Salmonella Typhimurium, and a 5.0-log reduction in L. monocytogenes when compared with the control. After 1 h. the three pathogens were reduced in number of CFU by 4.4. 5.3, and 5.2 log, respectively. After 3 h, the reductions were 6.9, 5.4, and 5.4 log, respectively. A similar pattern emerged when injured cells were enumerated. The ClO2, gas sachet was effective at killing pathogens on lettuce without deteriorating visual quality. Therefore, this product can be used during storage and transport of lettuce to improve its microbial safety.     

Evaluation of peroxyacetic acid as a potential pre-grinding treatment for control of Escherichia coli O157:H7 and Salmonella Typhimurium on beef trimmings

Peroxyacetic acid was evaluated in four separate trials for ability to reduce populations of Escherichia coli O157:H7 and Salmonella serotype Typhimurium on fresh beef trim. Trial 1 examined the effectiveness of peroxyacetic acid on individual pieces of fresh beef trim. Trial 2 evaluated the efficacy of peroxyacetic acid at low levels of contamination on batches of fresh beef trim. Trial 3 studied a washing effect of water. Lastly, Trial 4 compared the effectiveness of peroxyacetic acid to lactic acid. At various inoculation levels, peroxyacetic acid reduced populations of both pathogens by approximately 1.0log(10)CFU/cm(2) on fresh beef trim. Trial 3 showed that approximately half of the reductions found in Trials 1 and 2 were due to a washing effect of the water dip. In addition, as shown in Trial 1, increases in concentrations (>200ppm) did not significantly increase log(10) reductions of both pathogens. Following a water dip in Trial 4, peroxyacetic acid caused a reduction of 0.7log(10)CFU/cm(2) in E. coli O157:H7 and 1.0log(10)CFU/cm(2) in Salmonella Typhimurium, whereas lactic acid caused a reduction of 1.3log(10)CFU/cm(2) in E. coli O157:H7 and 2.1log(10)CFU/cm(2) in S. Typhimurium following the water dip. These results show that peroxyacetic acid was not more effective than 2% l-lactic acid in reducing pathogens on fresh beef trim.     

Evaluation of universal preenrichment broth for growth of heat-injured pathogens.

Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis. and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2 degrees C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37 degrees C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (> 10(3) CFU/ ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37 degrees C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation-but not at 6 h-sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.     

Interactions of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes plants cultivated in a gnotobiotic system

The growth and persistence of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes on a diverse range of plant types over extended cultivation periods was studied. When introduced on the seed of carrot, cress, lettuce, radish, spinach and tomato all the pathogens became rapidly established shortly after germination, attaining cell densities of the order of 5.5-6.5 log cfu/g. In general, Es. coli O157:H7 and L. monocytogenes became established and persisted at significantly higher levels on seedlings (9 days post-germination) than Salmonella. Es. coli O157:H7 became internalized in cress, lettuce, radish and spinach seedlings but was not recovered within the tissues of mature plants. Internalization of Salmonella was also observed in lettuce and radish but not cress or spinach seedlings. In contrast, L. monocytogenes did not internalize within seedlings but did persist on the surface of plants throughout the cultivation period. Co-inoculation of isolates recovered from the rhizosphere of plants did not significantly affect the numbers or persistence of human pathogens. The only exception was with Enterobacter cloacae, which reduced Es. coli O157:H7 Ph1 and L. monocytogenes levels by ca. 1 log cfu/g on lettuce. With the bioluminescent phenotype of Es. coli O157:H7 Ph1, it was demonstrated that the human pathogen became established on the roots of growing plants. Scanning electron micrographs of root seedlings suggested that Es. coli O157:H7 Ph1 preferentially colonized the root junctions of seedlings. It is proposed that such colonization sites enhanced the persistence of Es. coli O157:H7 on plants and facilitated internalization within developing seedlings. The results suggest that the risk associated with internalized human pathogens in salad vegetables at harvest is low. Nevertheless, the introduction of human pathogens at an early stage of plant development could enhance their persistence in the rhizosphere. The implications of the study with regards to on-farm food safety initiatives are discussed.     

Spread of bacterial pathogens during preparation of freshly squeezed orange juice

To study the potential of three bacterial pathogens to cross-contaminate orange juice during extraction, normal operation conditions during juice preparation at food service establishments were simulated. The spread of Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes from inoculated oranges to work surfaces and to the final product was determined. The transference of these three bacterial pathogens to orange juice made from uninoculated oranges with the use of contaminated utensils was also studied. Fresh oranges were inoculated with a marker strain of rifampicin-resistant Salmonella Typhimurium, E. coli O157:H7, or L. monocytogenes. Final pathogen levels in juice were compared as a function of the use of electric or mechanical juice extractors to squeeze orange juice from inoculated oranges. Pathogen populations on different contact surfaces during orange juice extraction were determined on sulfite-phenol red-rifampicin plates for Salmonella Typhimurium and E. coli O157:H7 and on tryptic soy agar supplemented with 0.1 g of rifampicin per liter for L. monocytogenes. After inoculation, the average pathogen counts for the orange rind surface were 2.3 log10 CFU/cm2 for Salmonella Typhimurium, 3.6 log10 CFU/cm2 for E. coli O157:H7, and 4.4 log10 CFU/cm2 for L. monocytogenes. This contamination was spread over all utensils used in orange juice squeezing. Mean pathogen counts for the cutting board, the knife, and the extractor ranged from -0.3 to 2.1 log10 CFU/cm2, and the juice contained 1.0 log10 CFU of Salmonella Typhimurium per ml, 2.3 log10 CFU of E. coli O157:H7 per ml, and 2.7 log10 CFU of L. monocytogenes per ml. Contact with contaminated surfaces resulted in the presence of all pathogens in orange juice made from uninoculated oranges. These results give emphasis to the importance of fresh oranges as a source of pathogens in orange juice.     

Autoinducer-2 activity of gram-negative foodborne pathogenic bacteria and its influence on biofilm formation

ABSTRACT:  This study evaluated whether autoinducer-2 (AI-2) activity would be associated with biofilm formation by Salmonella and Escherichia coli O157:H7 strains on food contact surfaces. In study I, a Salmonella Typhimurium DT104 strain and an E . coli O157:H7 strain, both AI-2 positive, were individually inoculated into 50 mL of Luria–Bertani (LB) or LB + 0.5% glucose (LBG) broth, without or with stainless steel or polypropylene ( Salmonella ) coupons. At 0, 14 ( Salmonella ), 24, 48, and 72 h of storage (25 °C), cells in suspension and detached cells from the coupons, obtained by vortexing, were enumerated on tryptic soy agar. In study II, a Salmonella Thompson AI-2-positive strain and an AI-2-negative strain, and an E . coli O157:H7 AI-2-positive strain and an AI-2-negative strain were inoculated into LB broth with stainless steel coupons. Cells were enumerated as in study I. In both studies, AI-2 activity was determined in cell-free supernatants. Cell numbers of S . Typhimurium DT104 on biofilms were higher ( P < 0.05) in LB than those in LBG, while the E . coli O157:H7 strain showed no difference ( P ≥ 0.05) in biofilm cell counts between LB and LBG after storage for 72 h. Both S . Typhimurium DT104 and E . coli O157:H7 strains produced higher ( P < 0.05) AI-2 activity in LBG than LB cell suspensions. Cell counts of AI-2-positive and-negative S . Thompson and E . coli O157:H7 strains were not different ( P ≥ 0.05) within suspensions or coupons (study II). The results indicated that, under the conditions of this study, AI-2 activity of the pathogen strains tested may not have a major influence on biofilm formation on food contact surfaces, which was similar between AI-2-positive and -negative strains.