Viable but Nonculturable Escherichia coli O157:H7 and Salmonella enterica in Fresh Produce: Rapid Determination by Loop-Mediated Isothermal Amplification Coupled with a Propidium Monoazide Treatment.

Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the "viable but nonculturable" (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce.IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

Endemic bacteriophages: a cautionary tale for evaluation of bacteriophage therapy and other interventions for infection control in animals.

BACKGROUND: One of the most effective targets for control of zoonotic foodborne pathogens in the farm to fork continuum is their elimination in food animals destined for market. Phage therapy for Escherichia coli O157:H7 in ruminants, the main animal reservoir of this pathogen, is a popular research topic. Since phages active against this pathogen may be endemic in host animals and their environment, they may emerge during trials of phage therapy or other interventions, rendering interpretation of trials problematic. METHODS: During separate phage therapy trials, sheep and cattle inoculated with 109 to 1010 CFU of E. coli O157:H7 soon began shedding phages dissimilar in plaque morphology to the administered therapeutic phages. None of the former was previously identified in the animals or in their environment. The dissimilar "rogue" phage was isolated and characterized by host range, ultrastructure, and genomic and proteomic analyses. RESULTS: The "rogue" phage (Phage vB_EcoS_Rogue1) is distinctly different from the administered therapeutic Myoviridae phages, being a member of the Siphoviridae (head: 53 nm; striated tail: 152x8 nm). It has a 45.8 kb genome which is most closely related to coliphage JK06, a member of the "T1-like viruses" isolated in Israel. Detailed bioinformatic analysis reveals that the tail of these phages is related to the tail genes of coliphage lambda. The presence of "rogue" phages resulting from natural enrichments can pose problems in the interpretation of phage therapeutic studies. Similarly, evaluation of any interventions for foodborne or other bacterial pathogens in animals may be compromised unless tests for such phages are included to identify their presence and potential impact.

Mycoplasma genitalium: To treat or not to treat?

ABSTRACT: Many APRNs are not familiar with Mycoplasma genitalium, even if it is included on a sexually transmitted infection screening panel. This article briefly outlines prevalence, asymptomatic infection, clinical manifestations, diagnostics, and treatment.

How NPs can eliminate practice barriers to intrauterine device use.

ABSTRACT: Barriers to intrauterine device use include cost, absence of qualified providers, the lack of simplified insertion protocols, cultural hesitation, and misconceptions of appropriateness of use. This article outlines how NPs can eliminate practice barriers to intrauterine device use and prevent unintended pregnancy.

Induction of viable but nonculturable Escherichia coli O157:H7 in the phyllosphere of lettuce: a food safety risk factor.

Escherichia coli O157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate of E. coli O157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4°C) and selected two strains with different nonculturable responses for the construction of E. coli O157:H7 Tn7gfp transformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16°C), both E. coli O157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8°C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (10(9) and 10(6) E. coli O157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log(10) cells but did not detect culturable cells. These findings indicate that E. coli O157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.

Resurgence of syphilis in the US.

ABSTRACT: Despite advances in screening and treatment for syphilis, the US has seen increased incidence of the disease in the past decade. This article gives an overview of incidence, risk factors, clinical manifestations, assessment, screening, treatment, and prevention of syphilis.

Disinfection of Alfalfa and Radish Sprouting Seed Using Oxidizing Agents and Treatments Compliant with Organic Food Production Principles.

ABSTRACT: Antimicrobial seed treatments recommended by Canadian guidance for sprouted vegetable production (2,000 ppm of hypochlorite for 15 to 20 min or 6 to 10% hydrogen peroxide for 10 min at room temperature) are not fully compliant with organic production principles. We investigated the effect of a sequential treatment consisting of a 10-min soak at 50°C in water followed by exposure to a 2.0% H2O2 plus 0.1% AcOH sanitizing solution against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica inoculated onto alfalfa and radish seed. The sequential treatment was as effective as the recommended treatments and could reduce populations of all three species by a minimum of 3 log CFU/g using a reduced (1:2) ratio of seed to sanitizing solution and low concentrations of sanitizers approved for use in organic food production. However, the efficacy of all the treatments examined in this work was considerably reduced by storage of the seed for 4 weeks at either 11 or 75% relative humidity prior to treatment and assessment. None of the treatments could eradicate the target pathogens from seed, irrespective of time elapsed since inoculation. The results of this work suggest that the effect of storage should be considered in the assessment of antimicrobial treatments for sprouting vegetable seed.

Nonculturable response of animal enteropathogens in the agricultural environment and implications for food safety.

Concerns about animal enteropathogen contamination of fresh horticultural products have,increased worldwide and are mainly due to the ability of bacteria to survive under stress conditions in the agricultural environment and during raw-vegetable processing. This review challenges the idea that the viable but nonculturable phenomenon that has been proven to occur in plant-associated environments contributes to human pathogen survival and might be correlated with foodborne infection. Factors associated with the nonculturable response of bacteria in the field and during postharvest processing and distribution are discussed, specifically for the most common animal enteropathogens linked with the consumption of raw products: Escherichia coli O157:H7, Salmonella, Listeria monocytogenes, and Shigella spp. The accurate detection of live bacterial populations is essential for pathogen screening in food and environmental safety control and in epidemiological analysis and may have to be considered for identification of critical control points at the time of food inspection.

Genotype, serotype, and antibiotic resistance of sorbitol-negative Escherichia coli isolates from feedlot cattle.

Rectal fecal samples from 80 steers receiving Rumensin, Revalor-S, and Liquamycin alone or in combination for growth promotion and disease prevention were examined for the presence of non-O157:H7 Shiga toxin-producing Escherichia coli. All isolates were identified with the API 20E test, virulence genes were detected with a PCR assay, and antibiotic susceptibilities were determined with the Sensititre system. Of the 153 E. coli isolates recovered 126 (82.3%) were sorbitol negative. Isolates were classified into 14 biochemical E. coli groups; 51.6% were negative for arginine dihydrolase, ornithine decarboxylase, sorbitol, and saccharose reactions but positive for lysine decarboxylase, indole production, and rhamnose reactions. Twenty-one O:H serotypes were detected in the 153 E. coli isolates. The most frequent serotypes were O2:H42 (49.7% of isolates), O49:NM (13.7%), O?:H25 (9.2%), and O10:NM (7.2%). One isolate of E. coli O172:H25 and one of E. coli O157: H39 were found. The stx1 gene was found in the two E. coli O98:H25 isolates. The eaeA and e-hlyA genes were detected in 21, 14, and 10 isolates of serotypes O49:NM, O?:H25, and O10:NM, respectively, and in each isolate of serotype O156:H25 and O172:H25. Four E. coli O132:H18 isolates were multiresistant to ampicillin, chloramphenicol, kanamycin, streptomycin, and sulfisoxazole. Tetracycline resistance due to the tet(B) gene was observed in 74 of the 76 E. coli O2:H42 isolates. Except for one isolate, all tetracycline-resistant isolates were negative for the virulence genes eaeA and e-hlyA or stx1. Pulsed-field gel electrophoresis typing revealed that the tetracycline-resistant serotypes were genetically diverse. Our data illustrate that cattle are a potential source of some atypical antibiotic-resistant E. coli isolates that harbor virulence genes.

Oral and rectal administration of bacteriophages for control of Escherichia coli O157:H7 in feedlot cattle.

This study compared oral and rectal administration of O157-specific bacteriophages for mitigating the fecal shedding of Escherichia coli O157 by experimentally inoculated steers. Fecal shedding of nalidixic acid-resistant (Nal(R)) E. coli O157:H7 was monitored over 83 days after oral (ORL; 3.3 x 10(11) PFU), rectal (REC; 1.5 x 10(11) PFU), both oral and rectal (O+R; 4.8 x 10(11) PFU), or no (CON; control) treatment with a four-strain O157-specific bacteriophage cocktail in multiple doses. Bacteriophages were enumerated by plaque assay, and NalR E. coli O157:H7 by direct plating on sorbitol MacConkey agar supplemented with cefixime, potassium tellurite, and nalidixic acid. Orally treated steers produced the fewest Nal(R) E. coli O157:H7 culture-positive samples (P < 0.06) compared with REC and O+R steers, but this number was only nominally lower (P = 0.26) than that for the CON steers. The overall mean shedding level (log CFU per gram of feces) was higher for REC steers (P < 0.10) than for steers of the other treatment groups. Despite the shedding of higher mean bacteriophage levels (log PFU per gram of feces) by ORL and O+R than by CON and REC steers, there was no difference (P > 0.05) in the number of E. coli O157-positive samples among treatments. Bacteriophage was isolated from CON steers, indicating that these steers acquired the bacteriophage from the environment and shed the phage at a level similar to that of REC steers (P = 0.39). Continuous bacteriophage therapy may be an efficacious method for mitigating shedding of E. coli O157:H7 in cattle, providing that the host bacterium does not develop resistance. This therapy may be especially advantageous if nontreated cattle can acquire this biocontrol agent from the feedlot environment.

Reaction of Surrogate Escherichia coli Serotype O157:H7 and Non-O157 Strains to Nutrient Starvation: Variation in Phenotype and Transcription of Stress Response Genes and Behavior on Lettuce Plants in the Field.

Preharvest contamination with bacteria borne by irrigation water may result in leafy vegetables serving as vehicles for transmission of Shiga toxin-producing Escherichia coli (STEC) to humans. The influence of starvation-associated stress on the behavior of non-toxin-producing strains of E. coli serotype O157:H7 and serotypes O26, O103, O111, and O145 was examined subsequent to their introduction to the phyllosphere of field-grown romaine lettuce as inocula simulating starved (96 h in sterile deionized water) and nutrient-depleted (24 h broth culture) cells. As with E. coli O157:H7, leaf populations of the non-O157 strains declined rapidly during the first 72 h postinoculation, displaying the biphasic decay curve typical of serotype O157:H7 isolates. Preinoculation treatment appeared not to influence decay rates greatly (P > 0.5), but strain-specific differences (persistence period and attachment proficiency) indicated that serotype O103:H2 strain PARC445 was a better survivor. Also assessed was the impact of preinoculation treatment on phenotypes key to leaf colonization and survival and the expression of starvation stress-associated genes. The 96-h starvation period enhanced biofilm formation in one strain but reduced motility and autoinducer 2 formation in all five study strains relative to those characteristics in stationary-phase cells. Transcription of rpoS, dps, uspA, and gapA was reduced significantly (P < 0.05) in starvation-stressed cells relative to that for exponential- and stationary-phase cultures. Strain-specific differences were observed; serotype O103:H2 PARC445 had greater downturns than did serotype O157:H7 and other non-O157 strains. Within this particular cohort, the behavior of the representative serotype O157:H7 strain, PARC443 (ATCC 700728), was not predictive of behavior of non-O157 members of this STEC group.

Behavior of Escherichia coli O157:H7 in leafy vegetables.

Leafy vegetables, including lettuce and spinach, have been implicated in several outbreaks of foodborne disease caused by Escherichia coli O157:H7, a pathogen of increasing public health significance because of the severity of the gastrointestinal illness and long-term, chronic sequelae that can result from infection. A definitive association between the consumption of leafy vegetables and human disease provides implicit evidence of transfer from animal sources to field crops and retail commodities, including minimally processed or fresh-cut products. Understanding the behavior of E. coli O157:H7 in leafy vegetables during production, after harvest, in storage, during processing, and in packaged fresh-cut products is essential for the development of effective control measures. To this end, previous research on the fate of the species at each step in the production of market-ready leafy vegetables is reviewed in this study. Several critical gaps in knowledge are identified, notably uncertainty about the location of contaminating cells on or in plant tissues, behavior in packaged products stored at low temperatures, and the influence of environmental stresses on growth and infectivity.

Antibiotic resistance and virulence genes in commensal Escherichia coli and Salmonella isolates from commercial broiler chicken farms.

Antibiotic resistance patterns and the presence of antibiotic and virulence determinants in 74 sorbitol-negative Escherichia coli and 62 Salmonella isolates from nine different broiler chicken farms were investigated. Each farm was supplied by one of three companies that used different antimicrobial agents in feed for growth promotion. The isolates were identified by API 20E for E. coli and by serological tests for Salmonella. The susceptibility of the isolates to antibiotics was determined by Sensititre using the Clinical and Laboratory Standards Institute's breakpoints. Fifty-two E. coli isolates (70.3%) and nine Salmonella isolates (14.52%) were multiresistant to at least nine antibiotics. The multiresistant isolates were evaluated for the presence of tetracycline resistance, integron class 1, and blacMY 2 genes by PCR. Of the 74 E. coli isolates, 55 were resistant to amoxicillin and ceftiofur. Among these 55 resistant E. coli isolates, 45 (81.8%) and 22 (40.0%) were positive for blacMY-2 and qacEdeltal-Sull genes, respectively. Tetracycline resistance was found in 56 isolates (75.8%) among which 12 (21.4%) and 24 (42.9%) gave positive results for tetA and tetB, respectively. Virulence genes (iss, tsh, and traT), aerobactin operon (iucC), and the eaeA gene were detected in some E. coli strains. Among the 27 amoxicillin- and ceftiofur-resistant Salmonella isolates, the blacMY-2 gene was detected in 22 isolates. The class 1 integron gene (qacEdeltal-Sull) was not detected in any Salmonella isolates, whereas the invasin (inv) and virulence (spy) genes were found in 61 (98.4%) and 26 (42%) of the Salmonella isolates, respectively. This study indicated that multiple antibiotic-resistant commensal E. coli and Salmonella strains carrying virulence genes can be found on commercial broiler chicken farms and may provide a reservoir for these genes in chicken production facilities. Except for the presence of tetB, there was no significant effect of feed formulations on the phenotypic or genotypic characteristics of the isolates.

Changes in Gene Transcription Induced by Hydrogen Peroxide Treatment of Verotoxin-Producing Escherichia coli O157:H7 and Non-O157 Serotypes on Romaine Lettuce.

Disease outbreaks of verotoxin-producing Escherichia coli (VTEC) O157:H7 and non-O157 serotypes associated with leafy green vegetables are becoming a growing concern. A better understanding of the behavior of VTEC, particularly non-O157 serotypes, on lettuce under stress conditions is necessary for designing more effective control strategies. Hydrogen peroxide (H2O2) can be used as a sanitizer to reduce the microbial load in leafy green vegetables, particularly in fresh produce destined for the organic market. In this study, we tested the hypothesis that H2O2 treatment of contaminated lettuce affects in the same manner transcription of stress-associated and virulence genes in VTEC strains representing O157 and non-O157 serotypes. Six VTEC isolates representing serotypes O26:H11, O103:H2, O104:H4, O111:NM, O145:NM, and O157:H7 were included in this study. The results indicate that 50 mM H2O2 caused a population reduction of 2.4-2.8 log10 (compared to non-treated control samples) in all six VTEC strains present on romaine lettuce. Following the treatment, the transcription of genes related to oxidative stress (oxyR and sodA), general stress (uspA and rpoS), starvation (phoA), acid stress (gadA, gadB, and gadW), and virulence (stx1A, stx2A, and fliC) were dramatically downregulated in all six VTEC serotypes (P ≤ 0.05) compared to not treated control samples. Therefore, VTEC O157:H7 and non-O157 serotypes on lettuce showed similar survival rates and gene transcription profiles in response to 50 mM H2O2 treatment. Thus, the results derived from this study provide a basic understanding of the influence of H2O2 treatment on the survival and virulence of VTEC O157:H7 and non-O157 serotypes on lettuce.

Effect of severe weather events on the shedding of Shiga toxigenic Escherichia coli in slaughter cattle and phenotype of serogroup O157 isolates.

High-event periods (HEPs) occur sporadically when beef carcasses and meat have episodes of acute contamination with Shiga toxin-producing Escherichia coli (STEC). In this study, severe weather events were investigated as catalysts for HEPs based on PCR and isolate prevalence of seven E. coli serogroups in slaughter cattle feces. Winter ambient temperatures with daily means 10.5oC warmer or 12.3°C colder than seasonal norms (-10.4°C) most altered STEC shedding. Fecal samples yielded increased proportions (P < 0.05) of O26 and O157 isolates during winter warm periods, and reduced (P < 0.05) O45 isolates during cold periods compared to samplings during seasonal norms. Based on changing PCR prevalence and isolates collected, O157 was the serogroup most responsive to severe weather events. Consequently, O157 isolates (n = 219) were evaluated for heat resistance, biofilm-forming potential and virulence gene subtypes. Two isolates had heat-resistant phenotypes with thermal death time at 60°C (D60) > 10 min and one also had strong biofilm-forming potential. However, this isolate lacked eae and stx genes. Severe weather can influence STEC shedding, particularly of O157, and could possibly trigger HEPs. However, our data suggest that it is unlikely for isolates to carry virulence genes and possess phenotypes capable of evading post-harvest microbiological interventions.

Destruction of Escherichia coli O157:H7 by vanillic acid in unpasteurized juice from six apple cultivars.

The behavior of Escherichia coli O157:H7 in Granny Smith, Gala, Empire, McIntosh, Red Delicious, and Golden Delicious apple juice with or without supplementation with 5 or 10 mM vanillic acid was examined over a storage period of 7 days at 4 and 15 degrees C. The consequences of supplementation on sensory difference and preference were also determined by triangle testing. Juices made from the six apple cultivars had pH values ranging between pH 3.13 and 3.92. Vanillic acid exerted a concentration, pH, and time-dependent lethal effect toward E. coli O157:H7 in unpasteurized apple juice. Supplementation with 10 mM vanillic acid led to a 5-logarithm reduction in populations after 7 days at both temperatures, but sensory analysis revealed significant differences from and preference for unsupplemented juices. Supplementation with 5 mM vanillic acid accelerated death of E. coli O157:H7, but population reductions ranged from 5 log CFU/ml in low pH juices to none in high pH juices, particularly at 4 degrees C. No sensory difference or preference was detected in two of the six juices at this level of supplementation.

Phenotypic and Genotypic Characteristics of Shiga Toxin-Producing Escherichia coli Isolated from Surface Waters and Sediments in a Canadian Urban-Agricultural Landscape.

A hydrophobic grid membrane filtration-Shiga toxin immunoblot method was used to examine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in four watersheds located in the Lower Mainland of British Columbia, Canada, a region characterized by rapid urbanization and intensive agricultural activity. STEC were recovered from 21.6, 23.2, 19.5, and 9.2% of surface water samples collected monthly from five sites in each watershed over a period of 1 year. Overall prevalence was subject to seasonal variation however, ranging between 13.3% during fall months and 34.3% during winter months. STEC were also recovered from 23.8% of sediment samples collected in one randomly selected site. One hundred distinct STEC isolates distributed among 29 definitive and 4 ambiguous or indeterminate serotypes were recovered from water and sediments, including isolates from Canadian "priority" serogroups O157 (3), O26 (4), O103 (5), and O111 (7). Forty seven isolates were further characterized by analysis of whole genome sequences to detect Shiga toxin gene (stx 1 and stx 2), intimin gene (eaeA) allelic variants and acquired virulence factors. These analyses collectively showed that surface waters from the region support highly diverse STEC populations that include strains with virulence factors commonly associated with human pathotypes. The present work served to characterize the microbiological hazard implied by STEC to support future assessments of risks to public health arising from non-agricultural and agricultural uses of surface water resources in the region.

Survival of Escherichia coli O157:H7 in feces from corn- and barley-fed steers.

The survival of Escherichia coli O157:H7 in feces from steers fed corn (CO) or barley (BA) was evaluated at -10, +4 and +22 degrees C. Fecal pats were inoculated with a four-strain mixture of nalidixic-acid resistant E. coli O157:H7 at two levels: 10(3) CFU g(-1) (low, L) and 105 CFU g(-1) (high, H). At -10 degrees C, duration of survival of E. coli O157:H7 was reduced (p < 0.05) in CO-L (35 days) compared to BA-L (49 days), likely due to the effects of fecal volatile fatty acids in combination with a fecal pH of <6.5. At 4 degrees C, E. coli O157:H7 was detected in BA-H, CO-H, CO-L and BA-L for 77, 77, 56 and 63 days, respectively, with no difference (p > 0.05) observed in the duration of survival or rate of decline of E. coli O157:H7 among treatments. Survival of E. coli O157:H7 was twice as likely (p < 0.05) at 22 degrees C than at 4 degrees C and -10 degrees C. While pH and dry matter content increased, and volatile fatty acid concentrations decreased over 84 days at all three temperatures, these changes were most pronounced at 22 degrees C. Survival of E. coli O157:H7 for extended periods of time in feces from both corn- and barley-fed animals was demonstrated, thus fecal material may serve as a vector for the transmission of the organism. The greater survival of E. coli O157:H7 at 22 degrees C suggests that temperature may play a role in the seasonality of transmission and prevalence of this bacterium in feedlot cattle. The reported greater prevalence of E. coli O157:H7 in cattle fed barley as compared to those fed corn does not appear to be related to elevated risk of transmission arising from differential survival of the bacterium in feces.

Fate of Salmonella enterica in a mixed ingredient salad containing lettuce, cheddar cheese, and cooked chicken meat.

Food service and retail sectors offer consumers a variety of mixed ingredient salads that contain fresh-cut vegetables and other ingredients such as fruits, nuts, cereals, dairy products, cooked seafood, cooked meat, cured meats, or dairy products obtained from external suppliers. Little is known about the behavior of enteric bacterial pathogens in mixed ingredient salads. A model system was developed to examine the fate of Salmonella enterica (inoculum consisting of S. enterica serovars Agona, Typhimurium, Enteritidis, Brandenberg, and Kentucky) on the surface of romaine lettuce tissues incubated alone and in direct contact with Cheddar cheese or cooked chicken. S. enterica survived but did not grow on lettuce tissues incubated alone or in contact with Cheddar cheese for 6 days at either 6 or 14°C. In contrast, populations increased from 2.01 ± 0.22 to 9.26 ± 0.22 CFU/cm(2) when lettuce washed in water was incubated in contact with cooked chicken at 14°C. Populations on lettuce leaves were reduced to 1.28 ± 0.14 CFU/cm(2) by washing with a chlorine solution (70 ppm of free chlorine) but increased to 8.45 ± 0.22 CFU/cm(2) after 6 days at 14°C. Experimentation with a commercial product in which one third of the fresh-cut romaine lettuce was replaced with inoculated lettuce revealed that S. enterica populations increased by 4 log CFU/g during storage for 3 days at 14°C. These findings indicate that rapid growth of bacterial enteric pathogens may occur in mixed ingredient salads; therefore, strict temperature control during the manufacture, distribution, handling, and storage of these products is critical.