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A survey of cold-blooded vertebrates and associated surface waters in a produce-growing region on the Central California Coast was done between May and September 2011 to determine the diversity of Salmonella. Samples from 460 amphibians and reptiles and 119 water samples were collected and cultured for Salmonella. Animals sampled were frogs (n=331), lizards (n=59), newts (n=5), salamanders (n=6), snakes (n=39), and toads (n=20). Salmonella was isolated from 37 individual animals, including frogs, lizards, snakes, and toads. Snakes were the most likely to contain Salmonella, with 59% testing positive followed by 15.3% of lizards, 5% of toads, and 1.2% of frogs. Fifteen water samples (12.6%) were positive. Twenty-two different serovars were identified, and the majority of isolates were S. enterica subsp. IIIb, with subsp. I, II, and IIIa also found. The serovar isolated most frequently was S. enterica subsp. IIIb 16:z₁₀:e,n,x,z₁₅, from snakes and frogs in five different locations. S. enterica subsp. I serovar Typhimurium and the monophasic I 6,8:d:- were isolated from water, and subspecies I Duisburg and its variants were found in animals and water. Some samples contained more than one type of Salmonella. Analysis of pulsed-field gel electrophoresis pulsotypes indicated that some strains persisted in animals and water collected from the same location. Sixty-six isolates displayed antibiotic resistance, with 27 isolates resistant to more than one antibiotic, including a subspecies IIIb isolate from snake having resistance to five different antibiotics. Twenty-three isolates were resistant to more than one class of antibiotic, and six isolates were resistant to three classes. While these subspecies of IIIa and IIIb cause fewer instances of human illness, they may serve as reservoirs of antibiotic resistance, determinants in the environment, and be sources of contamination of leafy greens associated with product recalls.

Captive amphibians and reptiles are well-documented sources of human salmonellosis through direct contact with contaminated feces or fomites. However, the relative significance of wild cold-blooded vertebrates as hosts/reservoirs of zoonotic enteric pathogens in their natural habitat is not known. Wild amphibian and reptile populations are present in the leafy green produce production environment in the central California coast, and there are reports from growers that frogs and other species sometimes intrude into produce fields. These intrusions may result in destruction of the crop and economic losses, due to food safety and quality concerns. Environmental groups have also noted potential conflicts between conservation efforts and food safety practices that result in removal or damage to aquatic habitat (e.g., farm ponds, adjacent wetland areas). To address these concerns, we conducted a survey of foodborne pathogen prevalence in common amphibian and reptile populations during the 2011 produce growing season in the central California coast. Preliminary results indicate that Salmonella prevalence was higher among wild-caught reptile (33%) compared with amphibian (4%) taxa. In contrast, all samples were negative for E. coli O157. Wildlife damage management in fresh produce production fields is challenging, due to the diversity of potential hosts/reservoirs of foodborne pathogens and a limited number of mitigation strategies. Professionals working in vertebrate pest control could play an important role in assisting fresh produce growers with implementation of co-management approaches that promote public health and conservation goals in the central coast agricultural landscape.

The surveillance of foods for Salmonella is hindered by bias in common enrichment media where serovars implicated in human illness are outgrown by less virulent serovars. We examined four Salmonella serovars, two common in human illness (Enteritidis and Typhimurium) and two that often dominate enrichments (Give and Kentucky), for factors that might influence culture bias. The four serovars had similar growth kinetics in Tryptic Soy Broth and Buffered Peptone Water. Phenotype microarray analysis with 950 chemical substrates to assess nutrient utilization and stress resistance revealed phenotype differences between serovars. Strains of S. Enteritidis had better utilization of plant-derived sugars such as xylose, mannitol, rhamnose, and fructose, while S. Typhimurium strains were able to metabolize tagatose. Strains of S. Kentucky used more compounds as phosphorus sources and grew better with inorganic phosphate as the sole phosphorus source. The sequences of nine genes involved in phosphate metabolism were compared, and there were differences between serovars in the catalytic ATP-binding domain of the histidine kinase phoR. Analysis of the predicted PhoR amino acid sequences from additional Salmonella genomes indicated a conservation of sequences each within the Typhimurium, Give, and Enteritidis serovars. However, three different PhoR versions were observed in S. Kentucky. [ABSTRACT FROM AUTHOR]

Prevalence and serovar diversity of Salmonella enterica were measured during a 5-year survey of surface waters in a 500-mi2 agricultural region of the Central California Coast. Rivers, streams, lakes, and ponds were sampled bimonthly resulting in 2,979 samples. Overall prevalence was 56.4% with higher levels detected in spring than in fall. Small, but significant, differences in prevalence were detected based on sample locations. Detection of Salmonella was correlated positively with both significant rain events and, in some environments, levels of generic Escherichia coli. Analysis of 1,936 isolates revealed significant serovar diversity, with 91 different serovars detected. The most common isolated serovars were S. enterica subsp. enterica serovars I 6,8:d:- (406 isolates, 21.0%, and potentially monophasic Salmonella Muenchen), Give (334 isolates, 17.3%), Muenchen (158 isolates, 8.2%), Typhimurium (227 isolates, 11.7%), Oranienburg (106 isolates, 5.5%), and Montevideo (78 isolates, 4%). Sixteen of the 24 most common serovars detected in the region are among the serovars reported to cause the most human salmonellosis in the United States. Some of the serovars were associated with location and seasonal bias. Analysis of XbaI pulsed field gel electrophoresis (PFGE) patterns of strains of serovars Typhimurium, Oranienburg, and Montevideo showed significant intraserovar diversity. PFGE pulsotypes were identified in the region for multiple years of the survey, indicating persistence or regular reintroduction to the region. IMPORTANCE Nontyphoidal Salmonella is among the leading causes of bacterial foodborne illness, and increasing numbers of outbreaks and recalls are due to contaminated produce. High prevalence and 91 different serovars were detected in this leafy green growing region. Seventeen serovars that cause most of the human salmonellosis in the United States were detected, with 16 of those serovars detected in multiple locations and multiple years of the 5-year survey. Understanding the widespread prevalence and diversity of Salmonella in the region will assist in promoting food safety practices and intervention methods for growers and regulators. [ABSTRACT FROM AUTHOR]

Isolation of Salmonella from enrichment cultures of food or environmental samples is a complicated process. Numerous factors including fitness in various selective enrichment media, relative starting concentrations in pre-enrichment, and competition among multi-serovar populations and associated natural microflora, come together to determine which serovars are identified from a given sample. A recently developed approach for assessing the relative abundance (RA) of multi-serovar Salmonella populations (CRISPR-SeroSeq or Deep Serotyping, DST) is providing new insight into how these factors impact the serovars observed, especially when different selective enrichment methods are used to identify Salmonella from a primary enrichment sample. To illustrate this, we examined Salmonella -positive poultry pre-enrichment samples through the selective enrichment process in Tetrathionate (TT) and Rappaport Vassiliadis (RVS) broths and assessed recovery of serovars with each medium. We observed the RA of serovars detected post selective enrichment varied depending on the medium used, initial concentration, and competitive fitness factors, all which could result in minority serovars in pre-enrichment becoming dominant serovars post selective enrichment. The data presented provide a greater understanding of culture biases and lays the groundwork for investigations into robust enrichment and plating media combinations for detecting Salmonella serovars of greater concern for human health. • Different Salmonella serovars are enriched from selective enrichments in RVS and TT. • Choice of plating media influences serovars recovered from selective enrichments. • Experiments revealed starting serovar relative concentrations influence isolation. • Robust methods are vital for reliably isolating Salmonella serovars of concern. [ABSTRACT FROM AUTHOR]

Produce contaminated with enteric pathogens is a major source of foodborne illness in the United States. Lakes, streams, rivers, and ponds were sampled with Moore swabs bi-monthly for over 2 years at 30 locations in the vicinity of a leafy green growing region on the Central California Coast and screened for Shiga toxin producing Escherichia coli (STEC), Salmonella enterica, and Listeria monocytogenes to evaluate the prevalence and persistence of pathogen subtypes. The prevalence of STEC from 1386 samples was 11%; 110 samples (8%) contained E. coli O157:H7 with the highest prevalence occurring close to cattle operations. Non-O157 STEC isolates represented major clinical O-types and 57% contained both shiga toxin types 1 and 2 and intimin. Multiple Locus Variable Number Tandem Repeat Analysis of STEC isolates indicated prevalent strains during the period of study. Notably, Salmonella was present at high levels throughout the sampling region with 65% prevalence in 1405 samples resulting in 996 isolates with slightly lower prevalence in late autumn. There were 2, 8, and 14 sites that were Salmonella-positive over 90, 80, and 70% of the time, respectively. The serotypes identified most often were 6,8:d:-, Typhimurium, and Give. Interestingly, analysis by Pulsed Field Gel Electrophoresis indicated persistence and transport of pulsotypes in the region over several years. In this original study of L. monocytogenes in the region prevalence was 43% of 1405 samples resulting in 635 individual isolates. Over 85% of the isolates belonged to serotype 4b with serotypes 1/2a, 1/2b, 3a, 4d with 4e representing the rest, and there were 12 and 2 sites that were positive over 50 and 80% of the time, respectively. Although surface water is not directly used for irrigation in this region, transport to the produce can occur by other means. This environmental survey assesses initial contamination levels toward an understanding of transport leading to produce recalls or outbreaks. [ABSTRACT FROM AUTHOR]

For foodborne outbreak investigations it can be difficult to isolate the relevant strain from food and/or environmental sources. If the sample is contaminated by more than one strain of the pathogen the relevant strain might be missed. In this study mixed cultures of Salmonella enterica were grown in one set of standard enrichment media to see if culture bias patterns emerged. Nineteen strains representing four serogroups and ten serotypes were compared in four-strain mixtures in Salmonella-only and in cattle fecal culture enrichment backgrounds using Salmonella enrichment media. One or more strain(s) emerged as dominant in each mixture. No serotype was most fit, but strains of serogroups C2 and E were more likely to dominate enrichment culture mixtures than strains of serogroups B or C1. Different versions of Rappaport- Vassiliadis (RV) medium gave different patterns of strain dominance in both Salmonella-only and fecal enrichment culture backgrounds. The fittest strains belonged to serogroups C1, C2, and E, and included strains of S. Infantis, S. Thompson S. Newport, S. 6,8:d:-, and S. Give. Strains of serogroup B, which included serotypes often seen in outbreaks such as S. Typhimurium, S. Saintpaul, and S. Schwarzengrund were less likely to emerge as dominant strains in the mixtures when using standard RV as part of the enrichment. Using a more nutrient-rich version of RV as part of the protocol led to a different pattern of strains emerging, however some were still present in very low numbers in the resulting population. These results indicate that outbreak investigations of food and/or other environmental samples should include multiple enrichment protocols to ensure isolation of target strains of Salmonella. [ABSTRACT FROM AUTHOR]

Three enrichment broths commonly used for detection of Salmonella (buffered peptone water [BPW], tryptic soy broth [TSB]. and universal preenrichment broth [UPB]) were compared for use in real-time SYBR Green PCR detection of Salmonella introduced into enrichment cultures made from store-bought lettuce and tomatoes. The produce served as a source of normal plant microbiota to measure how well DNA-based detection methods for Salmonella work in a suspension of plant-associated bacteria that may be closely related to Salmonella. A qualitative assessment of the background microbiota that grew in the three enrichment broths cultures from tomato and lettuce samples revealed that different bacteria predominated in the different broths. Results obtained with live produce-related outbreak Salmonella strains and PCR primers directed toward three different Salmonella genes suggest that the ability to detect Salmonella from these enrichment cultures by real-time PCR was 10 to 1,000 times better with TSB enrichment cultures. Detection levels were similar between the different enrichment media when an immunomagnetic separation method was used; however, the immunological technique did not enhance detection from TSB enrichment cultures. Detection could be affected by the medium and by the background microbiota. An immunomagnetic separation method may be useful in BPW and UPB enrichment cultures but not in TSB enrichment cultures. [ABSTRACT FROM AUTHOR]

Numerous Salmonella enterica food-borne illness outbreaks have been associated with contaminated vegetables, in particular sprouted seeds, and the incidence of reported contamination has steadily risen. In order to understand the physiology of S. enterica serovar Newport on plants, a screen was developed to identity transposon mutants that were defective in attachment to alfalfa sprouts. Twenty independent mutants from a pool of 6,000 were selected for reduced adherence to alfalfa sprouts. Sixty-five percentage of these mutants had insertions in uncharacterized genes. Among the characterized genes were strains with insertions in the intergenic region between agfB, the surface-exposed aggregative fimbria (curli) nucleator, and agfD, a transcriptional regulator of the LuxR superfamily, and rpoS, the stationary-phase sigma factor. Both AgfD and RpoS have been reported to regulate curli and cellulose production and RpoS regulates other adhesins such as pili. The intergenic and rpoS mutants were reduced in initial attachment to alfalfa sprouts by 1 log unit compared to the wild type. Mutations of agfA, curli subunit, and agfB in S. enterica serovar Enteritidis differentially affected attachment to plant tissue. The agfA mutation was not reduced in ability to attach to or colonize alfalfa sprouts, whereas the agfB mutation was reduced. Thus, agfB alone can play a role in attachment of S. enterica to plant tissue. These results reveal that S. enterica genes important for virulence in animal systems are also required for colonization of plants, a secondary host that can serve as a vector of S. enterica from animal to animal. [ABSTRACT FROM AUTHOR]

A correction to the article "Prevalence of shiga toxin producing Escherichia coli, Salmonella enterica and Listeria monocytogenes at public access watershed sites in a California Central Coast agricultural region" that was published in 2014 issue is presented.

Abstract: Irrigation with water of poor microbiological quality can elevate levels of bacteria on produce. This study aimed to identify climate and management variables associated with generic Escherichia coli in irrigation water on leafy green produce farms and to measure the prevalence of E. coli O157 and Salmonella spp. in irrigation and non-irrigation water sources on these farms. Water and sediment samples collected from various points along irrigation systems, as well as from streams and ponds on farms on the Central California coast between May 27th, 2008 and October 26th, 2010 were cultured for generic E. coli (MPN/100mL or cfu 100g) (n=436), E. coli O157 (n=437), and (n=163) Salmonella. Variables were based on grower's management practices, landscape features in proximity to samples (e.g., distance to roads and ranches/livestock), and climate data accessed from an online database. Negative binomial regression models were constructed to test associations between generic E. coli (MPN/100mL) in water from farms and variables. Arithmetic mean concentration of E. coli for water, not including those from Moore swabs, and sediment samples, was 7.1×102 MPN/100mL and 1.0×104 cfu/100g, respectively. Matched by collection day, E. coli concentration in sediment (cfu/100g) was typically 10- to 1000-fold higher than the overlying water (MPN/100mL) for these irrigation systems. Generic E. coli concentration (MPN/100mL) increased by 60.1% for each 1m/s increase in wind speed and decreased by 3% for each 10m increase in the distance between the sample location and rangeland. Moore swabs detected a higher proportion of E. coli O157 (13.8%) positive water samples compared to grab samples (1.8%); 1.7% of sediment samples had detectable levels of this pathogen. Interestingly, season was not significantly associated with E. coli O157 presence in water or sediments from produce farms or water sources with public access. Salmonella was detected in 6% (6/96) water and 4.3% (3/67) sediment samples. Generic E. coli concentration was not significantly associated with the presence of either E. coli O157 or Salmonella in water or sediment samples, suggesting that, for this 2.5-year period and geographical location, generic E. coli would likely be an unreliable indicator bacteria for predicting the presence of these food- and waterborne pathogens in a key produce production environment. [Copyright &y& Elsevier]