Persistence of a Salmonella enterica serovar typhimurium DT12 clone in a piggery and in agricultural soil amended with Salmonella-contaminated slurry

Prevalence of Salmonella enterica on a Danish pig farm presenting recurrent infections was investigated. A comparison of the pulsed-field gel electrophoresis patterns of fecal isolates from piggeries, waste slurry, and agricultural soil amended with Salmonella-contaminated animal waste (slurry) and subclinical isolates from the same farm (collected in 1996 and later) showed identical patterns, indicating long-term persistence of the Salmonella enterica serovar Typhimurium DT12 clone in the herd environment. Furthermore, when Salmonella-contaminated slurry was disposed of on the agricultural soil (a common waste disposal practice), the pathogen was isolated up to 14 days after the spread, indicating potentially high risks of transmission of the pathogen in the environment, animals, and humans.     

Longitudinal Study of Distributions of Similar Antimicrobial-Resistant Salmonella Serovars in Pigs and Their Environment in Two Distinct Swine Production Systems

The aim of this longitudinal study was to determine and compare the prevalences and genotypic profiles of antimicrobial-resistant (AR) Salmonella isolates from pigs reared in antimicrobial-free (ABF) and conventional production systems at farm, at slaughter, and in their environment. We collected 2,889 pig fecal and 2,122 environmental (feed, water, soil, lagoon, truck, and floor swabs) samples from 10 conventional and eight ABF longitudinal cohorts at different stages of production (farrowing, nursery, finishing) and slaughter (postevisceration, postchill, and mesenteric lymph nodes [MLN]). In addition, we collected 1,363 carcass swabs and 205 lairage and truck samples at slaughter. A total of 1,090 Salmonella isolates were recovered from the samples; these were isolated with a significantly higher prevalence in conventionally reared pigs (4.0%; n = 66) and their environment (11.7%; n = 156) than in ABF pigs (0.2%; n = 2) and their environment (0.6%; n = 5) (P < 0.001). Salmonella was isolated from all stages at slaughter, including the postchill step, in the two production systems. Salmonella prevalence was significantly higher in MLN extracted from conventional carcasses than those extracted from ABF carcasses (P < 0.001). We identified a total of 24 different serotypes, with Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Anatum, Salmonella enterica serovar Infantis, and Salmonella enterica serovar Derby being predominant. The highest frequencies of antimicrobial resistance (AR) were exhibited to tetracycline (71%), sulfisoxazole (42%), and streptomycin (17%). Multidrug resistance (resistance to ≥3 antimicrobials; MDR) was detected in 27% (n = 254) of the Salmonella isolates from the conventional system. Our study reports a low prevalence of Salmonella in both production systems in pigs on farms, while a higher prevalence was detected among the carcasses at slaughter. The dynamics of Salmonella prevalence in pigs and carcasses were reciprocated in the farm and slaughter environment, clearly indicating an exchange of this pathogen between the pigs and their surroundings. Furthermore, the phenotypic and genotypic fingerprint profile results underscore the potential role played by environmental factors in dissemination of AR Salmonella to pigs.     

Survival of Salmonella enterica Serovar Newport in Manure and Manure-Amended Soils

Salmonella enterica serovar Newport has undergone a rapid epidemic spread in dairy cattle. This provides an efficient mechanism for pathogen amplification and dissemination into the environment through manure spreading on agricultural land. The objective of this study was to determine the survival characteristics of Salmonella serovar Newport in manure and manure-amended soils where the pathogen may be amplified. A multidrug-resistant (MDR) Salmonella serovar Newport strain and a drug-susceptible (DS) strain, both bovine isolates, were inoculated into dairy manure that was incubated under constant temperature and moisture conditions alone or after being mixed with sterilized or nonsterilized soil. Salmonella serovar Newport concentrations increased by up to 400% in the first 1 to 3 days following inoculation, and a trend of steady decline followed. With manure treatment, a sharp decline in cell concentration occurred after day 35, possibly due to microbial antagonism. For all treatments, decreases in Salmonella serovar Newport concentrations over time fit a first-order kinetic model. Log reduction time was 14 to 32 days for 1 log₁₀, 28 to 64 days for 2 log₁₀, and 42 to 96 days for 3 log₁₀ declines in the organisms' populations from initially inoculated concentrations. Most-probable-number monitoring data indicated that the organisms persisted for 184, 332, and 405 days in manure, manure-amended nonsterilized soil, and manure-amended sterilized soil, respectively. The MDR strain and the DS strain had similar survival patterns.     

Fecal Shedding of Zoonotic Food-Borne Pathogens by Wild Rodents in a Major Agricultural Region of the Central California Coast

Recent outbreaks of food-borne illness associated with the consumption of produce have increased concern over wildlife reservoirs of food-borne pathogens. Wild rodents are ubiquitous, and those living close to agricultural farms may pose a food safety risk should they shed zoonotic microorganisms in their feces near or on agricultural commodities. Fecal samples from wild rodents trapped on 13 agricultural farms (9 produce, 3 cow-calf operations, and 1 beef cattle feedlot) in Monterey and San Benito Counties, CA, were screened to determine the prevalence and risk factors for shedding of several food-borne pathogens. Deer mice (Peromyscus maniculatus) were the most abundant rodent species trapped (72.5%). Cryptosporidium species (26.0%) and Giardia species (24.2%) were the predominant isolates from rodent feces, followed by Salmonella enterica serovars (2.9%) and Escherichia coli O157:H7 (0.2%). Rodent trap success was significantly associated with detection of Salmonella in rodent feces, while farm type was associated with fecal shedding of Cryptosporidium and Giardia. Seasonal shedding patterns were evident, with rodents trapped during the spring and summer months being significantly less likely to be shedding Cryptosporidium oocysts than those trapped during autumn. Higher rodent species diversity tended to correlate with lower fecal microbial prevalence, and most spatiotemporal pathogen clusters involved deer mice. Rodents in the study area posed a minimal risk as environmental reservoirs of E. coli O157:H7, but they may play a role in environmental dissemination of Salmonella and protozoa. Rodent control efforts that potentially reduce biodiversity may increase pathogen shedding, possibly through promotion of intraspecific microbial transmission.     

Long-Term Survival of Pathogenic and Sanitation Indicator Bacteria in Experimental Biowaste Composts

For economic, agricultural, and environmental reasons, composting is frequently used for organic waste recycling. One approach to limiting the potential risk from bacterial food-borne illnesses is to ensure that soil amendments and organic fertilizers are disinfected. However, more knowledge concerning the microbiological safety of composted substrates other than sludge and manure is necessary. Experimental in-vessel biowaste composts were used to study the survival of seeded Listeria monocytogenes, Salmonella enterica subsp. enterica serotype Enteritidis, and Escherichia coli. Four organic waste mixtures, containing various proportions of paper and cardboard, fruits and vegetables, and green waste, were composted in laboratory reactors with forced aeration. The physicochemical and microbiological parameters were monitored for 12 weeks during composting. The survival of bacteria over a 3-month period at 25°C was assessed with samples collected after different experimental composting times. Strain survival was also monitored in mature sterilized composts. Nonsterile composts did not support pathogen growth, but survival of seeded pathogens was observed. Salmonella serovar Enteritidis survived in all composts, and longer survival (3 months) was observed in mature composts (8 and 12 weeks of composting). Mature biowaste composts may support long-term survival of Salmonella serovar Enteritidis during storage at room temperature. E. coli and L. monocytogenes survival was observed only in 4-week-old composts and never in older composts. Proper composting may prevent long-term survival of E. coli and L. monocytogenes. These results suggest that like composted sewage sludge or manure, domestic waste composts may support pathogen survival. Survival was not related to the physicochemical characteristics of the composts.     

Genome-Wide Methylation Patterns in Salmonella enterica Subsp. enterica Serovars

The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication. Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT) DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N⁶-methyladenine (m6A) methylated motifs, one N⁴-methylcytosine (m4C) motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases) associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems), indicating the high diversity of methylation patterns present in Salmonella.     

Characterization of Salmonella occurring at high prevalence in a population of the land iguana Conolophus subcristatus in Galápagos Islands, Ecuador

The aim of the study was to elucidate the association between the zoonotic pathogen Salmonella and a population of land iguana, Colonophus subcristatus, endemic to Galápagos Islands in Ecuador. We assessed the presence of Salmonella subspecies and serovars and estimated the prevalence of the pathogen in that population. Additionally, we investigated the genetic relatedness among isolates and serovars utilising pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA and determined the antimicrobial susceptibility to a panel of antimicrobials. The study was carried out by sampling cloacal swabs from animals (n = 63) in their natural environment on in the island of Santa Cruz. A high prevalence (62/63, 98.4%) was observed with heterogeneity of Salmonella subspecies and serovars, all known to be associated with reptiles and with reptile-associated salomonellosis in humans. Serotyping revealed 14 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 48), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 1), and S. enterica subsp. houtenae (n = 7). Four serovars were predominant: S. Poona (n = 18), S. Pomona (n = 10), S. Abaetetuba (n = 8), and S.Newport (n = 5). The S. Poona isolates revealed nine unique XbaI PFGE patterns, with 15 isolates showing a similarity of 70%. Nine S. Pomona isolates had a similarity of 84%. One main cluster with seven (88%) indistinguishable isolates of S. Abaetetuba was observed. All the Salmonella isolates were pan-susceptible to antimicrobials representative of the most relevant therapeutic classes. The high prevalence and absence of clinical signs suggest a natural interaction of the different Salmonella serovars with the host species. The interaction may have been established before any possible exposure of the iguanas and the biocenosis to direct or indirect environmental factors influenced by the use of antimicrobials in agriculture, in human medicine or in veterinary medicine.     

Survival and Transmission of Salmonella enterica Serovar Typhimurium in an Outdoor Organic Pig Farming Environment

It was investigated how organic rearing conditions influence the Salmonella enterica infection dynamics in pigs and whether Salmonella persists in the paddock environment. Pigs inoculated with S. enterica serovar Typhimurium were grouped with Salmonella-negative tracer pigs. Bacteriological and serological testing indicated that organic pigs were susceptible to Salmonella infections, as 26 of 46 (56%) tracer pigs turned culture positive. An intermittent and mainly low-level excretion of Salmonella (<100 cells g⁻¹) partly explains why the bacteriological prevalence appeared lower than the seroprevalence. Salmonella persisted in the paddock environment, as Salmonella was isolated from 46% of soil and water samples (n = 294). After removal of pigs, Salmonella was found in soil samples for up to 5 weeks and in shelter huts during the entire test period (7 weeks). Subsequent introduction of Salmonella-negative pigs into four naturally Salmonella-contaminated paddocks caused Salmonella infections of pigs in two paddocks. In one of these paddocks, all tracer pigs (n = 10) became infected, coinciding with a previous high Salmonella infection rate and high Salmonella excretion level. Our results showed that pigs reared under organic conditions were susceptible to Salmonella infections (just like conventional pigs) and that Salmonella persisting in the paddock environment could pose an infection risk. A driving force for these infections seemed to be pigs with a high Salmonella excretion level, which caused substantial contamination of the environment. This suggests that isolation of animals as soon as a Salmonella infection is indicated by clinical symptoms of diarrhea could be a means of reducing and controlling the spread and persistence of Salmonella in outdoor organic pig production environments.     

lac Repressor Is an Antivirulence Factor of Salmonella enterica: Its Role in the Evolution of Virulence in Salmonella

The genus Salmonella includes many pathogens of great medical and veterinary importance. Bacteria belonging to this genus are very closely related to those belonging to the genus Escherichia. lacZYA operon and lacI are present in Escherichia coli, but not in Salmonella enterica. It has been proposed that Salmonella has lost lacZYA operon and lacI during evolution. In this study, we have investigated the physiological and evolutionary significance of the absence of lacI in Salmonella enterica. Using murine model of typhoid fever, we show that the expression of LacI causes a remarkable reduction in the virulence of Salmonella enterica. LacI also suppresses the ability of Salmonella enterica to proliferate inside murine macrophages. Microarray analysis revealed that LacI interferes with the expression of virulence genes of Salmonella pathogenicity island 2. This effect was confirmed by RT-PCR and Western blot analysis. Interestingly, we found that SBG0326 of Salmonella bongori is homologous to lacI of Escherichia coli. Salmonella bongori is the only other species of the genus Salmonella and it lacks the virulence genes of Salmonella pathogenicity island 2. Overall, our results demonstrate that LacI is an antivirulence factor of Salmonella enterica and suggest that absence of lacI has facilitated the acquisition of virulence genes of Salmonella pathogenicity island 2 in Salmonella enterica making it a successful systemic pathogen.     

Uptake and Replication of Salmonella enterica in Acanthamoeba rhysodes

The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized by Acanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation of hilA, a key regulator in the expression of the invasion-associated Salmonella pathogenicity island 1. Microscopy of infected A. rhysodes revealed that S. enterica resided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated with hilA and the Salmonella virulence plasmid. The data show that Acanthamoeba spp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by defined Salmonella virulence loci.     

Comparison of Salmonella enterica Serovar Typhimurium LT2 and Non-LT2 Salmonella Genomic Sequences, and Genotyping of Salmonellae by Using PCR

Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.     

Diversity and Antimicrobial Resistance of Salmonella enterica Isolates from Surface Water in Southeastern United States

A study of prevalence, diversity, and antimicrobial resistance of Salmonella enterica in surface water in the southeastern United States was conducted. A new scheme was developed for recovery of Salmonella from irrigation pond water and compared with the FDA''s Bacteriological Analytical Manual (8th ed., 2014) (BAM) method. Fifty-one isolates were recovered from 10 irrigation ponds in produce farms over a 2-year period; nine Salmonella serovars were identified by pulsed-field gel electrophoresis analysis, and the major serovar was Salmonella enterica serovar Newport (S. Newport, n = 29), followed by S. enterica serovar Enteritidis (n = 6), S. enterica serovar Muenchen (n = 4), S. enterica serovar Javiana (n = 3), S. enterica serovar Thompson (n = 2), and other serovars. It is noteworthy that the PulseNet patterns of some of the isolates were identical to those of the strains that were associated with the S. Thompson outbreaks in 2010, 2012, and 2013, S. Enteritidis outbreaks in 2011 and 2013, and an S. Javiana outbreak in 2012. Antimicrobial susceptibility testing confirmed 16 S. Newport isolates of the multidrug resistant-AmpC (MDR-AmpC) phenotype, which exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT), and to the 1st, 2nd, and 3rd generations of cephalosporins (cephalothin, amoxicillin-clavulanic acid, and ceftriaxone). Moreover, the S. Newport MDR-AmpC isolates had a PFGE pattern indistinguishable from the patterns of the isolates from clinical settings. These findings suggest that the irrigation water may be a potential source of contamination of Salmonella in fresh produce. The new Salmonella isolation scheme significantly increased recovery efficiency from 21.2 (36/170) to 29.4% (50/170) (P = 0.0002) and streamlined the turnaround time from 5 to 9 days with the BAM method to 4 days and thus may facilitate microbiological analysis of environmental water.     

Distribution and Characterization of Salmonella enterica Isolates from Irrigation Ponds in the Southeastern United States

Irrigation water has been implicated as a likely source of produce contamination by Salmonella enterica. Therefore, the distribution of S. enterica was surveyed monthly in irrigation ponds (n = 10) located within a prime agricultural region in southern Georgia and northern Florida. All ponds and 28.2% of all samples (n = 635) were positive for Salmonella, with an overall geometric mean concentration (0.26 most probable number [MPN]/liter) that was relatively low compared to prior reports for rivers in this region. Salmonella peaks were seasonal; the levels correlated with increased temperature and rainfall (P < 0.05). The numbers and occurrence were significantly higher in water (0.32 MPN/liter and 37% of samples) than in sediment (0.22 MPN/liter and 17% of samples) but did not vary with depth. Representative isolates (n = 185) from different ponds, sample types, and seasons were examined for resistance to 15 different antibiotics; most strains were resistant to streptomycin (98.9%), while 20% were multidrug resistant (MDR) for 2 to 6 antibiotics. DiversiLab repetitive extragenic palindromic-element sequence-based PCR (rep-PCR) revealed genetic diversity and showed 43 genotypes among 191 isolates, as defined by >95% similarity. The genotypes did not partition by pond, season, or sample type. Genetic similarity to known serotypes indicated Hadar, Montevideo, and Newport as the most prevalent. All ponds achieved the current safety standards for generic Escherichia coli in agricultural water, and regression modeling showed that the E. coli level was a significant predictor for the probability of Salmonella occurrence. However, persistent populations of Salmonella were widely distributed in irrigation ponds, and the associated risks for produce contamination and subsequent human exposure are unknown, supporting continued surveillance of this pathogen in agricultural settings.     

Detection of a Salmonella enterica Serovar California Strain Spreading in Spanish Feed Mills and Genetic Characterization with DNA Microarrays

We performed an epidemiological study on Salmonella isolated from raw plant-based feed in Spanish mills. Overall, 32 different Salmonella serovars were detected. Despite its rare occurrence in humans and animals, Salmonella enterica serovar California was found to be the predominant serovar in Spanish feed mills. Different typing techniques showed that isolates of this serovar were genetically closely related, and comparative genomic hybridization using microarray technology revealed 23 S. enterica serovar Typhimurium LT2 gene clusters that are absent from serovar California.     

Prevalence of Salmonella spp. in Oysters in the United States

Food-borne diseases such as salmonellosis can be attributed, in part, to the consumption of raw oysters. To determine the prevalence of Salmonella spp. in oysters, oysters harvested from 36 U.S. bays (12 each from the West, East, and Gulf coasts in the summer of 2002, and 12 bays, four per coast, in the winter of 2002-2003) were tested. Salmonella was isolated from oysters from each coast of the United States, and 7.4% of all oysters tested contained Salmonella. Isolation tended to be bay specific, with some bays having a high prevalence of Salmonella, while other bays had none. Differences in the percentage of oysters from which Salmonella was isolated were observed between the summer and winter months, with winter numbers much lower probably due to a variety of weather-related events. The vast majority (78/101) of Salmonella isolates from oysters were Salmonella enterica serovar Newport, a major human pathogen, confirming the human health hazard of raw oyster consumption. Contrary to previous findings, no relationship was found between the isolation of fecal coliforms and Salmonella from oysters, indicating a necessity for specific monitoring for Salmonella and other pathogens rather than the current reliance on fecal coliform testing.     

Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates

The accurate sub-typing of Salmonella enterica isolates is essential for epidemiological investigations and surveillance of Salmonella infections. Salmonella isolates are currently identified using the Kauffman-White serotyping scheme. Multilocus sequence typing (MLST) schemes have been developed for the major bacterial pathogens including Salmonella and have assisted in understanding the molecular epidemiology and population biology of these organisms. Recently, the DiversiLab rep-PCR system has been developed using micro-fluidic chips to provide standardized, semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 serovars, were analyzed using MLST and the DiversiLab rep-PCR system. MLST was able to identify 31 sequence types (STs), while the DiversiLab system revealed 38 DiversiLab types (DTs). The rep-PCR distinguished isolates of different serovars and showed greater discriminatory power (0.95) than MLST typing (0.89). Rep-PCR exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST typing with serotyping was 96%, representing a strong association. Comparison of rep-PCR profiles with those held in an online library database led to the accurate prediction of serovar in 63% of cases and resulted in inaccurate predictions for 10% of profiles. MLST and the rep-PCR system may provide useful additional informative techniques for the molecular identification of S. enterica. We conclude that the DiversiLab rep-PCR system may provide a rapid (less than 4 h) and standardized method for sub-typing isolates of S. enterica.     

Geographical and Temporal Dissemination of Salmonellae Isolated from Domestic Animal Hosts in the Culiacan Valley,Mexico

The prevalence and diversity of salmonellae from domestic animal hosts were investigated in the Culiacan Valley, Mexico. A total of 240 farm animal feces (cows, chicken, and sheep) were evaluated for Salmonella spp. presence from July 2008 to June 2009. Salmonella enterica subsp. enterica strains were isolated from 76 samples (31.7%), and 20 serotypes were identified being Salmonella Oranienburg (25%), Salmonella Give (14%), Salmonella Saintpaul (12%), and Salmonella Minnesota (11%) the most frequent isolates. Twenty-four percent (18/76) of the isolates were resistant to ampicillin. Salmonella Oranienburg, Salmonella Minnesota, Salmonella Give, Salmonella Agona, Salmonella Weltevreden, and Salmonella Newport serotypes showed multiple pulsed-field electrophoresis patterns. Salmonella Oranienburg was the dominant serotype in the Culiacan Valley; however, no specific distribution patterns were detected in animal sources or sampling sites. The genetic diversity of salmonellae could be an evidence of the continuous animal exposition to the bacteria. Also, Salmonella adaptation in asymptomatic animals could be justified by the development of natural host immunity. This study provides novel information about Salmonella population distribution in domestic animals living at tropical areas. The presence of asymptomatic carriers may be critical to understand the routes of transmission of Salmonella in areas of high disease prevalence.     

Molecular phylogeny of the salmonellae: relationships among Salmonella species and subspecies determined from four housekeeping genes and evidence of lateral gene transfer events

The salmonellae are a diverse group of bacteria within the family Enterobacteriaceae that includes two species, Salmonella enterica and Salmonella bongori. In order to characterize the phylogenetic relationships of the species and subspecies of Salmonella, we analyzed four housekeeping genes, gapA, phoP, mdh and recA, comprising 3,459 bp of nucleotide sequence data for each isolate sequenced. Sixty-one isolates representing the most common serotypes of the seven subspecies of Salmonella enterica and six isolates of Salmonella bongori were included in this study. We present a robust phylogeny of the Salmonella species and subspecies that clearly defines the lineages comprising diphasic and monophasic subspecies. Evidence of intersubspecies lateral gene transfer of the housekeeping gene recA, which has not previously been reported, was obtained.     

Fitness of Salmonella enterica serovar Thompson in the Cilantro Phyllosphere

The epiphytic fitness of Salmonella enterica was assessed on cilantro plants by using a strain of S. enterica serovar Thompson that was linked to an outbreak resulting from cilantro. Salmonella serovar Thompson had the ability to colonize the surface of cilantro leaves, where it was detected by confocal laser scanning microscopy (CLSM) at high densities on the veins and in natural lesions. The population sizes of two common colonizers of plant surfaces, Pantoea agglomerans and Pseudomonas chlororaphis, were 10-fold higher than that of the human pathogen on cilantro incubated at 22°C. However, Salmonella serovar Thompson achieved significantly higher population levels and accounted for a higher proportion of the total culturable bacterial flora on cilantro leaves when the plants were incubated at warm temperatures, such as 30°C, after inoculation, indicating that the higher growth rates exhibited by Salmonella serovar Thompson at warm temperatures may increase the competitiveness of this organism in the phyllosphere. The tolerance of Salmonella serovar Thompson to dry conditions on plants at 60% relative humidity was at least equal to that of P. agglomerans and P. chlororaphis. Moreover, after exposure to low humidity on cilantro, Salmonella serovar Thompson recovered under high humidity to achieve its maximum population size in the cilantro phyllosphere. Visualization by CLSM of green fluorescent protein-tagged Salmonella serovar Thompson and dsRed-tagged P. agglomerans inoculated onto cilantro revealed that the human pathogen and the bacterial epiphyte formed large heterogeneous aggregates on the leaf surface. Our studies support the hypothesis that preharvest contamination of crops by S. enterica plays a role in outbreaks linked to fresh fruits and vegetables.     

Identification of Specific Gene Sequences Conserved in Contemporary Epidemic Strains of Salmonella enterica

Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes.