Towards an understanding of Salmonella enterica serovar Typhimurium persistence in swine.

Salmonella enterica is an important food borne pathogen that is frequently carried by swine. Carrier animals pose a food safety risk because they can transmit S. enterica to finished food products in the processing plant or by contamination of the environment. Environmental contamination has become increasingly important as non-animal foods (plant-based) have been implicated as sources of S. enterica. The prevalence of S. enterica in swine is high and yet carrier animals remain healthy. S. enterica has developed a highly sophisticated set of virulence factors that allow it to adapt to host environments and to cause disease. It is assumed that S. enterica also has developed unique ways to maintain itself in animals and yet not cause disease. Here we describe our research to understand persistence. Specifically, data are presented that demonstrates that detection of most carrier animals requires specific stresses that cause S. enterica to be shed from pigs. As well, we describe a phenotypic phase variation process that appears to be linked to the carrier state and a complex set of factors that control phenotypic phase variation. Finally, we describe how the composition of the gut bacterial microbiome may contribute to persistence and at the least how S. enterica might alter the composition of the gut bacterial microbiome.

Characterization and differential gene expression between two phenotypic phase variants in Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium strain 798 has previously been shown to undergo phenotypic phase variation. One of the phenotypes expresses virulence traits such as adhesion, while the other phenotype does not. Phenotypic phase variation appears to correlate with the ability of this strain to cause persistent, asymptomatic infections of swine. A new method to detect cells in either phenotypic phase was developed using Evans Blue-Uranine agar plates. Using this new assay, rates of phenotypic phase variation were obtained. The rate of phase variation from non-adhesive to adhesive phenotype was approximately 10(-4) per cell per generation while phase variation from the adhesive to the non-adhesive phenotype was approximately 10(-6) per cell per generation. Two highly virulent S. Typhimurium strains, SL1344 and ATCC 14028, were also shown to undergo phase variation. However, while the rate from adhesive to non-adhesive phenotype was approximately the same as for strain 798, the non-adhesive to adhesive phenotype shift was 37-fold higher. Differential gene expression was measured using RNA-Seq. Eighty-three genes were more highly expressed by 798 cells in the adhesive phenotype compared to the non-adhesive cells. Most of the up-regulated genes were in virulence genes and in particular all genes in the Salmonella pathogenicity island 1 were up-regulated. When compared to the virulent strain SL1344, expression of the virulence genes was approximately equal to those up-regulated in the adhesive phenotype of strain 798. A comparison of invasive ability demonstrated that strain SL1344 was the most invasive followed by the adhesive phenotype of strain 798, then the non-adhesive phenotype of strain 798. The least invasive strain was ATCC 14028. The genome of strain 798 was sequenced and compared to SL1344. Both strains had very similar genome sequences and gene deletions could not readily explain differences in the rates of phase variation from non-adhesive to the adhesive phenotype.

Evaluation of a commercially available modified-live Streptococcus equi subsp equi vaccine in ponies.

OBJECTIVE: To evaluate a commercially available modified-live Streptococcus equi subsp equi vaccine for safety and persistence in vaccinated ponies and to detect recombination or reversion events in the vaccine strain. ANIMALS: 5 ponies that were 1.5 to 8 years old (group 1) and 4 ponies that were 6 months old (group 2). PROCEDURES: Ponies were vaccinated, with a subsequent booster vaccination 2 to 3 weeks later, and monitored for 50 days. At booster vaccination, an equal amount of a tetracycline-resistant wild-type strain of S equiwas administered. Recovery of all strains was performed by use of bacteriologic culture and PCR assays. RESULTS: Ponies in group 1 had background antibody titers against S equi antigen before vaccination despite the lack of known exposure to S equi. Ponies in group 2 were immunologically naïve. Increases in anti-S equi antibody titers were detected in both groups. Ponies in group 1 did not have clinical signs of disease caused by S equi. In group 2, all ponies developed abscesses in retropharyngeal lymph nodes; 1 pony developed severe clinical disease and was euthanized. The vaccine strain was recovered from ponies in group 2 for up to 24 days after vaccination. CONCLUSIONS AND CLINICAL SIGNIFICANCE: Although the vaccine was successful in inducing IgG antibodies against S equi in all ponies, findings suggested that the vaccine may have caused substantial morbidity and some deaths in the young ponies. In young ponies, the vaccine strain persisted in tissues for weeks; however, no evidence of recombination was detected.

A multiphasic typing approach to subtype Streptococcus equi subspecies equi.

The objective of the present investigation was to differentiate between strains of Streptococcus equi subspecies equi implicated in abscess formation in vaccinated horses. Streptococcus equi isolates recovered from clinical specimens associated with equine strangles cases submitted to the University of Illinois Veterinary Diagnostic Laboratory were compared with S. equi isolates representing at least 12 lots of a commercial modified live vaccine (MLV) to determine whether the isolates obtained from the abscesses were vaccine or wild type. Genotyping techniques evaluated included enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR), repetitive extragenic palindrome PCR, BOX element PCR, ribotyping, and pulsed-field gel electrophoresis (PFGE). Phenotypic evaluations were performed using the Biolog GP2 Microplate (hereafter, Biolog). In cases where Biolog and PFGE results did not coincide, a single nucleotide polymorphism located in the upstream regulatory region of szp gene was used to identify the S. equi strains. PFGE and Biolog successfully differentiated wild-type S. equi strains isolated from clinical submissions from isolates of the MLV. PFGE genotyping enabled further subtyping of the wild-type strains, whereas Biolog combined with szp sequencing was useful in differentiating the MLV strain from its wild-type progenitor. Deletion of a single guanine residue located in the upstream regulatory region of the szp gene appears to be conserved among vaccine isolates, and shows a 98.5% correlation to Biolog identification. This multiphasic approach can be used to answer specific diagnostic questions pertaining to the source of infection and/or outbreak, or to address quarantine concerns.

Effects of therapeutic ceftiofur administration to dairy cattle on Escherichia coli dynamics in the intestinal tract.

The goal of this study was to follow ceftiofur-treated and untreated cattle in a normally functioning dairy to examine enteric Escherichia coli for changes in antibiotic resistance profiles and genetic diversity. Prior to treatment, all of the bacteria cultured from the cows were susceptible to ceftiofur. Ceftiofur-resistant E. coli was only isolated from treated cows during and immediately following the cessation of treatment, and the 12 bla(CMY-2)-positive isolates clustered into two genetic groups. E. coli bacterial counts dropped significantly in the treated animals (P < 0.027), reflecting a disappearance of the antibiotic-susceptible strains. The resistant bacterial population, however, did not increase in quantity within the treated cows; levels stayed low and were overtaken by a returning susceptible population. There was no difference in the genetic diversities of the E. coli between the treated and untreated cows prior to ceftiofur administration or after the susceptible population of E. coli returned in the treated cows. A cluster analysis of antibiotic susceptibility profiles resulted in six clusters, two of which were multidrug resistant and were comprised solely of isolates from the treated cows immediately following treatment. The antibiotic treatment provided a window to detect the presence of ceftiofur-resistant E. coli but did not appear to cause its emergence or result in its amplification. The finding of resistant isolates following antibiotic treatment is not sufficient to estimate the strength of selection pressure nor is it sufficient to demonstrate a causal link between antibiotic use and the emergence or amplification of resistance.

Development of a polymerase chain reaction assay for the detection of antibiotic resistance genes in community DNA.

Many methods are used to detect antibiotic resistance genes in samples. The objective of the study reported here was to compare polymerase chain reaction (PCR) analysis of community DNA with fecal culturing for detecting antibiotic resistance genes in cattle samples. In the laboratory-based portion of this study, known concentrations of an Escherichia coli strain with 3 antibiotic resistance genes (cmy-2, flo, and cat) were added to feces from dairy cattle. These genes were used to assess the effect of various primer pairs, chromosomally versus plasmid-encoded genes, and gene copy number on the sensitivity of PCR amplification. Gene-specific PCR amplification was performed on the community DNA extracted from the feces. Feces were cultured for the inoculated strain. In the field-based portion of the study, 80 cattle fecal samples of unknown gene status were compared by use of similar methods. Culture and PCR amplification from community DNA extractions produced variable results, and this variability was most noticeable at dilutions that approached the detection limit of the assay. Typically, PCR amplification had a higher sensitivity than did culture for detecting the gene of interest. However, the sensitivity of culture was improved by plating on selective media containing antibiotics. The community DNA approach enables assessment of bacterial communities in complex samples such as feces, a task that can be prohibitive by budget or time constraints associated with culture methods. Through a strategic combination of culture and community DNA approaches, the relationship between specific selection pressures and the persistence and dissemination of specific resistance genes can be elucidated.

Relationship between phenotypic and genotypic florfenicol resistance in Escherichia coli.

This study evaluated the relationship between florfenicol resistance and flo genotypes in 1,987 Escherichia coli isolates from cattle. The flo gene was detected in 164 isolates, all of which expressed resistance to florfenicol at MICs of >/=256 microg/ml. The florfenicol MICs for all isolates that lacked flo were