In vivo passage of Salmonella Typhimurium results in minor mutations in the bacterial genome and increases in vitro invasiveness

International audienceAbstractEggs and raw or undercooked egg-containing food items are frequently identified as the bacterial source during epidemiolocal investigation of Salmonella outbreaks. Multi-locus variable number of tandem repeats analysis (MLVA) is a widely used Salmonella typing method enabling the study of diversity within populations of the same serotype. In vivo passage, however, has been linked with changes in MLVA type and more broadly the Salmonella genome. We sought to investigate whether in vivo passage through layer hens had an effect on MLVA type as well as the bacterial genome and whether any mutations affected bacterial virulence. Layer hens were infected with either Salmonella Typhimurium DT9 (03-24-11-11-523) as part of a single infection or were co-infected with an equal amount of Salmonella Mbandaka. Salmonella shedding in both single and co-infected birds was variable over the course of the 16-week experiment. Salmonella Typhimurium and Salmonella Mbandaka were identified in feces of co-infected birds. Salmonella colonies isolated from fecal samples were subtyped using MLVA. A single change in SSTR-6 was observed in Salmonella Typhimurium strains isolated from co-infected birds. Isolates of Salmonella Typhimurium of both the parent (03-24-11-11-523) and modified (03-24-12-11-523) MLVA type were sequenced and compared with the genome of the parent strain. Sequence analysis revealed that in vivo passaging resulted in minor mutation events. Passaged isolates exhibited significantly higher invasiveness in cultured human intestinal epithelial cells than the parent strain. The microevolution observed in this study suggests that changes in MLVA may arise more commonly and may have clinical significance

Comparison of peroxyacetic acid and acidified sodium chlorite at reducing natural microbial contamination on chicken meat pieces

ABSTRACT: The spin-chill process at poultry processing plants involves the immersion of chicken carcasses in cold water (<5°C) often containing sodium hypochlorite which significantly contributes to the reduction of bacterial loads. Cutting carcasses into pieces, however, has been linked with increases in Campylobacter and Salmonella counts. Here, the efficacy of PAA and ASC on reducing bacteria on skin-on, bone-in thigh cuts was investigated. Three concentrations of ASC (60, 112, and 225 ppm) and PAA (50, 75, 100 ppm) were used. Thighs were dipped into sanitizer and tested for total viable bacterial counts, Campylobacter load, and prevalence of Salmonella. The efficacy of PAA and ASC was also compared with chlorine (8 ppm). All sanitizers exhibited a greater log reduction compared with water. PAA at both 75 and 100 ppm resulted in significantly higher log reductions compared with the water only. PAA at 100 ppm and 225 ppm ASC were the most effective at reducing Campylobacter. All wash treatments reduced the proportion of Salmonella positive samples, but the greatest reduction was observed for 225 ppm ASC. Both concentrations of ASC resulted in a greater reduction in total viable counts compared with chlorine

<i>Salmonella enterica</i> isolates from layer farm environments are able to form biofilm on eggshell surfaces

<p>This study examined the eggshell biofilm forming ability of <i>Salmonella enterica</i> isolates recovered from egg farms. Multicellular behaviour and biofilm production were examined at 22 and 37°C by Congo red morphology and the crystal violet staining assay. The results indicated that the biofilm forming behaviour of <i>Salmonella</i> isolates was dependent on temperature and associated with serovars. Significantly greater biofilm production was observed at 22°C compared with 37°C. The number of viable biofilm cells attached to eggshells after incubation for 48 h at 22°C was significantly influenced by serovar. Scanning electron microscopic examination revealed firm attachment of bacterial cells to the eggshell surface. The relative expression of <i>csg</i>D and <i>adr</i>A gene was significantly higher in eggshell biofilm cells of <i>S</i>. Mbandaka and <i>S</i>. Oranienburg. These findings demonstrate that <i>Salmonella</i> isolates are capable of forming biofilm on the eggshell surface and that this behaviour is influenced by temperature and serovar.</p

Murine cytomegalovirus strains co-replicate at multiple tissue sites and establish co-persistence in salivary glands in the absence of Ly49H-mediated competition

Infection with multiple genetically distinct strains of pathogen is common and can lead to positive (complementation) or negative (competitive) within-host interactions. These interactions can alter aspects of the disease process and help shape pathogen evolution. Infection of the host with multiple strains of cytomegalovirus (CMV) occurs frequently in humans and mice. Profound, NK-cell-mediated (apparent) competition has been identified in C57BL/6 mice, and prevented the replication and shedding of certain co-infecting CMV strains. However, the frequency of such strong competition has not been established. Other within-host interactions such as complementation or alternative forms of competition remain possible. Moreover, high rates of recombination in both human CMV and murine CMV (MCMV) suggest prolonged periods of viral co-replication, rather than strong competitive suppression. An established model was employed to investigate the different possible outcomes of multi-strain infection in other mouse strains. In this study, co-replication of up to four strains of MCMV in the spleen, liver and salivary glands was observed in both MCMV-susceptible and MCMV-resistant mice. In the absence of apparent competition, no other forms of competition were unmasked. In addition, no evidence of complementation between viral strains was observed. Importantly, co-replication of MCMV strains was apparent for up to 90 days in the salivary glands. These data indicated that competition was not the default outcome of multi-strain CMV infection. Prolonged, essentially neutral, co-replication may be the norm, allowing for multi-strain transmission and prolonged opportunities for recombination

Competition is evident early, before the maturation of acquired immunity.

<p>B6 mice were inoculated i.p. with 1×10⁴ pfu of either C4C, K181 or K181^Δm157 (closed symbols) or a total of 1×10⁴ pfu of a mixed inoculum of equal proportions of with C4C and K181 or C4C and K181^Δm157 (open symbols). <b>A</b>. Viral DNA levels in the spleens of B6 mice three days after single strain infection. <b>B</b>. Viral DNA levels in the spleens of mice co-infected with C4C and K181. <b>C</b>. Viral DNA levels in the spleens of mice co-infected with C4C and K181^Δm157<b>D</b>. Viral DNA levels in the livers of B6 mice three days after single strain infection. <b>E</b>. Viral DNA levels in the livers of mice co-infected with C4C and K181. <b>F</b>. Viral DNA levels in the spleen of mice co-infected with C4C and K181^Δm157. Note competition was evident in the spleen (B) but not in the liver (E). Data are mean ± SEM for 5 animals per group. Dotted line indicates limit of detection. DNA levels in mice following single strain infection were compared by one-way Anova, asterisks indicate level of significance after Tukey's post hoc analysis (*** P<0.001, ** P<0.01, * P<0.05).</p

Blocking the NK cell receptor Ly49H eliminates viral competition.

<p>B6 mice were treated with 200 µg of the monoclonal antibody 3D10 (anti-Ly49H) or 200 µg of isotype control antibody 9E10 (anti-cMyc) on day −2, 0, 4, 8, 12 and 16 of infection. On day 0 mice were inoculated i.p. with 1×10⁴ pfu of either a single MCMV strain (closed symbols) or a total of 1×10⁴ pfu of a mixed inoculum of C4A and C4C (half filled diamonds - plaque assay or open symbols – strain specific PCR). <b>A</b>. Titers of infectious virus were determined by plaque assay in salivary glands of B6 mice infected with either C4A or C4C (closed symbols), or in anti-Ly49H or isotype control treated mice that were also co-infected with C4A and C4C (half filled diamonds). <b>B</b>. Viral DNA levels in B6 mice treated with anti-Ly49H and co-infected with C4A and C4C. Note both C4A and C4C DNA were detectable following anti-Ly49H treatment. <b>C</b>. Viral DNA levels in B6 mice treated with isotype control mAb and co-infected with C4A and C4C. Note competition was retained in these mice with C4A undetectable in the salivary glands. The x-axis represents the limit of detection for plaque assays (100 pfu/g of tissue) and the multiplex qPCR (1×10⁴ copy number/g of tissue), n = 5 co-infected mice/group, 3 single strain infected mice/group.</p