Detection of Campylobacter jejuni and Campylobacter coli from broiler chicken-related samples using BAX PCR and conventional International Organization for Standardization culture.

In this study, the conventional International Organization for Standardization (ISO) culture method was compared with the DuPont Qualicon BAX system, a high-throughput, rapid molecular assay that can be used to detect several bacterial species, including Campylobacter jejuni and Campylobacter coli in diverse sample types. Standard enrichment culture is a time-consuming process, taking up to 6 days to obtain a confirmed result. Rapid molecular assays have been developed that provide results within 24 h. Naturally contaminated samples from the poultry production chain were examined for the presence of Campylobacter spp. Samples from broiler chicken ceca (n = 100), fresh chicken carcass rinses (n = 60), and bootsocks (gauze sock walked through a broiler chicken house; n = 50) were enriched according to the ISO 10272 method in Bolton broth specifically designed to detect Campylobacter spp. in complex sample types. Samples were enriched without blood for use with the BAX system using the Campylobacter BAX kits for the detection of C. jejuni and C. coli. Samples also were directly plated onto modified charcoal cefperazone deoxycholate agar, and results were compared with those from the enriched samples for the ability to detect Campylobacter spp. Campylobacter spp. were isolated from 49% of samples with conventional enrichment cultures, from 48% with direct culture, from 68% with the BAX system and enrichment cultures, and from 62% with the BAX system used directly with samples. Overall, the BAX system detected more positive samples than did the conventional culture method and is an effective methodology for the rapid and reliable detection of Campylobacter spp. from diverse sample types.

Inactivation of ppk differentially affects virulence and disrupts ATP homeostasis in Salmonella enterica serovars Typhimurium and Gallinarum.

Polyphosphate is involved in resistance to stress in a number of bacterial species; however, its role in the virulence of Salmonella enterica serovars which differ in their host range has not been described. We examined the role of polyphosphate kinase in infection, growth and survival of S. Typhimurium (broad-host range) and S. Gallinarum (avian-adapted). We also used ppk mutants to assess the downstream effects on intracellular ATP levels. ppk mutants had significantly (P<0.05) elevated ATP in stationary phase compared to the wild-type and, depending on the serovar, were defective in growth, survival and virulence. The virulence of S. Typhimurium ppk::SpcStr was significantly (P<0.05) attenuated following oral infection of both Rhode Island Red chickens and BALB/c mice. In contrast, inactivation of the ppk gene of S. Gallinarum did not affect growth or virulence. The differential contribution of polyphosphate to the virulence of S. Typhimurium and S. Gallinarum may reflect aspects of the pathogenesis and host range of these serovars. The ppk mutant of both serovars survived significantly less well (P<0.05) in a saline starvation-survival model, relative to the respective parent. The effect of ppk mutation on survival was formally described by fitting the data to the Weibull model and by estimation of k(max). Measurement of rpoS promoter activity using a lacZ transcriptional fusion demonstrated repression of rpoS in a ppk background, confirming a role for polyphosphate in RpoS induction. Together the data indicate the crucial importance of maintaining stable intracellular ATP during infection and nutritional stress. We suggest that polyphosphate plays a central role in homeostasis during growth and stress.

Role of the alternative sigma factors sigmaE and sigmaS in survival of Salmonella enterica serovar Typhimurium during starvation, refrigeration and osmotic shock.

The ability of Salmonella enterica serovar Typhimurium to survive environmental stress requires specific, coordinated, responses, which induce resistance to the stress condition. This study investigated the relative contribution of sigmaE and sigmaS, the sigma factors regulating extracytoplasmic and general stress response functions, respectively, to survival at low temperature and also in media of differing osmotic strength, conditions relevant to food preservation. To determine if low-temperature storage is a signal for sigmaE- and sigmaS-mediated survival, the ability of S. Typhimurium rpoE, rpoS and rpoE/rpoS mutants to survive in a saline starvation-survival model at a refrigeration temperature (4.5 degrees C) was examined. Under these conditions, the rpoE mutant was significantly (P<0.05) compromised compared to the parent and to an rpoS mutant. The double mutant in rpoE and rpoS displayed a cumulative defect in survival. In hyperosmotic environments (low aw) containing 6 % NaCl and at refrigeration temperature, both sigma factors were important for maximum survival but sigmaS played the dominant role. Analysis of the metabolic activity of starved populations at 4.5 and 37 degrees C revealed significantly (P<0.001) elevated electron-transport system activity in mutants in rpoE and rpoS, indicating a role for sigmaE- and sigmaS-regulated genes in maintaining energy homeostasis. Together these data demonstrate that sigmaE and sigmaS are important for survival of S. Typhimurium in conditions encountered during food processing and that the relative contribution of sigmaE and sigmaS is critically dependent on the precise nature of the stress.

Glycogen production by different Salmonella enterica serotypes: contribution of functional glgC to virulence, intestinal colonization and environmental survival.

In enteric bacteria, the contribution of endogenous energy sources to survival both inside and outside the host is poorly understood. The contribution of glycogen production to the virulence, colonization and environmental survival of different Salmonella enterica serotypes was assessed. Of 19 serotypes (339 strains) tested for glycogen production, 17 (256 strains) were positive. The avian-specific serovars S. Gallinarum (62 strains) and S. Pullorum (21 strains) did not produce glycogen. The sequence of glgC in three S. Gallinarum strains tested revealed an identical deletion of 11 consecutive bases, which was not present in S. Pullorum, and a CCC insertion after position 597. Transduction of S. Gallinarum and S. Pullorum to a glycogen-positive phenotype did not change the ability to colonize the intestine or affect virulence in the chicken. Mortality rates in chickens following oral infection with a S. Typhimurium glycogen mutant (glgC : : km) were not significantly reduced, although colonization of the intestine was reduced over the first 4 weeks of the trial. Growth and yield of the glgC : : km mutant were comparable to the parent. The glgC mutant survived less well in faeces and in water at 4 degrees C when the strain was grown in LB broth containing 0.5 % glucose, and in saline it died off more rapidly after 7 days. The data suggest that glycogen has a complex but comparatively minor role in virulence and colonization, but a more significant role in survival.