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.

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.

Host genes affect intestinal colonisation of newly hatched chickens by Campylobacter jejuni.

Campylobacter jejuni is the leading cause of food-borne gastro-enteritis and infection can be followed by severe clinical complications, such as the autoimmune neuropathy Guillain-Barré syndrome. Poultry meat is considered to be a common source of infection, with most flocks infected from 2 to 3 weeks of age. We have examined the effect of host genetics on the colonisation levels of C. jejuni in chickens. Chicks from different inbred lines were challenged with 10(7) to 10(8) cfu of C. jejuni 14N or C. jejuni 81-176 on the day of hatch and levels of bacterial colonisation measured over a period of 2-3 weeks. We consistently observed a 10- to 100-fold difference between four inbred lines in the number of C. jejuni organisms present in the cloaca or in the caeca, with the greatest differences detected between line N, which carried relatively high bacterial levels, and line 6(1), which carried relatively low numbers of bacteria. Amongst the four lines studied, major histocompatibility complex did not appear to be a major factor in determining the resistance. The difference in numbers of cloacal bacteria was observed as soon as 24 h after challenge and was still present at the end of the experiment. Lines N and 6(1) were chosen to analyse the mode of inheritance of the genetic differences in response to this infection. Challenge of progeny from reciprocal (6(1) female x N male) and (6(1) female x N male) F1 crosses and from (N female x 6(1) male) F1 female x N male backcrosses with C. jejuni 14N revealed that the difference in bacterial numbers was inherited in a manner consistent with the resistance (low bacterial numbers) controlled by a single autosomal dominant locus. These data suggest that it might be possible to identify the genes responsible by genetic mapping and candidate gene analysis.

Adaptation of Campylobacter jejuni NCTC11168 to high-level colonization of the avian gastrointestinal tract.

The genome sequence of the human pathogen Campylobacter jejuni NCTC11168 has been determined recently, but studies on colonization and persistence in chickens have been limited due to reports that this strain is a poor colonizer. Experimental colonization and persistence studies were carried out with C. jejuni NCTC11168 by using 2-week-old Light Sussex chickens possessing an acquired natural gut flora. After inoculation, NCTC11168 initially colonized the intestine poorly. However, after 5 weeks we observed adaptation to high-level colonization, which was maintained after in vitro passage. The adapted strain exhibited greatly increased motility. A second strain, C. jejuni 11168H, which had been selected under in vitro conditions for increased motility (A. V. Karlyshev, D. Linton, N. A. Gregson, and B. W. Wren, Microbiology 148:473-480, 2002), also showed high-level intestinal colonization. The levels of colonization were equivalent to those of six other strains, assessed under the same conditions. There were four mutations in C. jejuni 11168H that reduced colonization; maf5, flaA (motility and flagellation), and kpsM (capsule deficiency) eliminated colonization, whereas pglH (general glycosylation system deficient) reduced but did not eliminate colonization. This study showed that there was colonization of the avian intestinal tract by a Campylobacter strain having a known genome sequence, and it provides a model for colonization and persistence studies with specific mutations.