Patterns of genome evolution that have accompanied host adaptation in Salmonella.

Many bacterial pathogens are specialized, infecting one or few hosts, and this is often associated with more acute disease presentation. Specific genomes show markers of this specialization, which often reflect a balance between gene acquisition and functional gene loss. Within Salmonella enterica subspecies enterica, a single lineage exists that includes human and animal pathogens adapted to cause infection in different hosts, including S. enterica serovar Enteritidis (multiple hosts), S. Gallinarum (birds), and S. Dublin (cattle). This provides an excellent evolutionary context in which differences between these pathogen genomes can be related to host range. Genome sequences were obtained from ∼ 60 isolates selected to represent the known diversity of this lineage. Examination and comparison of the clades within the phylogeny of this lineage revealed signs of host restriction as well as evolutionary events that mark a path to host generalism. We have identified the nature and order of events for both evolutionary trajectories. The impact of functional gene loss was predicted based upon position within metabolic pathways and confirmed with phenotyping assays. The structure of S. Enteritidis is more complex than previously known, as a second clade of S. Enteritidis was revealed that is distinct from those commonly seen to cause disease in humans or animals, and that is more closely related to S. Gallinarum. Isolates from this second clade were tested in a chick model of infection and exhibited a reduced colonization phenotype, which we postulate represents an intermediate stage in pathogen-host adaptation.

Bacteriophages Isolated from Chicken Meat and the Horizontal Transfer of Antimicrobial Resistance Genes.

Antimicrobial resistance in microbes poses a global and increasing threat to public health. The horizontal transfer of antimicrobial resistance genes was thought to be due largely to conjugative plasmids or transposons, with only a minor part being played by transduction through bacteriophages. However, whole-genome sequencing has recently shown that the latter mechanism could be highly important in the exchange of antimicrobial resistance genes between microorganisms and environments. The transfer of antimicrobial resistance genes by phages could underlie the origin of resistant bacteria found in food. We show that chicken meat carries a number of phages capable of transferring antimicrobial resistance. Of 243 phages randomly isolated from chicken meat, about a quarter (24.7%) were able to transduce resistance to one or more of the five antimicrobials tested into Escherichia coli ATCC 13706 (DSM 12242). Resistance to kanamycin was transduced the most often, followed by that to chloramphenicol, with four phages transducing tetracycline resistance and three transducing ampicillin resistance. Phages able to transduce antimicrobial resistance were isolated from 44% of the samples of chicken meat that we tested. The statistically significant (P = 0.01) relationship between the presence of phages transducing kanamycin resistance and E. coli isolates resistant to this antibiotic suggests that transduction may be an important mechanism for transferring kanamycin resistance to E. coli. It appears that the transduction of resistance to certain antimicrobials, e.g., kanamycin, not only is widely distributed in E. coli isolates found on meat but also could represent a major mechanism for resistance transfer. The result is of high importance for animal and human health.

Monophasic expression of FliC by Salmonella 4,[5],12:i:- DT193 does not alter its pathogenicity during infection of porcine intestinal epithelial cells.

Non-typhoidal serotypes of Salmonella enterica remain important food-borne pathogens worldwide and the frequent emergence of epidemic strains in food-producing animals is a risk to public health. In recent years, Salmonella 4,[5],12:i:- isolates, expressing only phase 1 (FliC) of the two flagellar antigens, have emerged and increased in prevalence worldwide. In Europe, the majority of 4,[5],12:i:- isolates belong to phage types DT193 and DT120 of Salmonella Typhimurium and pigs have been identified as the reservoir species. In this study we investigated the ability of pig-derived monophasic (4,[5],12:i:-) and biphasic DT193 isolates to invade a porcine intestinal epithelial cell line (IPEC-1) and activate TLR-5, IL-8 and caspases. We found that the 4,[5],12:i:- isolates exhibited comparable adhesion and invasion to that of the virulent S. Typhimurium isolate 4/74, suggesting that these strains could be capable of colonizing the small intestine of pigs in vivo. Infection with 4,[5],12:i:- and biphasic DT193 isolates resulted in approximately the same level of TLR-5 (a flagellin receptor) and IL-8 (a proinflammatory chemokine) mRNA upregulation. The monophasic variants also elicited similar levels of caspase activation and cytotoxicity to the phase-variable DT193 isolates. These findings suggest that failure of 4,[5],12:i:- DT193 isolates to express a second phase of flagellar antigen (FljB) is unlikely to hamper their pathogenicity during colonization of the porcine intestinal tract.

Dynamics of dual infection with Campylobacter jejuni strains in chickens reveals distinct strain-to-strain variation in infection ecology.

Although multiple genotypes of Campylobacter jejuni may be isolated from the same commercial broiler flock, little is known about the infection dynamics of different genotypes within individuals or their colonization sites within the gut. Single experimental infections with C. jejuni M1 (sequence type 137, clonal complex 45) and C. jejuni 13126 (sequence type 21, clonal complex 21) revealed that 13126 colonized the ceca at significantly higher levels. The dissemination and colonization sites of the two C. jejuni strains then were examined in an experimental broiler flock. Two 33-day-old broiler chickens were infected with M1 and two with 13126, and 15 birds were left unchallenged. Cloacal swabs were taken postinfection to determine the colonization and shedding of each strain. By 2 days postinfection (dpi), 8/19 birds were shedding M1 whereas none were shedding 13126. At 8 dpi, all birds were shedding both strains. At 18 dpi, liver and cecal levels of each isolate were quantified, while in 10 birds they also were quantified at nine sites throughout the gastrointestinal (GI) tract. 13126 was found throughout the GI tract, while M1 was largely restricted to the ceca and colon. The livers of 7/19 birds were culture positive for 13126 only. These data show that 13126 has a distinctly different infection biology than strain M1. It showed slower colonization of the lower GI tract but was more invasive and able to colonize at a high level throughout the GI tract. The finding that C. jejuni strains have markedly different infection ecologies within the chicken has implications for control in the poultry industry and suggests that the contamination risk of edible tissues is dependent on the isolate involved.

Salmonella bongori provides insights into the evolution of the Salmonellae.

The genus Salmonella contains two species, S. bongori and S. enterica. Compared to the well-studied S. enterica there is a marked lack of information regarding the genetic makeup and diversity of S. bongori. S. bongori has been found predominantly associated with cold-blooded animals, but it can infect humans. To define the phylogeny of this species, and compare it to S. enterica, we have sequenced 28 isolates representing most of the known diversity of S. bongori. This cross-species analysis allowed us to confidently differentiate ancestral functions from those acquired following speciation, which include both metabolic and virulence-associated capacities. We show that, although S. bongori inherited a basic set of Salmonella common virulence functions, it has subsequently elaborated on this in a different direction to S. enterica. It is an established feature of S. enterica evolution that the acquisition of the type III secretion systems (T3SS-1 and T3SS-2) has been followed by the sequential acquisition of genes encoding secreted targets, termed effectors proteins. We show that this is also true of S. bongori, which has acquired an array of novel effector proteins (sboA-L). All but two of these effectors have no significant S. enterica homologues and instead are highly similar to those found in enteropathogenic Escherichia coli (EPEC). Remarkably, SboH is found to be a chimeric effector protein, encoded by a fusion of the T3SS-1 effector gene sopA and a gene highly similar to the EPEC effector nleH from enteropathogenic E. coli. We demonstrate that representatives of these new effectors are translocated and that SboH, similarly to NleH, blocks intrinsic apoptotic pathways while being targeted to the mitochondria by the SopA part of the fusion. This work suggests that S. bongori has inherited the ancestral Salmonella virulence gene set, but has adapted by incorporating virulence determinants that resemble those employed by EPEC.

The effect of growth temperature on the pathogenicity of Campylobacter.

Control of Campylobacter in the food chain requires a better understanding of the behaviour of the bacteria in relevant environments. Campylobacter species are largely non-pathogenic in poultry, the body temperature of which is 42 °C. However, the bacteria are highly pathogenic in humans whose body temperature is 37 °C. The aim of this study was to examine if switching from commensal to pathogenic behaviour was related to temperature. We examined the growth, motility and invasion of T84 cells by three species of Campylobacter: C. jejuni 81116, C. jejuni M1, C. coli 1669, C. coli RM2228 and C. fetus fetus NC10842 grown at 37 and 42 °C. Our results suggest that C. jejuni isolates grow similarly at both temperatures but some are more motile at 42 °C and some are more invasive at 37 °C, which may account for its rapid spread in poultry flocks and for infection in humans, respectively. C. coli, which are infrequent causes of Campylobacter infections in humans, is less able to grow and move at 37 °C compared to 42 °C but was significantly more invasive at the lower temperature. C. fetus fetus, which is infrequently found in poultry, is less able to grow and invade at 42 °C.

Enhanced recovery of Salmonella Typhimurium DT104 from exposure to stress at low temperature.

Salmonella enterica serovar Typhimurium (S. Typhimurium) remains an important cause of food-borne infection in the developed world. In order to establish infection within a host, Salmonella must survive and recover from a range of environmental stresses. S. Typhimurium strain SL1344 is among the most extensively studied pathogenic Salmonella strains, while S. Typhimurium phage type DT104 is an important type that has been associated with pandemic spread and a high number of food-borne disease outbreaks over the last two decades. In this study, we have compared the abilities of these two S. Typhimurium types to recover from stress exposures commonly encountered in food production, including 685 mM NaCl, pH 3.8, low temperature (6 °C) and combinations thereof. Following removal from prolonged (8 days) stress, DT104 cultures that had been exposed to low temperature, with or without additional stress, resumed exponential growth more rapidly than SL1344 cultures exposed to the same conditions. SL1344 showed higher levels of filamentation than DT104 in response to NaCl exposure at low temperature. Further, SL1344 incurred higher levels of membrane damage in response to elevated NaCl and pH 3.8 at both temperatures compared with DT104. However, both strains recovered normal cell division and membrane integrity within 6 h when all stresses were removed. Expression of the Salmonella pathogenicity island 1 gene prgH, the first gene in the prg/org operon, was monitored using a chromosomal reporter in which gfp(+) expression was driven by the prgH promoter. Recovery of prgH expression was comparable for SL1344 and DT104 exposed to stress at 22 °C. However, DT104 cultures exposed to pH 3.8 or combined NaCl and low-pH stress at low temperature resumed prgH expression more rapidly than SL1344. Both strains recovered maximal levels of prgH expression after 6 h recovery from all stresses and, interestingly, maximal levels of prgH expression were significantly higher in SL1344, consistent with prgH expression in late-exponential, non-stressed SL1344 and DT104 cultures. Together, these data show that S. Typhimurium is capable of rapid recovery from environmental and food-related stresses, and give insight into the enhanced ability of DT104 compared with SL1344 to adapt to such stresses, which may contribute to the success of this globally disseminated pathogenic phage type.

Differences in Salmonella enterica serovar Typhimurium strain invasiveness are associated with heterogeneity in SPI-1 gene expression.

Most studies on Salmonella enterica serovar Typhimurium infection focus on strains ATCC SL1344 or NTCC 12023 (ATCC 14028). We have compared the abilities of these strains to induce membrane ruffles and invade epithelial cells. S. Typhimurium strain 12023 is less invasive and induces smaller membrane ruffles on MDCK cells compared with SL1344. Since the SPI-1 effector SopE is present in SL1344 and absent from 12023, and SL1344 sopE mutants have reduced invasiveness, we investigated whether 12023 is less invasive due to the absence of SopE. However, comparison of SopE(+) and SopE(-) S. Typhimurium strains, sopE deletion mutants and 12023 expressing a sopE plasmid revealed no consistent relationship between SopE status and relative invasiveness. Nevertheless, absence of SopE was closely correlated with reduced size of membrane ruffles. A PprgH-gfp reporter revealed that relatively few of the 12023 population (and that of the equivalent strain ATCC 14028) express SPI-1 compared to other S. Typhimurium strains. Expression of a PhilA-gfp reporter mirrored that of PprgH-gfp in 12023 and SL1344, implicating reduced signalling via the transcription factor HilA in the heterogeneous SPI-1 expression of these strains. The previously unrecognized strain heterogeneity in SPI-1 expression and invasiveness has important implications for studies of Salmonella infection.

Exposure of Campylobacter jejuni to 6 degrees C: effects on heat resistance and electron transport activity.

Human infection with Campylobacter jejuni is frequently associated with the consumption of foods, especially chicken meat, which have been exposed to a range of temperatures during processing, storage, and cooking. Despite the public health importance of C. jejuni, little is known about the effects of cold exposure (refrigeration) on the subsequent ability of this pathogen to survive heat challenge. This work examined the effect of rapid exposure to 6 degrees C for 24 h on the heat resistance at 52 degrees C of 19 C. jejuni strains originally isolated from various sources. The resulting death curves were analyzed with the Weibull model. Unlike cold-exposed cells of Escherichia coli and Salmonella, which have been reported to show significant increased sensitivity to heat, such exposure had only a marginal effect on heat resistance of the C. jejuni strains in this study. A possible explanation for this effect is that rapid chilling renders C. jejuni cells unable to adapt to reduced temperatures in an active manner. This hypothesis is supported by the observation that exposure to 6 degrees C for 24 h resulted in a significant and marked reduction in electron transport system activity when compared with controls at 37 degrees C.

Increasing prevalence of a fluoroquinolone resistance mutation amongst Campylobacter jejuni isolates from four human infectious intestinal disease studies in the United Kingdom.

BACKGROUND: Campylobacter jejuni is the most common bacterial cause of human infectious intestinal disease. METHODS: We genome sequenced 601 human C. jejuni isolates, obtained from two large prospective studies of infectious intestinal disease (IID1 [isolates from 1993-1996; n = 293] and IID2 [isolates from 2008-2009; n = 93]), the INTEGRATE project [isolates from 2016-2017; n = 52] and the ENIGMA project [isolates from 2017; n = 163]. RESULTS: There was a significant increase in the prevalence of the T86I mutation conferring resistance to fluoroquinolone between each of the three later studies (IID2, INTEGRATE and ENIGMA) and IID1. Although the distribution of major multilocus sequence types (STs) was similar between the studies, there were changes in both the abundance of minority STs associated with the T86I mutation, and the abundance of clones within single STs associated with the T86I mutation. DISCUSSION: Four population-based studies of community diarrhoea over a 25 year period revealed an increase over time in the prevalence of the T86I amongst isolates of C. jejuni associated with human gastrointestinal disease in the UK. Although associated with many STs, much of the increase is due to the expansion of clones associated with the resistance mutation.

Amoxicillin therapy of poultry flocks: effect upon the selection of amoxicillin-resistant commensal Campylobacter spp.

BACKGROUND: The aim of this study was to investigate the effect of amoxicillin therapy of poultry flocks upon the persistence of commensal Campylobacter spp. and the incidence of antibiotic resistance. METHODS: Four poultry flocks naturally colonized with Campylobacter were treated with amoxicillin and monitored before, during and up to 4 weeks post-treatment. The numbers of Campylobacter were determined and the isolates speciated and typed by flaA short variable region (SVR) sequence analysis and PFGE. The susceptibility of the isolates to antibiotics, presence of the Cj0299 gene encoding a beta-lactamase and beta-lactamase production (nitrocefin hydrolysis) were also determined. RESULTS: Amoxicillin-resistant Campylobacter were isolated from Flock 1 before and during treatment, but Campylobacter were not detected afterwards. Flock 2 was colonized by amoxicillin-susceptible strains throughout sampling. No amoxicillin-resistant isolates arose during or after treatment. Flock 3 contained amoxicillin-susceptible and -resistant types pre-treatment. Resistant isolates were detected during treatment, while antibiotic-susceptible isolates re-emerged at 3 weeks post-treatment. All Campylobacter isolates from Flock 4 were amoxicillin resistant, irrespective of sampling time. All but one of the 82 amoxicillin-resistant (MICs 16 to >128 mg/L) Campylobacter jejuni and Campylobacter coli tested for the presence of Cj0299 carried the gene and all of these produced beta-lactamase. Co-amoxiclav remained active against amoxicillin-resistant isolates. CONCLUSIONS: Amoxicillin therapy had little effect on the numbers of amoxicillin-resistant commensal Campylobacter except for one flock where amoxicillin-resistant Campylobacter temporarily dominated. Amoxicillin therapy did not select amoxicillin-resistant isolates from a previous susceptible strain. Co-amoxiclav remained active against amoxicillin-resistant isolates.

The response of Campylobacter jejuni to low temperature differs from that of Escherichia coli.

Human infection with Campylobacter jejuni is often associated with the consumption of foods that have been exposed to both chilling and high temperatures. Despite the public health importance of this pathogen, little is known about the effects of cold exposure on its ability to survive a subsequent heat challenge. This work examined the effect of rapid exposure to chilling, as would occur in poultry processing, on the heat resistance at 56 degrees C of two C. jejuni strains, 11168 and 2097e48, and of Escherichia coli K-12. Unlike E. coli K-12, whose cold-exposed cells showed increased sensitivity to 56 degrees C, such exposure had only a marginal effect on subsequent heat resistance in C. jejuni. This may be explained by the finding that during rapid chilling, unlike E. coli cells, C. jejuni cells are unable to alter their fatty acid composition and do not adapt to cold exposure. However, their unaltered fatty acid composition is more suited to survival when cells are exposed to high temperatures. This hypothesis is supported by the fact that in C. jejuni, the ratio of unsaturated to saturated fatty acids was not significantly different after cold exposure, but it was in E. coli. The low-temperature response of C. jejuni is very different from that of other food-borne pathogens, and this may contribute to its tolerance to further heat stresses.

Bacteria isolated from parasitic nematodes--a potential novel vector of pathogens?

Bacterial pathogens are ubiquitous in soil and water - concurrently so are free-living helminths that feed on bacteria. These helminths fall into two categories; the non-parasitic and the parasitic. The former have been the focus of previous work, finding that bacterial pathogens inside helminths are conferred survival advantages over and above bacteria alone in the environment, and that accidental ingestion of non-parasitic helminths can cause systemic infection in vertebrate hosts. Here, we determine the potential for bacteria to be associated with parasitic helminths. After culturing helminths from fecal samples obtained from livestock the external bacteria were removed. Two-hundred parasitic helminths from three different species were homogenised and the bacteria that were internal to the helminths were isolated and cultured. Eleven different bacterial isolates were found; of which eight were indentified. The bacteria identified included known human and cattle pathogens. We concluded that bacteria of livestock can be isolated in parasitic helminths and that this suggests a mechanism by which bacteria, pathogenic or otherwise, can be transmitted between individuals. The potential for helminths to play a role as pathogen vectors poses a potential livestock and human health risk. Further work is required to assess the epidemiological impact of this finding.

Phenotypic and proteomic characterization of multiply antibiotic-resistant variants of Salmonella enterica serovar Typhimurium selected following exposure to disinfectants.

In previous work, Salmonella enterica serovar Typhimurium strain SL1344 was exposed to sublethal concentrations of three widely used farm disinfectants in daily serial passages for 7 days in an attempt to investigate possible links between the use of disinfectants and antimicrobial resistance. Stable variants OXCR1, QACFGR2, and TOPR2 were obtained following treatment with an oxidizing compound blend, a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde, and a tar acid-based disinfectant, respectively. All variants exhibited ca. fourfold-reduced susceptibility to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin. This coincided with reduced levels of outer membrane proteins for all strains and high levels of AcrAB-TolC for OXCR1 and QACFGR2, as demonstrated by two-dimensional high-performance liquid chromatography-mass spectrometry. The protein profiles of OXCR1 and QACFGR2 were similar, but they were different from that of TOPR2. An array of different proteins protecting against oxidants, nitroaromatics, disulfides, and peroxides were overexpressed in all strains. The growth and motility of variants were reduced compared to the growth and motility of the parent strain, the expression of several virulence proteins was altered, and the invasiveness in an enteric epithelial cell line was reduced. The colony morphology of OXCR1 and QACFGR2 was smooth, and both variants exhibited a loss of modal distribution of the lipopolysaccharide O-antigen chain length, favoring the production of short O-antigen chain molecules. Metabolic changes were also detected, suggesting that there was increased protein synthesis and a shift from oxidative phosphorylation to substrate level phosphorylation. In this study, we obtained evidence that farm disinfectants can select for strains with reduced susceptibility to antibiotics, and here we describe changes in protein expression in such strains.

Campylobacter and IFNgamma interact to cause a rapid loss of epithelial barrier integrity.

BACKGROUND: The intestinal epithelium is a single layer of polarized cells and is the primary barrier separating foreign antigen and underlying lymphoid tissue. IFNgamma alters epithelial barrier function during inflammation by disrupting tight cell junctions and facilitating the paracellular transport of luminal antigens. The aim of this work was to determine whether Campylobacter infection of cells exposed to IFNgamma would lead to greater disruption of cell monolayers and hence increased bacterial translocation. METHODS: Monolayers were polarized on Transwell polycarbonate membranes for 14 days and then cultured in the presence or absence of 100 U/mL IFNgamma. Campylobacter was added to the apical side of the monolayer at an MOI of 30. Transepithelial electrical resistance (TEER) was recorded and bacteria in the basal well counted every 2 hours. Cells were stained for occludin, actin, and nuclear DNA, and cell viability determined by measurement of apoptosis. RESULTS: In the presence of IFNgamma, TEER dropped significantly after 18 hours, indicating a reduction in barrier function. A further significant decrease was seen in the presence of both IFNgamma and Campylobacter, indicating a synergistic effect, and cellular morphology and viability were affected. Bacterial translocation across the monolayer was also significantly greater in the presence of IFNgamma. CONCLUSIONS: These combined effects indicate that Campylobacter infection concomitant with intestinal inflammation would result in a rapid and dramatic loss of epithelial barrier integrity, which may be a key event in the pathogenesis of Campylobacter-mediated colitis and the development of bloody diarrhea.

Role of outer membrane lipopolysaccharides in the protection of Salmonella enterica serovar Typhimurium from desiccation damage.

The ability to survive desiccation between hosts is often essential to the success of pathogenic bacteria. The bacterial outer membrane is both the cellular interface with hostile environments and the focus of much of the drying-induced damage. This study examined the contribution of outer membrane-associated polysaccharides to the survival of Salmonella enterica serovar Typhimurium in air-dried blood droplets following growth in high and low osmolarity medium and under conditions known to induce expression of these polysaccharides. Strains lacking the O polysaccharide (OPS) element of the outer membrane lipopolysaccharide were more sensitive to desiccation. Lipopolysaccharide core mutation further to OPS loss did not result in increased susceptibility to drying. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed lipopolysaccharide profiles that supported the hypothesis that OPS expression is required for optimal drying resistance in S. Typhimurium. The role of O antigen in Salmonella spp. in maintaining a hydrated layer around the dried cell or in slowing the rate of dehydration and rehydration is discussed.

Campylobacter colonization of the chicken induces a proinflammatory response in mucosal tissues.

Campylobacter jejuni is a major cause of human inflammatory enteritis, but colonizes the gastrointestinal tract of poultry to a high level in a commensal manner. In vitro, C. jejuni induces the production of cytokines from both human and avian-model epithelial cell and macrophage infections. This suggests that, in vivo, Campylobacter could induce proinflammatory signals in both hosts. We investigated whether a proinflammatory cytokine response can be measured in both day-of-hatch and 2-week-old Light Sussex chickens during infection with C. jejuni. A significant induction of proinflammatory chemokine transcript was observed in birds of both ages, compared with levels in mock-infected controls. This correlated with an influx of heterophils but was not associated with any pathology. These results suggest that in poultry there may be a controlled inflammatory process during colonization.

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.

Campylobacters as zoonotic pathogens: a food production perspective.

Campylobacters remain highly important zoonotic pathogens worldwide which infect an estimated 1% of the population of Western Europe each year. Certain campylobacters are also important in infections of animals, particularly of the reproductive tract, and some are involved in periodontal disease. This paper focuses, however, on the two species which are most important in food-borne infections of humans, Campylobacter (C.) jejuni and C. coli. Infection with these campylobacters is serious in its own right but can also have long-term sequelae such as reactive arthritis and Guillain-Barré syndrome. The pathogens are ubiquitous in nature and in domestic animals and, as a consequence, are found frequently in the environment and on many raw foods, of both plant and animal origin and bacterial numbers can be very high on certain key foods like raw poultry meat. Although all commercial poultry species can carry campylobacters, the risk is greater from chicken because of the high levels of consumption. Campylobacters are relatively 'new' zoonotic pathogens as routine culture from clinical specimens only became possible in the late 1970s. As a consequence there is much that still needs to be understood about the behaviour and pathogenicity of these highly important bacteria. In particular, and from a food industry/food safety perspective, it is important to better understand the behaviour of C. jejuni and C. coli in the food production environment, and how this affects their ability to survive certain food production processes. There is a belief that campylobacters are much more sensitive to hostile conditions than either salmonellas or Escherichia coli. Much of data to support this view have been derived from laboratory experiments and may not fully represent the natural situation. Studies are showing that campylobacters may be more robust than previously thought and thus may represent a greater challenge to food safety. We recommend that research is undertaken to better understand how campylobacters behave in the food chain and how responses to relevant conditions affect their ability to survive processing and their virulence. There is also a need to better understand the reasons why campylobacters are capable of frequent change, particularly in the expression of surface antigens.