Campylobacter spp., Yersinia enterocolitica, and Salmonella enterica and their simultaneous occurrence in German fattening pig herds and their environment.

Campylobacter spp., Salmonella enterica, and Yersinia enterocolitica are common causes of foodborne infections in humans with pork as a potential source. Monitoring programs at farm level are, to date, only implemented for S. enterica, while epidemiological knowledge of the other two pathogens is still lacking. This study aimed to assess the pathogen load (in the pigs' environment) in fattening pig herds, their simultaneous occurrence, and the occurrence of Campylobacter spp. and Y. enterocolitica in herds in different Salmonella risk categories. In 50 fattening pig herds in northern Germany, four pooled fecal samples and 10 swab samples from the pigs' direct environment (pen walls, nipple drinkers), indirect environment (hallways, drive boards), and flies and rodent droppings were collected from each herd and submitted for cultural examination. Campylobacter spp. were detected in 38.1% of fecal, 32.7% of direct environment, 5.3% of indirect environment, and 4.6% of flies/pests samples collected, and Y. enterocolitica in 17.1, 8.1, 1.2, and 3.1% and S. enterica in 11.2, 7.7, 4.1, and 1.5%, respectively. For Campylobacter spp., Y. enterocolitica, and S. enterica, 80, 48, and 32% of herds were positive, respectively; 22 herds were positive for both Campylobacter spp. and Y. enterocolitica, 12 for Campylobacter spp. and S. enterica, and 7 for Y. enterocolitica and S. enterica. There was no significant association between the pathogens at herd level. Campylobacter spp. and Y. enterocolitica were found more often in samples from the low Salmonella risk category (odds ratio, 0.51; confidence interval, 0.36 to 0.73, and 0.3, 0.17 to 0.57), and this was also the case for Y. enterocolitica at herd level (odds ratio, 0.08; confidence interval, 0.02 to 0.3). This study provides evidence that the pigs' environment should be accounted for when implementing control measures on farms against Campylobacter spp. and Y. enterocolitica. An extrapolation from the current Salmonella monitoring to the other two pathogens does not seem feasible.

OXA-48 carbapenemase-producing Salmonella enterica serovar Kentucky isolate of sequence type 198 in a patient transferred from Libya to Switzerland

Here, we report a case of OXA-48-producing Salmonella enterica serovar Kentucky of sequence type 198 (ST198) from perianal screening cultures of a patient transferred from Libya to Switzerland. The blaOXA-48 gene was carried by Tn1999.2 and located on an ∼60-kb IncL/M plasmid. This Salmonella strain also possessed the blaVEB-8, aac(6)-Ib, tet(A), sul1, and mphA resistance genes and substitutions in GyrA (Ser83Phe and Asp87Asn) and ParC (Ser80Ile). This finding emphasizes that prompt screening strategies are essential to prevent the dissemination of carbapenemase producers imported from countries where they are endemic.

Virulence of broad- and narrow-host-range Salmonella enterica serovars in the streptomycin-pretreated mouse model.

Salmonella enterica subspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a given S. enterica serovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying other S. enterica subspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of various S. enterica subspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject of Salmonella genome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.

Comparison of Salmonella enterica serovar Typhimurium colitis in germfree mice and mice pretreated with streptomycin.

Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of bacterial enterocolitis. Mice are generally protected from Salmonella serovar Typhimurium colonization and enterocolitis by their resident intestinal microflora. This phenomenon is called "colonization resistance" (CR). Two murine Salmonella serovar Typhimurium infection models are based on the neutralization of CR: (i) in specific-pathogen-free mice pretreated with streptomycin (StrSPF mice) antibiotics disrupt the intestinal microflora; and (ii) germfree (GF) mice are raised without any intestinal microflora, but their intestines show distinct physiologic and immunologic characteristics. It has been unclear whether the same pathogenetic mechanisms trigger Salmonella serovar Typhimurium colitis in GF and StrSPF mice. In this study, we compared the two colitis models. In both of the models Salmonella serovar Typhimurium efficiently colonized the large intestine and triggered cecum and colon inflammation starting 8 h postinfection. The type III secretion system encoded in Salmonella pathogenicity island 1 was essential in both disease models. Thus, Salmonella serovar Typhimurium colitis is triggered by similar pathogenetic mechanisms in StrSPF and GF mice. This is remarkable considering the distinct physiological properties of the GF mouse gut. One obvious difference was more pronounced damage and reduced regenerative response of the cecal epithelium in GF mice. Overall, StrSPF mice and GF mice provide similar but not identical models for Salmonella serovar Typhimurium colitis.

Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice.

Salmonella enterica subspecies 1 serovar Typhimurium is a common cause of gastrointestinal infections. The host's innate immune system and a complex set of Salmonella virulence factors are thought to contribute to enteric disease. The serovar Typhimurium virulence factors have been studied extensively by using tissue culture assays, and bovine infection models have been used to verify the role of these factors in enterocolitis. Streptomycin-pretreated mice provide an alternative animal model to study enteric salmonellosis. In this model, the Salmonella pathogenicity island 1 type III secretion system has a key virulence function. Nothing is known about the role of other virulence factors. We investigated the role of flagella in murine serovar Typhimurium colitis. A nonflagellated serovar Typhimurium mutant (fliGHI) efficiently colonized the intestine but caused little colitis during the early phase of infection (10 and 24 h postinfection). In competition assays with differentially labeled strains, the fliGHI mutant had a reduced capacity to get near the intestinal epithelium, as determined by fluorescence microscopy. A flagellated but nonchemotactic cheY mutant had the same virulence defects as the fliGHI mutant for causing colitis. In competitive infections, both mutants colonized the intestine of streptomycin-pretreated mice by day 1 postinfection but were outcompeted by the wild-type strain by day 3 postinfection. Together, these data demonstrate that flagella are required for efficient colonization and induction of colitis in streptomycin-pretreated mice. This effect is mostly attributable to chemotaxis. Recognition of flagellar subunits (i.e., flagellin) by innate immune receptors (i.e., Toll-like receptor 5) may be less important.

InvB is required for type III-dependent secretion of SopA in Salmonella enterica serovar Typhimurium.

The Salmonella effector protein SopA is translocated into host cells via the SPI-1 type III secretion system (TTSS) and contributes to enteric disease. We found that the chaperone InvB binds to SopA and slightly stabilizes it in the bacterial cytosol and that it is required for its transport via the SPI-1 TTSS.

Role of the Salmonella pathogenicity island 1 effector proteins SipA, SopB, SopE, and SopE2 in Salmonella enterica subspecies 1 serovar Typhimurium colitis in streptomycin-pretreated mice.

Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium) induces enterocolitis in humans and cattle. The mechanisms of enteric salmonellosis have been studied most extensively in calf infection models. The previous studies established that effector protein translocation into host cells via the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (TTSS) is of central importance in serovar Typhimurium enterocolitis. We recently found that orally streptomycin-pretreated mice provide an alternative model for serovar Typhimurium colitis. In this model the SPI-1 TTSS also plays a key role in the elicitation of intestinal inflammation. However, whether intestinal inflammation in calves and intestinal inflammation in streptomycin-pretreated mice are induced by the same SPI-1 effector proteins is still unclear. Therefore, we analyzed the role of the SPI-1 effector proteins SopB/SigD, SopE, SopE2, and SipA/SspA in elicitation of intestinal inflammation in the murine model. We found that sipA, sopE, and, to a lesser degree, sopE2 contribute to murine colitis, but we could not assign an inflammation phenotype to sopB. These findings are in line with previous studies performed with orally infected calves. Extending these observations, we demonstrated that in addition to SipA, SopE and SopE2 can induce intestinal inflammation independent of each other and in the absence of SopB. In conclusion, our data corroborate the finding that streptomycin-pretreated mice provide a useful model for studying the molecular mechanisms of serovar Typhimurium colitis and are an important starting point for analysis of the molecular events triggered by SopE, SopE2, and SipA in vivo.

Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host.

Salmonella enterica subspecies 1 serovar Typhimurium is a principal cause of human enterocolitis. For unknown reasons, in mice serovar Typhimurium does not provoke intestinal inflammation but rather targets the gut-associated lymphatic tissues and causes a systemic typhoid-like infection. The lack of a suitable murine model has limited the analysis of the pathogenetic mechanisms of intestinal salmonellosis. We describe here how streptomycin-pretreated mice provide a mouse model for serovar Typhimurium colitis. Serovar Typhimurium colitis in streptomycin-pretreated mice resembles many aspects of the human infection, including epithelial ulceration, edema, induction of intercellular adhesion molecule 1, and massive infiltration of PMN/CD18(+) cells. This pathology is strongly dependent on protein translocation via the serovar Typhimurium SPI1 type III secretion system. Using a lymphotoxin beta-receptor knockout mouse strain that lacks all lymph nodes and organized gut-associated lymphatic tissues, we demonstrate that Peyer's patches and mesenteric lymph nodes are dispensable for the initiation of murine serovar Typhimurium colitis. Our results demonstrate that streptomycin-pretreated mice offer a unique infection model that allows for the first time to use mutants of both the pathogen and the host to study the molecular mechanisms of enteric salmonellosis.

Protection from superoxide damage associated with an increased level of the YggX protein in Salmonella enterica

The deleterious effect of superoxide radicals on cell growth and survival is predominately caused by rapid oxidation of labile [Fe-S] clusters in proteins. Oxidation of these clusters releases Fe(II) ions, which participate in Fenton chemistry that damages DNA. Here it is shown that elevated levels of the YggX protein increase the resistance of Salmonella enterica to superoxide stress, reverse enzymatic defects attributed to oxidized [Fe-S] clusters, and decrease the spontaneous mutation frequency. The data are consistent with a model in which YggX protects protein [Fe-S] clusters from oxidation.

Relationship between evolutionary rate and cellular location among the Inv/Spa invasion proteins of Salmonella enterica.

For 21 strains of Salmonella enterica, nucleotide sequences were obtained for three invasion genes, spaO, spaP, and spaQ, of the chromosomal inv/spa complex, the products of which form a protein export system required for entry of the bacteria into nonphagocytic host cells. These genes are present in all eight subspecies of the salmonellae, and homologues occur in a variety of other bacteria, including the enteric pathogens Shigella and Yersinia, in which they are plasmid borne. Evolutionary diversification of the invasion genes among the subspecies of S. enterica has been generally similar in pattern and average rate to that of housekeeping genes. However, the range of variation in evolutionary rate among the invasion genes is unusually large, and there is a relationship between the evolutionary rate and cellular location of the invasion proteins, possibly reflecting diversifying selection on exported proteins in adaptation to variable host factors in extracellular environments. The SpaO protein, which is hypervariable in S. enterica and exhibits only 24% sequence identity with its homologues in Shigella and Yersinia, is secreted. In contrast, the membrane-associated proteins SpaP, SpaQ, and InvA are weakly polymorphic and have > 60% sequence identity with the corresponding proteins of other enteric bacteria. Acquisition of the inv/spa genes may have been a key event in the evolution of the salmonellae as pathogens, following which the invention of flagellar phase shifting facilitated niche expansion to include warm-blooded vertebrates.

Investigation of ansB and sspA derived promoters for multi- and single-copy antigen expression in attenuated Salmonella enterica var. typhimurium

Five candidate promoters were examined to determine their utility in directing immunogenic levels of expression of the C fragment from tetanus toxin in attenuated S. enterica used as an oral vaccine in mice. Promoters derived from the genes encoding the stringent starvation protein (sspA) from E. coli and S. enterica, but not ansB derived promoters, expressed immunogenic levels of C fragment from multi-copy plasmids in attenuated S. enterica in vivo and, following oral immunization, induced high titre specific anti-tetanus toxoid serum antibodies. We also demonstrate that not only the choice of promoter, replicon and growth conditions but also how expression constructs are assembled in the chosen plasmid is critical for the successful development of plasmid-based antigen delivery systems using attenuated S. enterica. In addition, the S. enterica sspA promoter is able to elicit anti-tetanus toxoid antibodies in mice when the psspA-tetC expression cassette is integrated in single copy on the S. enterica chromosome.

Long-term persistence of multi-drug-resistant Salmonella enterica serovar Newport in two dairy herds

Objective - To evaluate the association between maintaining joint hospital and maternity pens;and persistence of multi-drug-resistant (MDR) Salmonella enterica serovar Newport on 2 dairy farms. Design - Observational study. Sample Population - Feces and environmental samples from 2 dairy herds. Procedure - Herds were monitored for fecal shedding of S enterica Newport after outbreaks of clinical disease. Fecal and environmental samples were collected approximately monthly from pens housing sick cows and calving cows and from pens containing lactating cows. Cattle shedding the organism were tested serially on subsequent visits to determine carrier status. One farm was resampled after initiation of interventional procedures, including separation of hospital and maternity pens. Isolates were characterized via serotyping, determination of antimicrobial resistance phenotype, detection of the CMY-2 gene, and DNA fingerprinting. Results - The prevalence (32.4% and 33.3% on farms A and B, respectively) of isolating Salmonella from samples from joint hospital-maternity pens was significantly higher than the prevalence in samples from pens housing preparturient cows (0.8%, both farms) and postparturient cows on Farm B (8.8%). Multi-drug-resistant Salmonella Newport was isolated in high numbers from bedding material, feed refusals, lagoon slurry, and milk filters. One cow excreted the organism for 190 days. Interventional procedures yielded significant reductions in the prevalences of isolating the organism from fecal and environmental samples. Most isolates were of the C2 serogroup and were resistant to third-generation cephalosporins. Conclusions and Clinical Relevance - Management practices may be effective at reducing the persistence of MDR Salmonella spp in dairy herds, thus mitigating animal and public health risk.

The SEF14 fimbrial antigen of Salmonella enterica serovar Enteritidis is encoded within a pathogenicity islet

The DNA sequence of the chromosomal gene cluster encoding the SEF14 fimbriae of Salmonella enterica serovar Enteritidis was determined. Five contiguous open reading frames, sefABCDE, were identified. The sefE gene shared significant homology with araC-like positive regulators. Serovar-associated virulence plasmid (SAP) genes orf7,8,9 and pefI were identified immediately adjacent to the sef operon. The pefI gene encoded a putative regulator of the Plasmid-encoded fimbrial antigen (PEF) expression. The entire sef--pef region, flanked by two IS-like elements, was inserted adjacent to leuX that encoded a transfer RNA molecule. The organisation of this region was suggestive of a classic pathogenicity islet. Southern hybridisation confirmed two copies of the SAP derived orf7,8,9 and pefI region in S. Enteritidis, one in the chromosome and one on the SAP. Of other group D Salmonella, only S. Blegdam and S. Moscow harboured both chromosomal and plasmid copies of pefI--orf9 region although polymorphism was evident.

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

Antibiotic and biocide resistance in Salmonella enterica and Escherichia coli 0157

Bacterial resistance to antibiotics and biocides is a prevalent problem, which may be exacerbated by the commonplace and often unnecessary inclusion of biocides into domestic products. Addition of antimicrobials, to domestic disinfectants has raised concern about promoting microbial resistance and potential cross-resistance to therapeutic antibiotics. This study investigated the potential for resistance in Salmonella enterica serovars Enteritidis, Typhimurium, Virchow and Escherichia call 0157 to commonly used biocides, to identify mechanisms underlying resistance and whether these provided cross-resistance to antibiotics. Salmonella enterica and E. coli 0157 strains were serially exposed to sub-inhibitory. concentrations of erythromycin (ERY), benzalkonium chloride (BKC), chlorhexidine hydrochloride (CHX)and triclosan (TLN). Once resistance was achieved permeability changes in the outer membrane, including LPS, cell surface charge and hydrophobicityand the presence of,an active efflux were investigated as possible resistance candidates. Thin layer chromatography (TLC) and Gas chromatography (GC) were carried out to examine fatty acid and lipid changes in E. coli 0157 isolates with reduced susceptibility to TLN. Cross-resistance was studied by the Stoke's method and standard microdilution assays. Examination of the outer membrane proteins and LPS did not reveal any significant changes between parent and resistant strains. The hydrophobicity of the cells increased as the cells were passaged and became less. susceptible. An active efflux system was the most likely mechanism of resistance in all strains tested and a fab1 mutation was associated with E. coli 0157 resistant to TLN isolates. In all isolates investigated the resistance was stable for over 30 passages in biocide-free media. A high degree of cross-resistance was obtained in TLN-resjstant Escherichia coli 0157 strains, which repeatedly exerted decreased susceptibility to various antimicrobials, including chloramphenicol, erythromycin, imipenem, tetracycline and trimethoprirn:, as well as to various biocides. The results of this laboratory-based investigation suggest that it is possible for microorganisms to become resistant to biocides when repeatedly exposed to sublethal concentrations. This may be especially the case in the domestic environment where administration of biocides is poorly controlled. Eventually it could lead to the undesirable situation of resident strains becoming resistant to disinfection and cross resistant to other antimicrobials.