The efficacy of Salenvac, a Salmonella enterica subsp. Enterica serotype Enteritidis iron-restricted bacterin vaccine, in laying chickens

The protective effect of two vaccination regimes using Salenvac, a commercially available iron-restricted Salmonella enterica subsp. Enterica serotype Enteritidis PT4 bacterin vaccine, was verified in laying birds. Immunization was intramuscular at 1 day old and again at 4 weeks of age (V2), or at 1 day and 4 weeks with a third dose at 18 weeks of age (V3). Challenge S. Enteritidis (5 to 7.5) x 10(7) colony forming units) was given intravenously at 8, 17, 23, 30 and 59 weeks of age. For all age groups, both vaccination regimes reduced significantly the number of tissues and faecal samples that were culture positive for the challenge strain. For laying birds, fewer eggs (P < 0.001) were culture positive for S. Enteritidis after challenge from vaccinated laying birds ( 56/439 batches of eggs) than unvaccinated birds (99/252 batches). The data give compelling evidence that the vaccine is efficacious and may contribute to the reduction of layer infection and egg contamination.

The effect of chlortetracycline treatment and its subsequent withdrawal on multi-resistant Salmonella enterica serovar Typhimurium DT104 and commensal Escherichia coli in the pig

Aims: To investigate the effect of a therapeutic and sub-therapeutic chlortetracycline treatment on tetracyclineresistant Salmonella enterica serovar Typhimurium DT104 and on the commensal Escherichia coli in pig. Methods and Results: Salmonella Typhimurium DT104 was orally administered in all pigs prior to antibiotic treatment, and monitored with the native E. coli. Higher numbers of S. Typhimurium DT104 were shed from treated pigs than untreated pigs. This lasted up to 6 weeks post-treatment in the high-dose group. In this group, there was a 30% increase in E. coli with a chlortetracycline minimal inhibitory concentration (MIC) > 16 mg l(-1) and a 10% increase in E. coli with an MIC > 50 mg l(-1) during and 2 weeks post-treatment. This effect was less-pronounced in the low-dose group. PCR identified the predominant tetracycline resistance genes in the E. coli as tetA, tetB and tetC. The concentration of chlortetracycline in the pig faeces was measured by HPLC and levels reached 80 mug g(-1) faeces during treatment. Conclusion: Chlortetracycline treatment increases the proportion of resistant enteric bacteria beyond the current withdrawal time. Significance and Impact of the Study: Treated pigs are more likely to enter abattoirs with higher levels of resistant bacteria than untreated pigs promoting the risk of these moving up the food chain and infecting man.

Membrane ruffling and invasion of human and avian cell lines is reduced for aflagellate mutants of Salmonella enterica serotype Enteritidis

Independent studies have demonstrated that flagella are associated with the invasive process of Salmonella enterica serotypes, and aflagellate derivatives of Salmonella enterica serotype Enteritidis are attenuated in murine and avian models of infection. One widely held view is that the motility afforded by flagella, probably aided by chemotactic responses, mediates the initial interaction between bacterium and host cell. The adherence and invasion properties of two S. Enteritidis wild-type strains and isogenic aflagellate mutants were assessed on HEp-2 and Div-1 cells that are of human and avian epithelial origin, respectively. Both aflagellate derivatives showed a significant reduction of invasion compared with wild type over the three hours of the assays. Complementation of the defective fliC allele recovered partially the wild-type phenotype. Examination of the bacterium-host cell interaction by electron and confocal microscopy approaches showed that wild-type bacteria induced ruffle formation and significant cytoskeletal rearrangements on HEp-2 cells within 5 minutes of contact. The aflagellate derivatives induced fewer ruffles than wild type. Ruffle formation on the Div-1 cell line was less pronounced than for HEp-2 cells for wild-type S. Enteritidis. Collectively, these data support the hypothesis that flagella play an active role in the early events of the invasive process.

Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens

Cost effective control of avian diseases and food borne pathogens remains a high priority for all sectors of the poultry industry with cleansing and disinfection, vaccination and competitive exclusion approaches being used widely. Previous studies showed that Bacillus subtilis PY79(hr) was an effective competitive exclusion agent for use in poultry to control avian pathogenic Escherichia coli serotype O78:K80. Here we report experiments that were undertaken to test the efficacy of B. subtilis PY79(hr) in the control of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens. To do this, 1-day-old and 20-day-old specific pathogen free (SPF) chicks were dosed with a suspension of B. subtilis spores prior to challenge with S. Enteritidis (S1400) and C. perfringens, respectively. For both challenge models, a single oral inoculum of 1 x 10(9) spores given 24 h prior to challenge was sufficient to suppress colonisation and persistence of both S. Enteritidis and C perfringens. In particular, the faecal shedding of S. Enteritidis, as measured by a semi-quantitative cloacal swabbing technique, was reduced significantly for the 36 days duration of the experiment. B. subtilis persisted in the intestine although with decreasing numbers over the same period. These data add further evidence that B. subtilis spores may be effective agents in the control of avian diseases and food borne pathogens.

Lack of flagella disadvantages Salmonella enterica serovar Enteritidis during the early stages of infection in the rat

The roles of flagella and five fimbriae (SEF14, SEF17, SEF21, pef, lpf) in the early stages (up to 3 days) of Salmonella enterica serovar Enteritidis (S. Enteritidis) infection have been investigated in the rat. Wild-type strains LA5 and S1400 (fim+/fla+) and insertionally inactivated mutants unable to express the five fimbriae (fim-/fla+), flagella (fim+/fla-) or fimbriae and flagella (fim-/fla-) were used. All wild-type and mutant strains were able to colonize the gut and spread to the mesenteric lymph nodes, liver and spleen. There appeared to be little or no difference between the fim-/fla+ and wild-type (fim+/fla+) strains. In contrast, the numbers of aflagellate (fim+/fla- or fim-/fla-) salmonella in the liver and spleen were transiently reduced. In addition, fim+/fla- or fim-/fla-strains were less able to persist in the upper gastrointestinal tract and the inflammatory responses they elicited in the gut were less severe. Thus, expression of SEF14, SEF17, SEF21, pef and lpf did not appear to be a prerequisite for induction of S. Enteritidis infection in the rat. Deletion of flagella did, however, disadvantage the bacterium. This may be due to the inability to produce or release the potent immunomodulating protein flagellin.

The O-antigen of Salmonella enterica serotype Enteritidis PT4: a significant factor in gastrointestinal colonisation of young but not newly hatched chicks

The lipopolysaccharide of Salmonella and other Gram negative pathogenic species has been implicated as a major virulence determinant and in this study we report the role of LPS of S. Enteritidis in the colonisation and persistent gastrointestinal infection of young poultry. The gene encoding the unique O-antigen ligase, waaL, was mutated by insertional inactivation in a well characterised S. Enteritidis strain, S1400/94. The waaL mutant, designated PCP, produced rough colonies on agar medium, did not agglutinate O9 antiserum, did not produce an LPS ladder on silver stained gels and was serum sensitive. PCP and a nalidixic acid marked derivative of S1400/94 (S1400/94 Nal(r)) were used to orally challenge young chicks, separately and together in competitive index experiments. At post-mortem examination of 1-day-old chicks challenged S1400/94 Nal(r) and PCP separately there were no significant differences in the numbers of S1400/94 Nal(r) and PCP bacteria in tissues sampled on days 1, 2. and 5. By day 42 after challenge S1400/94 Nal(r) bacteria were recovered in significantly higher numbers than PCP from the caecal contents (P < 0.001). In competitive index studies in the 1-day-old chick PCP colonised, invaded and persisted in lower numbers than S1400/94 Nal(r). In 4-week-old chicks challenged separately, PCP bacteria were recovered from all tissues examined in significantly lower numbers than S1400/94 Nal(r). In competitive index experiments in 4-week-old chicks, PCP was not detected at any site and at any time point. Therefore, the O-antigen of S. Enteritidis plays art important role in poultry infections although this role is less important in the newly hatched chick. Crown Copyright (C) 2004 Published by Elsevier B.V. All rights reserved.

Flagella and curli fimbriae are important for the growth of Salmonella enterica serovars in hen eggs

Salmonella enterica serovar Enteritidis is unable to multiply in the albumen of fresh eggs and must gain access to the yolk contents in order to multiply to a high level (> 10(6) c.f.u. per ml egg contents). As human Salmonella infections resulting from the consumption of infected eggs more frequently involve serovar Enteritidis phage type (PT) 4 than other serovars or PTs, a number of isolates of various S. enterica serovars were examined for their ability to multiply to a high level in eggs over a period of 8 days storage at 20 degreesC. Their behaviour was compared to that of a range of defined fimbrial and flagella mutants of S. Enteritidis. Strains that did not express flagella were unable to multiply in eggs, and those deficient for curli fimbriae, including strains of S. Enteritidis PT6, displayed high-level growth in significantly fewer eggs than those able to express curli. Most S. Enteritidis strains multiplied to a high level in between 5 and 10 % of eggs during 8 days storage. One PT4 strain, though, showed high levels of growth in more than 25 % of eggs over this period, significantly higher than the other PTs or the two other isolates of PT4 tested. This ability may be important for the association of PT4 infection with the consumption of eggs.

Effect of a 5 day enrofloxacin treatment on Salmonella enterica serotype Typhimurium DT104 in the pig

Objectives: There are concerns that the use of enrofloxacin in livestock production may contribute to the development of fluoroquinolone resistance in zoonotic bacteria. The objective of our study was to investigate the effect of a single 5 day enrofloxacin treatment on Salmonella enterica serotype Typhimurium DT104 in a pig model. Results: Our results showed that a single treatment failed to eradicate S. Typhimurium DT104, which continued to be isolated up to 35 days after treatment. We also provide evidence that treatment positively selects for S. Typhimurium DT104 strains that are already nalidixic acid resistant (gyrA Asn-87) or cyclohexane resistant, the latter being indicative of an up-regulated efflux pump. Emergence of fluoroquinolone resistance was not detected during treatment or post-treatment in any of the Salmonella strains monitored. However, the effect of enrofloxacin on the nalidixic acid-resistant and cyclohexane-resistant S. Typhimurium DT104 outlasted the current withdrawal time of 10 days for Baytril (commercial veterinary formulation of enrofloxacin). Conclusions: In conclusion, our study has provided direct evidence that enrofloxacin-treated pigs could be entering abattoirs with higher numbers of quinolone-resistant zoonotic bacteria than untreated pigs, increasing the risk of these entering the food chain.

Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica

Salmonella enterica isolates (n = 182) were examined for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE. The frequency, location, and type of GyrA substitution varied with the serovar. Mutations were found in parC that encoded Thr57-Ser, Thr66-Ile, and Ser80-Arg substitutions. Mutations in the gyrB quinolone resistance-determining region were located at codon Tyr420-Cys or Arg437-Len. Novel mutations were also found in parE encoding Glu453-Gly, His461-Tyr, Ala498-Thr, Val512-Gly, and Ser518-Cys. Although it is counterintuitive, isolates with a mutation in both gyrA and parC were more susceptible to ciprofloxacin than were isolates with a mutation in gyrA alone.

Antibiotic resistance genes, integrons and multiple antibiotic resistance in thirty-five serotypes of Salmonella enterica isolated from humans and animals in the UK

Objectives: To examine 397 strains of Salmonella enterica of human and animal origin comprising 35 serotypes for the presence of aadB, aphAI-IAB, aadA1, aadA2, bla(Carb(2)) or pse1, bla(Tem), cat1, cat2, dhfr1, floR, strA, sul1, sul2, tetA(A), tetA(B) and tetA(G) genes, the presence of class 1 integrons and the relationship of resistance genes to integrons and antibiotic resistance. Results: Some strains were resistant to ampicillin (91), chloramphenicol (85), gentamicin (2), kanamycin (14), spectinomycin (81), streptomycin (119), sulfadiazine (127), tetracycline (108) and trimethoprim (45); 219 strains were susceptible to all antibiotics. bla(Carb(2)), floR and tetA(G) genes were found in S. Typhimurium isolates and one strain of S. Emek only. Class 1 integrons were found in S. Emek, Haifa, Heidelberg, Mbandaka, Newport, Ohio, Stanley, Virchow and in Typhimurium, mainly phage types DT104 and U302. These strains were generally multi-resistant to up to seven antibiotics. Resistance to between three and six antibiotics was also associated with class 1 integron-negative strains of S. Binza, Dublin, Enteritidis, Hadar, Manhattan, Mbandaka, Montevideo, Newport, Typhimurium DT193 and Virchow. Conclusion: The results illustrate specificity of some resistance genes to S. Typhimurium or non- S. Typhimurium serotypes and the involvement of both class 1 integron and non-class 1 integron associated multi-resistance in several serotypes. These data also indicate that the bla(Carb(2)), floR and tetA(G) genes reported in the SG1 region of S. Typhimurium DT104, U302 and some other serotypes are still predominantly limited to S. Typhimurium strains.

Effect of triclosan or a phenolic farm disinfectant on the selection of antibiotic-resistant Salmonella enterica

Objective: To determine the effect of growth of five strains of Salmonella enterica and their isogenic multiply antibiotic-resistant (MAR) derivatives with a phenolic farm disinfectant or triclosan (biocides) upon the frequency of mutation to resistance to antibiotics or cyclohexane. Methods: Strains were grown in broth with or without the biocides and then spread on to agar containing ampicillin, ciprofloxacin or tetracycline each at 4x MIC or agar overlaid with cyclohexane. Incubation was for 24 and 48 h and the frequency of mutation to resistance was calculated for strains with and without prior growth with the biocides. MICs were determined and the presence of mutations in the acrR and marR regions was determined by sequencing and the presence of mutations in gyrA by light-cycler analysis, for a selection of the mutants that arose. Results: The mean frequency of mutation to antibiotic or cyclohexane resistance was increased similar to10- to 100-fold by prior growth with the phenolic disinfectant or triclosan. The increases were statistically significant for all antibiotics and cyclohexane following exposure to the phenolic disinfectant (P less than or equal to 0.013), and for ampicillin and cyclohexane following exposure to triclosan (P less than or equal to 0.009). Mutants inhibited by >1 mg/L ciprofloxacin arose only from strains that were MAR. Reduced susceptibility to ciprofloxacin (at 4x MIC for parent strains) alone was associated with mutations in gyrA. MAR mutants did not contain mutations in the acrR or marR region. Conclusions: These data renew fears that the use of biocides may lead to an increased selective pressure towards antibiotic resistance.

Mutant prevention concentrations of ciprofloxacin and enrofloxacin for Salmonella enterica

Objectives: To determine the mutant prevention concentrations (MPCs) of ciprofloxacin and enrofloxacin against four strains of Salmonella enterica serovar Enteritidis and four strains of S. Typhimurium including one fully susceptible, one multiply resistant (MAR), one GyrA mutant and one GyrA/MAR mutant. Further, to examine mutants arising after exposure to sub-MPC concentrations of the antibiotics for susceptibility to ciprofloxacin and enrofloxacin, and cyclohexane tolerance. Methods: MICs were determined using the agar dilution method of the BSAC. The MPC was recorded as the lowest concentration of antibiotic to inhibit growth from an inoculum of 10(10) cfu. Results: The MPCs and resulting MPC/MIC ratios of enrofloxacin were generally two- to four-fold higher than for ciprofloxacin. At 24 h for both antibiotics, MPCs were lowest for the fully susceptible strains (0.25-0.5 mg/L), similar for the MAR (1-4 mg/L) and GyrA (2-4 mg/L) mutants and highest for the GyrA/MAR mutants (1-8 mg/L). MPC/MIC ratios at 24 h were 2-16 for all strains except those for the MAR strains without mutation in gyrA where the ratios were 8-64. Conclusions: The ability to eradicate Salmonella in vivo depends on many factors such as antibiotic susceptibility of the strain, dose and route of administration. It is suggested that these MPC values will be useful when considering dosing strategies. In view of the high MPC/MIC ratio, MAR strains with wild-type gyrA, although susceptible to ciprofloxacin (MICs 0.06-0.13 mg/L), may give rise to treatment failures.

The role of prophage-like elements in the diversity of Salmonella enterica serovars

The Salmonella enterica serovar Typhi CT18 (S. Typhi) chromosome harbours seven distinct prophage-like elements, some of which may encode functional bacteriophages. In silico analyses were used to investigate these regions in S. Typhi CT18, and ultimately compare these integrated bacteriophages against 40 other Salmonella isolates using DNA microarray technology. S. Typhi CT18 contains prophages that show similarity to the lambda, Mu, P2 and P4 bacteriophage families. When compared to other S. Typhi isolates, these elements were generally conserved, supporting a clonal origin of this serovar. However, distinct variation was detected within a broad range of Salmonella serovars; many of the prophage regions are predicted to be specific to S. Typhi. Some of the P2 family prophage analysed have the potential to carry non-essential "cargo" genes within the hyper-variable tail region, an observation that suggests that these bacteriophage may confer a level of specialisation on their host. Lysogenic bacteriophages therefore play a crucial role in the generation of genetic diversity within S. enterica. (C) 2004 Elsevier Ltd. All rights reserved.

Identification of core and variable components of the Salmonella enterica subspecies I genome by microarray

We have performed microarray hybridization studies on 40 clinical isolates from 12 common serovars within Salmonella enterica subspecies I to identify the conserved chromosomal gene pool. We were able to separate the core invariant portion of the genome by a novel mathematical approach using a decision tree based on genes ranked by increasing variance. All genes within the core component were confirmed using available sequence and microarray information for S. enterica subspecies I strains. The majority of genes within the core component had conserved homologues in Escherichia coli K-12 strain MG1655. However, many genes present in the conserved set which were absent or highly divergent in K-12 had close homologues in pathogenic bacteria such as Shigella flexneri and Pseudomonas aeruginosa. Genes within previously established virulence determinants such as SPI1 to SPI5 were conserved. In addition several genes within SPI6, all of SPI9, and three fimbrial operons (fim, bcf, and stb) were conserved within all S. enterica strains included in this study. Although many phage and insertion sequence elements were missing from the core component, approximately half the pseudogenes present in S. enterica serovar Typhi were conserved. Furthermore, approximately half the genes conserved in the core set encoded hypothetical proteins. Separation of the core and variant gene sets within S. enterica subspecies I has offered fundamental biological insight into the genetic basis of phenotypic similarity and diversity across S. enterica subspecies I and shown how the core genome of these pathogens differs from the closely related E. coli K-12 laboratory strain.