Effect of fluoroquinolone exposure on the proteome of Salmonella enterica serovar Typhimurium

Objectives: The physiological response of Salmonella enterica serovar Typhimurium to fluoroquinolone antibiotics was investigated using proteomic methods. Methods: Proteomes were prepared from strain SL1344 following treatment of broth cultures with ciprofloxacin (0.03 and 0.008 mg/L; 2x and 0.5x MIC) and enrofloxacin (0.03 mg/L) and from a multiple antibiotic resistant (MAR) mutant. Protein expression was determined by two-dimensional HPLC-MSn and also after exposure to ciprofloxacin by two-dimensional gel electrophoresis (2D-GE). Results: The number of proteins (mean +/- SD) detected by 2D-GE derived from control cultures of the wild-type strain was significantly (P < 0.05) reduced from 296 +/- 77 to 153 +/- 36 following treatment with ciprofloxacin (0.03 mg/L). Raised expression (P < 0.05) of 17 proteins was also detected, and increases of up to 8-fold (P < 0.0001) were observed for subunits of F1F0-ATP synthase, TolC and Imp. Analysis by two-dimensional HPLC-MSn provided higher proteome coverage with 787 +/- 50 proteins detected, which was reduced (P < 0.005) to 560 +/- 14 by ciprofloxacin (0.03 mg/L). Increased expression of 43 proteins was observed which included those detected by 2D-GE and additionally the efflux pump protein AcrB. The basal expression of the AcrAB/TolC efflux pump was elevated in the MAR mutant compared with the untreated wild-type and augmented following treatment with ciprofloxacin (0.03 mg/L). F1F0-ATP synthase and Imp were only elevated in the mutant when treated with ciprofloxacin. Conclusions: These studies suggest that increased expression of AcrAB/TolC was associated with resistance while other increases, such as in F1F0-ATP synthase and Imp, were a response to fluoroquinolone.

Modification of enrofloxacin treatment regimens for poultry experimentally infected with Salmonella enterica serovar Typhimurium DT104 to minimize selection of resistance

We hypothesized that higher doses of fluoroquinolones for a shorter duration could maintain efficacy (as measured by reduction in bacterial count) while reducing selection in chickens of bacteria with reduced susceptibility. Chicks were infected with Salmonella enterica serovar Typhimurium DT104 and treated 1 week later with enrofloxacin at the recommended dose for 5 days (water dose adjusted to give 10 mg/kg of body weight of birds or equivalence, i.e., water at 50 ppm) or at 2.5 or 5 times the recommended dose for 2 days or 1 day, respectively. The dose was delivered continuously (ppm) or pulsed in the water (mg/kg) or by gavage (mg/kg). In vitro in sera, increasing concentrations of 0.5 to 8 mu g/ml enrofloxacin correlated with increased activity. In vivo, the efficacy of the 1-day treatment was significantly less than that of the 2- and 5-day treatments. The 2-day treatments showed efficacy similar to that of the 5-day treatment in all but one repeat treatment group and significantly (P < 0.01) reduced the Salmonella counts. Dosing at 2.5x the recommended dose and pulsed dosing both increased the peak antibiotic concentrations in cecal contents, liver, lung, and sera as determined by high-pressure liquid chromatography. There was limited evidence that shorter treatment regimens (in particular the 1-day regimen) selected for fewer strains with reduced susceptibility. In conclusion, the 2-day treatment would overall require a shorter withholding time than the 5-day treatment and, in view of the increased peak antibiotic concentrations, may give rise to improved efficacy, in particular for treating respiratory and systemic infections. However, it would be necessary to validate the 2-day regimen in a field situation and in particular against respiratory and systemic infections to validate or refute this hypothesis.

Overexpression of marA, soxS and acrB in veterinary isolates of Salmonella enterica rarely correlates with cyclohexane tolerance

Objectives: To determine the contribution of the AcrAB efflux system to cyclohexane tolerance in Salmonella enterica. Methods: The expression of the efflux pump gene, acrB, and regulators marA and soxS from 46 isolates of S. enterica of 14 different serovars was determined by comparative RT-PCR and denaturing HPLC analysis. Results: Twenty-one of the 46 isolates were cyclohexane tolerant, a phenotype associated with multiple antibiotic resistance (MAR) and overexpression of efflux pumps. Of the cyclohexane-tolerant isolates 81% were MAR, whereas only 44% of the cyclohexane-susceptible isolates were MAR, confirming the association between cyclohexane tolerance and MAR. However, there was no correlation between cyclohexane tolerance or MAR and overexpression of acrB, soxS or marA. Conclusions: These data suggest that cyclohexane tolerance in S. enterica can be mediated by an acrB-independent mechanism.

Prolonged treatment of Salmonella enterica serovar Typhimurium with commercial disinfectants selects for multiple antibiotic resistance, increased efflux and reduced invasiveness

Objectives: To study how disinfectants affect antimicrobial susceptibility and phenotype of Salmonella enterica serovar Typhimurium SL1344. Methods: Wild-type strain SL1344 and its isogenic gyrA mutant were passaged daily for 7 days in subinhibitory concentrations, and separately for 16 days in gradually increasing concentrations of a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde (QACFG), an oxidizing compound blend (OXC), a phenolic tar acids-based disinfectant (TOP) and triclosan. The MICs of antimicrobials and antibiotics for populations and representative isolates and the proportion of cells resistant to the MICs for the wild-type were determined. Expression of acrB gene, growth at 37 degrees C and invasiveness of populations in Caco-2 intestinal epithelial cells were assessed. Results: QACFG and triclosan showed the highest selectivity for variants with reduced susceptibility to chloramphenicol, tetracycline, ampicillin, acriflavine and triclosan. Populations treated with the above biocides had reduced invasiveness in Caco-2 cells, and altered growth kinetics. Resistance to disinfectants was observed only after exposure to gradually increasing concentrations of triclosan, accompanied with a 2000-fold increase in its MIC. Growth in OXC and TOP did not affect the MICs of antibiotics, but resulted in the appearance of a proportion of cells resistant to the MIC of acriflavine and triclosan for the wild-type. Randomly selected stable variants from all populations, except the one treated with TOP, over-expressed acrB. Conclusions: In vitro exposure to QACFG and triclosan selects for Salmonella Typhimurium cells with reduced susceptibility to several antibiotics. This is associated with overexpression of AcrAB efflux pump, but accompanied with reduced invasiveness.

Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence

Objectives: To determine if one passage of Salmonella enterica serovar Typhimurium in the presence of farm disinfectants selected for mutants with decreased susceptibility to disinfectants and/or antibiotics. Methods: Eight Salmonella Typhimurium strains including field isolates and laboratory mutants were exposed to either a tar oil phenol (PFD) disinfectant, an oxidizing compound disinfectant (OXC), an aldehyde based disinfectant (ABD) or a dairy sterilizer disinfectant (based on quaternary ammonium biocide) in agar. The susceptibility of mutants obtained after disinfectant exposure to antibiotics and disinfectants was determined as was the accumulation of norfloxacin. The proteome of SL1344 after exposure to PFD and OXC was analysed using two-dimensional liquid chromatography mass spectrometry. Results: Strains with either acrB or tolC inactivated were more susceptible to most disinfectants than other strains. The majority (3/5) of mutants recovered after disinfectant exposure required statistically significantly longer exposure times to disinfectants than their parent strains to generate a 5 log kill. Small decreases in antibiotic susceptibility were observed but no mutants were multiply antibiotic-resistant (MAR). Notably exposure to ABD decreased susceptibility to ciprofloxacin in some strains. Mutants with increased disinfectant tolerance were able to survive and persist in chicks as well as in parent strains. Analysis of proteomes revealed significantly increased expression of the AcrAB-TolC efflux system after PFD exposure. Conclusions: Data presented demonstrate that efflux pumps are required for intrinsic resistance to some disinfectants and that exposure to disinfectants can induce expression of the AcrAB-TolC efflux system, but that single exposure was insufficient to select for MAR strains.

Antipathogenic activity of probiotics against Salmonella Typhimurium and Clostridium difficile in anaerobic batch culture systems: is it due to synergies in probiotic mixtures or the specificity of single strains?

Probiotics are currently being investigated for prevention of infections caused by enteric pathogens. The aim of this in vitro study was to evaluate the influence of three single probiotics: Lactobacillus casei NCIMB 30185 (PXN 37), Lactobacillus acidophilus NCIMB 30184 (PXN 35), Bifidobacterium breve NCIMB 30180 (PXN 25) and a probiotic mixture containing the above strains plus twelve other strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera on the survival of Salmonella Typhimurium and Clostridium difficile using pH-controlled anaerobic batch cultures containing mixed fecal bacteria. Changes in relevant bacterial groups and effects of probiotic addition on survival of the two pathogens were assessed over 24 h. Quantitative analysis of bacterial populations revealed that there was a significant increase in lactobacilli and/or bifidobacteria numbers, depending on probiotic addition, compared with the control (no added probiotic). There was also a significant reduction in S. Typhimurium and C. difficile numbers in the presence of certain probiotics compared with controls. Of the probiotic treatments, two single strains namely L. casei NCIMB 30185 (PXN 37), and B. breve NCIMB 30180 (PXN 25) were the most potent in reducing the numbers of S. Typhimurium and C. difficile. In addition, the supplementation with probiotics into the systems influenced some fermentations parameters. Acetate was found in the largest concentrations in all vessels and lactate and formate were generally detected in higher amounts in vessels with probiotic addition compared to controls.

Comparative genomics of Salmonella enterica serovars Derby and Mbandaka, two prevalent serovars associated with different livestock species in the UK

Background Despite the frequent isolation of Salmonella enterica sub. enterica serovars Derby and Mbandaka from livestock in the UK and USA little is known about the biological processes maintaining their prevalence. Statistics for Salmonella isolations from livestock production in the UK show that S. Derby is most commonly associated with pigs and turkeys and S. Mbandaka with cattle and chickens. Here we compare the first sequenced genomes of S. Derby and S. Mbandaka as a basis for further analysis of the potential host adaptations that contribute to their distinct host species distributions. Results Comparative functional genomics using the RAST annotation system showed that predominantly mechanisms that relate to metabolite utilisation, in vivo and ex vivo persistence and pathogenesis distinguish S. Derby from S. Mbandaka. Alignment of the genome nucleotide sequences of S. Derby D1 and D2 and S. Mbandaka M1 and M2 with Salmonella pathogenicity islands (SPI) identified unique complements of genes associated with host adaptation. We also describe a new genomic island with a putative role in pathogenesis, SPI-23. SPI-23 is present in several S. enterica serovars, including S. Agona, S. Dublin and S. Gallinarum, it is absent in its entirety from S. Mbandaka. Conclusions We discovered a new 37 Kb genomic island, SPI-23, in the chromosome sequence of S. Derby, encoding 42 ORFS, ten of which are putative TTSS effector proteins. We infer from full-genome synonymous SNP analysis that these two serovars diverged, between 182kya and 625kya coinciding with the divergence of domestic pigs. The differences between the genomes of these serovars suggest they have been exposed to different stresses including, phage, transposons and prolonged externalisation. The two serovars possess distinct complements of metabolic genes; many of which cluster into pathways for catabolism of carbon sources.

Sequence analysis of a CTX-M-1 IncI1 plasmid found in Salmonella enterica 4,5,12,i:-, Escherichia coli and Klebsiella pneumoniae on a single UK pig farm

OBJECTIVES: In 2009, CTX-M Enterobacteriaceae and Salmonella isolates were recovered from a UK pig farm, prompting studies into the dissemination of the resistance and to establish any relationships between the isolates. METHODS: PFGE was used to elucidate clonal relationships between isolates whilst plasmid profiling, restriction analysis, sequencing and PCR were used to characterize the CTX-M-harbouring plasmids. RESULTS: Escherichia coli, Klebsiella pneumoniae and Salmonella 4,5,12:i:- and Bovismorbificans resistant to cefotaxime (n = 65) were recovered and 63 were shown by PCR to harbour a group 1 CTX-M gene. The harbouring hosts were diverse, but the group 1 CTX-M plasmids were common. Three sequenced CTX-M plasmids from E. coli, K. pneumoniae and Salmonella enterica serotype 4,5,12:i:- were identical except for seven mutations and highly similar to IncI1 plasmid ColIb-P9. Two antimicrobial resistance regions were identified: one inserted upstream of yacABC harbouring ISCR2 transposases, sul2 and floR; and the other inserted within shfB of the pilV shufflon harbouring the ISEcp1 transposase followed by blaCTX-M-1. CONCLUSIONS: These data suggest that an ST108 IncI1 plasmid encoding a blaCTX-M-1 gene had disseminated across multiple genera on this farm, an example of horizontal gene transfer of the blaCTX-M-1 gene.

Identification of potential drug targets in Salmonella Typhimurium using metabolic modelling and experimental validation

almonella enterica serovar Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of Salmonella Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in energy demand, while growing in glucose minimal medium. By grouping reactions with similar flux responses, a sub-network of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions, that when removed from the genome-scale model interfered with energy and biomass generation. 11 such sets were found to be essential for the production of biomass precursors. Experimental investigation of 7 of these showed that knock-outs of the associated genes resulted in attenuated growth for 4 pairs of reactions, while 3 single reactions were shown to be essential for growth.

Temperature and oxygen dependent metabolite utilization by Salmonella enterica Serovars Derby and Mbandaka

Salmonella enterica is a zoonotic pathogen of clinical and veterinary significance, with over 2500 serovars. In previous work we compared two serovars displaying host associations inferred from isolation statistics. Here, to validate genome sequence data and to expand on the role of environmental metabolite constitution in host range determination we use a phenotypic microarray approach to assess the ability of these serovars to metabolise ~500 substrates at 25°C with oxygen (aerobic conditions) to represent the ex vivo environment and at 37°C with and without oxygen (aerobic/anaerobic conditions) to represent the in vivo environment. A total of 26 substrates elicited a significant difference in the rate of metabolism of which only one, D-galactonic acid-g-lactone, could be explained by the presence (S. Mbandaka) or the absence (S. Derby) of metabolic genes. We find that S. Mbandaka respires more efficiently at ambient temperatures and under aerobic conditions on 18 substrates including: glucosominic acid, saccharic acid, trehalose, fumaric acid, maltotriose, N-acetyl-D-glucosamine, N-acetyl-beta-D-mannosamine, fucose, L-serine and dihydroxy-acetone; whereas S. Derby is more metabolically competent anaerobically at 37°C for dipeptides, glutamine-glutamine, alanine-lysine, asparagine-glutamine and nitrogen sources glycine and nitrite. We conclude that the specific phenotype cannot be reliably predicted from the presence of metabolic genes directly relating to the metabolic pathways under study.

Population structure and associated phenotypes of Salmonella enterica serovars Derby and Mbandaka overlap with host range

BACKGROUND:The Salmonella enterica serovar Derby is frequently isolated from pigs and turkeys whereas serovar Mbandaka is frequently isolated from cattle, chickens and animal feed in the UK. Through comparative genomics, phenomics and mutant construction we previously suggested possible mechanistic reasons why these serovars demonstrate apparently distinct host ranges. Here, we investigate the genetic and phenotypic diversity of these two serovars in the UK. We produce a phylogenetic reconstruction and perform several biochemical assays on isolates of S. Derby and S. Mbandaka acquired from sites across the UK between the years 2000 and 2010. RESULTS:We show that UK isolates of S. Mbandaka comprise of one clonal lineage which is adapted to proficient utilisation of metabolites found in soya beans under ambient conditions. We also show that this clonal lineage forms a biofilm at 25 °C, suggesting that this serovar maybe well adapted to survival ex vivo, growing in animal feed. Conversely, we show that S. Derby is made of two distinct lineages, L1 and L2. These lineages differ genotypically and phenotypically, being divided by the presence and absence of SPI-23 and the ability to more proficiently invade porcine jejunum derived cell line IPEC-J2. CONCLUSION:The results of this study lend support to the hypothesis that the differences in host ranges of S. Derby and S. Mbandaka are adaptations to pathogenesis, environmental persistence, as well as utilisation of metabolites abundant in their respective host environments.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19 kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FIiC), a Toll-like receptor 5 (TLR5) agonist. FHC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by Sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P vivax MSPI 19 in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1 (19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSPI 19-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malaria antigens and the innate immunity agonist FliC. it contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants. (C) 2008 Elsevier Ltd. All rights reserved.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Salmonella enterica Serovar Typhimurium Vaccine Strains Expressing a Nontoxic Shiga-Like Toxin 2 Derivative Induce Partial Protective Immunity to the Toxin Expressed by Enterohemorrhagic Escherichia coli

Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2 Delta AB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2 Delta AB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2 Delta AB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.

Paracoccidioides brasiliensis Vaccine Formulations Based on the gp43-Derived P10 Sequence and the Salmonella enterica FliC Flagellin

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43) or the derived P10 sequence containing a CD4(+) T-cell-specific epitope have shown promising results. In the present study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P. brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innate immunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunized with gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) and IL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused with P10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10 peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secreted type 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeast cells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimeric flagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lung damage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P. brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.