Importance of sigma factor mutations in increased triclosan resistance in <i>Salmonella</i> Typhimurium

BACKGROUND: Salmonella enterica is the second most common foodborne pathogen. The use of biocides is crucial to prevent spread of foodborne pathogens, and it would be devastating for food safety if Salmonella would become resistant to the disinfectants used. Another concern is that exposure to disinfectants might lead to decreased susceptibility to antibiotics. The current study aimed to identify genetic changes causing high level triclosan resistance in S. enterica serovar Typhimurium and evaluate how these affected antibiotic resistance and efflux pump activity. RESULTS: Wild type strains S. Typhimurium 4/74 and DTU3 were adapted to increasing concentrations of the biocide triclosan by serial passage. High level triclosan resistant isolates (MIC > 1000 μg/ml) were obtained. Strains were genome sequenced, and SNPs in fabI, rpoS and rpoD were found to be associated with high level resistance. However, work with defined mutants revealed that a SNP in fabI was not sufficient to obtain high level resistance. This required additional mutations in the sigma factors rpoS or rpoD. The adapted strains showed triclosan-dependent increased efflux, increased fabI expression and reduced susceptibility towards the antibiotics enrofloxacin and sulphamethoxazole/trimethoprim. CONCLUSIONS: Medium level triclosan resistance could be obtained by fabI mutations in S. Typhimurium, however, high level resistance was found to require sigma factor mutations in addition to a fabI mutation. Reduced antibiotic sensitivity was observed for the adapted strains, which could be associated with increased efflux

Antimicrobial peptides effectively kill a broad spectrum of Listeria monocytogenes and Staphylococcus aureus strains independently of origin, sub-type, or virulence factor expression

<p>Abstract</p> <p>Background</p> <p>Host defense peptides (HDPs), or antimicrobial peptides (AMPs), are important components of the innate immune system that bacterial pathogens must overcome to establish an infection and HDPs have been suggested as novel antimicrobial therapeutics in treatment of infectious diseases. Hence it is important to determine the natural variation in susceptibility to HDPs to ensure a successful use in clinical treatment regimes.</p> <p>Results</p> <p>Strains of two human bacterial pathogens, <it>Listeria monocytogenes </it>and <it>Staphylococcus aureus</it>, were selected to cover a wide range of origin, sub-type, and phenotypic behavior. Strains within each species were equally sensitive to HDPs and oxidative stress representing important components of the innate immune defense system. Four non-human peptides (protamine, plectasin, novicidin, and novispirin G10) were similar in activity profile (MIC value spectrum) to the human β-defensin 3 (HBD-3). All strains were inhibited by concentrations of hydrogen peroxide between 0.1% – 1.0%. Sub-selections of both species differed in expression of several virulence-related factors and in their ability to survive in human whole blood and kill the nematode virulence model <it>Caenorhabditis elegans</it>. For <it>L. monocytogenes</it>, proliferation in whole blood was paralleled by high invasion in Caco-2 cells and fast killing of <it>C. elegans</it>, however, no such pattern in phenotypic behavior was observed for <it>S. aureus </it>and none of the phenotypic differences were correlated to sensitivity to HDPs.</p> <p>Conclusion</p> <p>Strains of <it>L. monocytogenes </it>and <it>S. aureus </it>were within each species equally sensitive to a range of HDPs despite variations in subtype, origin, and phenotypic behavior. Our results suggest that therapeutic use of HDPs will not be hampered by occurrence of naturally tolerant strains of the two species investigated in the present study.</p

The putative thiosulfate sulfurtransferases PspE and GlpE contribute to virulence of <em>Salmonella</em> Typhimurium in the mouse model of systemic disease.

The phage-shock protein PspE and GlpE of the glycerol 3-phosphate regulon of Salmonella enterica serovar Typhimurium are predicted to belong to the class of thiosulfate sulfurtransferases, enzymes that traffic sulfur between molecules. In the present study we demonstrated that the two genes contribute to S. Typhimurium virulence, as a glpE and pspE double deletion strain showed significantly decreased virulence in a mouse model of systemic infection. However, challenge of cultured epithelial cells and macrophages did not reveal any virulence-associated phenotypes. We hypothesized that their contribution to virulence could be in sulfur metabolism or by contributing to resistance to nitric oxide, oxidative stress, or cyanide detoxification. In vitro studies demonstrated that glpE but not pspE was important for resistance to H(2)O(2). Since the double mutant, which was the one affected in virulence, was not affected in this assay, we concluded that resistance to oxidative stress and the virulence phenotype was most likely not linked. The two genes did not contribute to nitric oxid stress, to synthesis of essential sulfur containing amino acids, nor to detoxification of cyanide. Currently, the precise mechanism by which they contribute to virulence remains elusive

The role of ClpP, RpoS and CsrA in growth and filament formation of Salmonella enterica serovar Typhimurium at low temperature

BACKGROUND: Salmonellae are food-borne pathogens of great health and economic importance. To pose a threat to humans, Salmonellae normally have to cope with a series of stressful conditions in the food chain, including low temperature. In the current study, we evaluated the importance of the Clp proteolytic complex and the carbon starvation protein, CsrA, for the ability of Salmonella Typhimurium to grow at low temperature. RESULTS: A clpP mutant was severely affected in growth and formed pin point colonies at 10°C. Contrary to this, rpoS and clpP/rpoS mutants were only slightly affected. The clpP mutant formed cold resistant suppressor mutants at a frequency of 2.5 × 10(−3) and these were found not to express RpoS. Together these results indicated that the impaired growth of the clpP mutant was caused by high level of RpoS. Evaluation by microscopy of the clpP mutant revealed that it formed filamentous cells when grown at 10°C, and this phenotype too, disappered when rpoS was mutated in parallel indicating a RpoS-dependency. A csrA (sup) mutant was also growth attenuated a low temperature. An rpoS/csrA (sup) double mutant was also growth attenuated, indicating that the phenotype of the csrA mutant was independent from RpoS. CONCLUSIONS: The cold sensitivity of clpP mutant was associated with increased levels of RpoS and probably caused by toxic levels of RpoS. Although a csrA mutant also accumulated high level of RpoS, growth impairment caused by lack of csrA was not related to RpoS levels in a similar way

Prevalence of feline haemoplasma in cats in Denmark

BACKGROUND: Infections with the three feline haemotropic mycoplasmas Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum and Candidatus Mycoplasma turicensis cause feline infectious anemia. The purpose of this study was to investigate the prevalence of carriage of feline haemoplasma in Danish cats in different age groups. The presence was detected by a conventional polymerase chain reaction (PCR) assay on blood samples as well as by real-time PCR (RT-PCR). RESULTS: The study revealed a prevalence of 14.9% Candidatus Mycoplasma haemominutum positive cats and 1.5% Mycoplasma haemofelis positive cats. No cats were found positive for Candidatus Mycoplasma turicensis. The results showed a statistically significant higher prevalence in older (>8 years) cats compared to younger cats and a higher prevalence among domestic cats compared to purebred cats. As part of this study, we developed a cloning strategy to obtain Danish positive controls of haemoplasma 16S rRNA. CONCLUSION: From convenience-sampled cats in Denmark, we found that 16.4% were carriers of feline haemotropic mycoplasmas. Haemoplasma was mostly found in older and domestic cats. The prevalence found in Denmark is similar to that found in several other European countries. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13028-016-0260-1) contains supplementary material, which is available to authorized users