8 resultados para S. Enteritidis
Resumo:
We reported previously that a Salmonella enterica serovar Enteritidis dam mutant expressing a truncated Dam protein does not agglutinate in the presence of specific antibodies against O9 polysaccharide. Here we investigate the participation of Dam in lipopolysaccharide (LPS) synthesis in Salmonella. The LPS O-antigen profiles of a dam null mutant (SEDeltadam) and the Salmonella serovar Enteritidis parental strain were examined by using electrophoresis and silver staining. Compared to the parental strain, SEDeltadam produced LPS with shorter O-antigen polysaccharide chains. Since Wzz is responsible for the chain length distribution of the O antigen, we investigated whether Dam methylation is involved in regulating wzz expression. Densitometry analysis showed that the amount of Wzz produced by SEDeltadam is threefold lower than the amount of Wzz produced by the parental strain. Concomitantly, the activity of the wzz promoter in SEDeltadam was reduced nearly 50% in logarithmic phase and 25% in stationary phase. These results were further confirmed by reverse transcription-PCR showing that wzz gene expression was threefold lower in the dam mutant than in the parental strain. Our results demonstrate that wzz gene expression is downregulated in a dam mutant, indicating that Dam methylation activates expression of this gene. This work indicates that wzz is a new target regulated by Dam methylation and demonstrates that DNA methylation not only affects the production of bacterial surface proteins but also the production of surface polysaccharides.
Resumo:
The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 3×FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.
Resumo:
Mechanisms of antibiotic resistance were examined in nalidixic acid-resistant Salmonella enterica serovar Enteritidis field isolates displaying decreased susceptibility to ciprofloxacin and in in vitro-derived ciprofloxacin-resistant mutants (104-cip and 5408-cip). All field isolates harbored a single gyrA mutation (D87Y). Deletion of acrB and complementation with wild-type gyrA increased quinolone susceptibility. Selection for ciprofloxacin resistance was associated with the development of an additional gyrA (S83F) mutation in 104-cip, novel gyrB (E466D) and parE (V461G) mutations in 5408-cip, overexpression of acrB and decreased susceptibility to nonquinolone antibiotics in both mutants, and decreased OmpF production and altered lipopoly- saccharide in 104-cip. Complementation of mutated gyrA and gyrB with wild-type alleles restored susceptibility to quinolones in 104-cip and significantly decreased the ciprofloxacin MIC in 5408-cip. Complementation of parE had no effect on quinolone MICs. Deletion of acrB restored susceptibility to ciprofloxacin and other antibiotics tested. Both soxS and marA were overexpressed in 104-cip, and ramA was overexpressed in 5408-cip. Inactivation of each of these global regulators lowered ciprofloxacin MICs, decreased expression of acrB, and restored susceptibility to other antibiotics. Mutations were found in soxR (R20H) and in soxS (E52K) in 104-cip and in ramR (G25A) in 5408-cip. In conclusion, both efflux activity and a single gyrA mutation contribute to nalidixic acid resistance and reduced ciprofloxacin sensitivity. Ciprofloxacin resistance and decreased susceptibility to multiple antibiotics can result from different genetic events leading to development of target gene mutations, increased efflux activity resulting from differential expression of global regulators associated with mutations in their regulatory genes, and possible altered membrane permeability.
Resumo:
The amount of lipopolysaccharide (LPS) O antigen (OAg) and its chain length distribution are important factors that protect bacteria from serum complement. Salmonella enterica serovar Typhi produces LPS with long chain length distribution (L-OAg) controlled by the wzz gene, whereas serovar Typhimurium produces LPS with two OAg chain lengths: an L-OAg controlled by Wzz(ST) and a very long (VL) OAg determined by Wzz(fepE). This study shows that serovar Enteritidis also has a bimodal OAg distribution with two preferred OAg chain lengths similar to serovar Typhimurium. It was reported previously that OAg production by S. Typhi increases at the late exponential and stationary phases of growth. The results of this study demonstrate that increased amounts of L-OAg produced by S. Typhi grown to stationary phase confer higher levels of bacterial resistance to human serum. Production of OAg by serovars Typhimurium and Enteritidis was also under growth-phase-dependent regulation; however, while the total amount of OAg increased during growth, the VL-OAg distribution remained constant. The VL-OAg distribution was primarily responsible for complement resistance, protecting the non-typhoidal serovars from the lytic action of serum irrespective of the growth phase. As a result, the non-typhoidal species were significantly more resistant than S. Typhi to human serum. When S. Typhi was transformed with a multicopy plasmid containing the S. Typhimurium wzz(fepE) gene, resistance to serum increased to levels comparable to the non-typhoidal serovars. In contrast to the relevant role for high-molecular-mass OAg molecules, the presence of Vi antigen did not contribute to serum resistance of clinical isolates of serovar Typhi.
Resumo:
This study presents a solid-phase PCR (SP-PCR) for rapid detection, identification, and sub-typing of various Salmonella species, the major food-borne cause of salmonellosis. The target DNA is firstly amplified with PCR primers (one primer is labeled with fluorophores) in the liquid phase. Simultaneously on the solid phase, the amplified PCR amplicons interact with the nested DNA probes immobilized on the solid substrate as an array. If the immobilized probes match the sequence of the DNA templates they are extended by the polymerase and serve as template for the second strand elongation primed by the liquid phase primer thus generating new templates for the SP-PCR. After the reaction, PCR products labeled with fluorophores remain attached to the substrate and can be visualized directly by fluorescence readout devices. Using this method, S. enteritidis, S. typhimurium and S. dublin can be detected at the same time. The method offers several advantages over conventional multiplex PCR: less competition between different primer pairs thus increasing multiplexing capability, only single wavelength optical readout needed for the multiplexing detection, and less time-consuming owing to reduction of the post-PCR gel electrophoresis. The method will be useful for development of point-of-care devices for rapid detection and identification of Salmonella spp. A solid-phase PCR for rapid detection and identification of S. enteritidis, S. typhimurium and S. dublin is developed. The method offers advantages such as better multiplexing capability, only single wavelength optical readout needed, and less time-consuming.
Resumo:
TiO<sub>2sub> photocatalysis has demonstrated efficacy as a treatment process for water contaminated with chemical pollutants. When exposed to UVA light TiO<sub>2sub> also demonstrates an effective bactericidal activity. The mechanism of this process has been reported to involve attack by valence band generated hydroxyl radicals. In this study when three common bacterial pathogens, Escherichia coli, Salmonella enterica serovar Enteritidis and Pseudomonas aeruginosa, were exposed to TiO<sub>2sub> and UVA light a substantial decrease in bacterial numbers was observed. Control experiments in which all three pathogens were exposed to UVA light only resulted in a similar reduction in bacterial numbers. Moreover, exposure to UVA light alone resulted in the production of a smaller than average colony phenotype among the surviving bacteria, for all three pathogens examined, a finding which was not observed following treatment with UVA and TiO<sub>2sub>. Small slow growing colonies have been described for several pathogenic bacteria and are referred to as small colony variants. Several studies have demonstrated an association between small colony variants and persistent, recurrent and antibiotic resistant infections. We propose that the production of small colony variants of pathogenic bacteria following UVA treatment of drinking water may represent a health hazard. As these small colony variants were not observed with the UVA/TiO<sub>2sub> system this potential hazard is not a risk when using this technology. It would also appear that the bactericidal mechanism is different with the UVA/TiO<sub>2sub> process compared to when UVA light is used alone.
Resumo:
Bdellovibrio bacteriovorus is a bacterium which preys upon and kills Gram-negative bacteria, including the zoonotic pathogens Escherichia coli and Salmonella. Bdellovibrio has potential as a biocontrol agent, but no reports of it being tested in living animals have been published, and no data on whether Bdellovibrio might spread between animals are available. In this study, we tried to fill this knowledge gap, using B. bacteriovorus HD100 doses in poultry with a normal gut microbiota or predosed with a colonizing Salmonella strain. In both cases, Bdellovibrio was dosed orally along with antacids. After dosing non-Salmonella-infected birds with Bdellovibrio, we measured the health and well-being of the birds and any changes in their gut pathology and culturable microbiota, finding that although a Bdellovibrio dose at 2 days of age altered the overall diversity of the natural gut microbiota in 28-day-old birds, there were no adverse effects on their growth and well-being. Drinking water and fecal matter from the pens in which the birds were housed as groups showed no contamination by Bdellovibrio after dosing. Predatory Bdellovibrio orally administered to birds that had been predosed with a gut-colonizing Salmonella enterica serovar Enteritidis phage type 4 strain (an important zoonotic pathogen) significantly reduced Salmonella numbers in bird gut cecal contents and reduced abnormal cecal morphology, indicating reduced cecal inflammation, compared to the ceca of the untreated controls or a nonpredatory ΔpilA strain, suggesting that these effects were due to predatory action. This work is a first step to applying Bdellovibrio therapeutically for other animal, and possibly human, infections.
Resumo:
BACKGROUND: Multiyear epidemics of Salmonella enterica serovar Typhi have been reported from countries across eastern and southern Africa in recent years. In Blantyre, Malawi, a dramatic increase in typhoid fever cases has recently occurred, and may be linked to the emergence of the H58 haplotype. Strains belonging to the H58 haplotype often exhibit multidrug resistance and may have a fitness advantage relative to other Salmonella Typhi strains.
METHODS: To explore hypotheses for the increased number of typhoid fever cases in Blantyre, we fit a mathematical model to culture-confirmed cases of Salmonella enterica infections at Queen Elizabeth Central Hospital, Blantyre. We explored 4 hypotheses: (1) an increase in the basic reproductive number (R0) in response to increasing population density; (2) a decrease in the incidence of cross-immunizing infection with Salmonella Enteritidis; (3) an increase in the duration of infectiousness due to failure to respond to first-line antibiotics; and (4) an increase in the transmission rate following the emergence of the H58 haplotype.
RESULTS: Increasing population density or decreasing cross-immunity could not fully explain the observed pattern of typhoid emergence in Blantyre, whereas models allowing for an increase in the duration of infectiousness and/or the transmission rate of typhoid following the emergence of the H58 haplotype provided a good fit to the data.
CONCLUSIONS: Our results suggest that an increase in the transmissibility of typhoid due to the emergence of drug resistance associated with the H58 haplotype may help to explain recent outbreaks of typhoid in Malawi and similar settings in Africa.