Expression of ompR gene in the acid adaptation and thermal resistance of Salmonella Enteritidis SE86.

INTRODUCTION: The objective of this study was to evaluate the involvement of the ompR gene in the acid adaptation and thermal resistance of S. Enteritidis SE86, responsible agent of more than 95 % of investigated food-borne diseases, throughout the last decade in Southern Brazil. In this study, we constructed a mutant strain of S. Enteritidis SE86 (ΔompR) that was attenuated by a knockout technique. The OmpR protein expression was determined in a tagged (3XFLAG) strain of S. Enteritidis SE86. METHODOLOGY: The mutant strains were cultivated separately in nutrient broth and nutrient broth supplemented with 1% glucose (NBG) to induce acid adapted cells. The organisms were exposed to different temperature such as 37 ºC, 52 ºC, and 60ºC. The survival of the SE86 wild type (WT) and attenuated strain was determined by bacterial count, and the tagged protein (ompR::3XFLAG cat::FLAG) was detected by SDS-PAGE and immunoblotting with anti-FLAG antibodies. RESULTS: Results showed that when exposed at 52ºC, the acid-adapted SE86 WT cells were completely inactivated after 300 minutes; however, non-adapted cells (WT and ΔompR) and acid-adapted ΔompR demonstrated higher thermal sensitivity, since they were completely inactivated in 240 minutes. At 60ºC, the acid-adapted SE86 ΔompR also demonstrated higher sensitivity that SE86 WT, being totally inactivated after 15 minutes, while the WT cells were inactivated in 20 minutes. CONCLUSION: The acid adapted cells showed increased expression of OmpR when exposed to 52 ºC and 60ºC, this confirmed the requirement of acid adaptation  for S. Enteritidis SE86 to resist elevated temperatures.

Investigation of rpoS and dps genes in sodium hypochlorite resistance of Salmonella Enteritidis SE86 isolated from foodborne illness outbreaks in southern Brazil.

In Rio Grande do Sul, southern Brazil, Salmonella Enteritidis is one of the principal microorganisms responsible for foodborne disease. The present study was conducted to compare the sodium hypochlorite resistance of Salmonella Enteritidis SE86 with that of other strains of Salmonella Enteritidis isolated from different regions of the world and to investigate the involvement of the rpoS and dps genes in resistance to this disinfectant. We tested five Salmonella Enteritidis wild-type (WT) strains isolated from different countries, two mutant strains of Salmonella Enteritidis SE86, and two tagged (3XFLAG) strains of Salmonella Enteritidis SE86 for their resistance to sodium hypochlorite (200 ppm). The survival of the WT and attenuated strains was determined based on bacterial counts, and tagged proteins (Dps and RpoS) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-FLAG antibodies. None of the WT strains of Salmonella Enteritidis were totally inactivated after 20 min. The SE86 strain lacking dps was more sensitive to sodium hypochlorite than was the WT SE86 strain, with a 2-log reduction in counts after 1 min. The RpoS and Dps proteins were actively expressed under the conditions tested, indicating that in Salmonella Enteritidis SE86 these genes, which are expressed when in contact with sodium hypochlorite, are related to oxidative stress.

Distribution of pathogenicity islands among Colombian isolates of Salmonella.

INTRODUCTION: Salmonella pathogenicity islands (SPIs) are regions scattered along the bacterial chromosome, with an acknowledged pivotal role during gastrointestinal and systemic infection. The distribution of SPIs has been investigated in reference strains. However, there is a lack of studies on their presence and/or assortment within the genomes of Salmonella enterica (S. enterica) serovars that circulate in different geographical regions. Therefore, in this study, we aimed to determine the presence of genes of the pathogenicity islands 1 to 5 (SPI-1 to 5), in Salmonella clinical isolates from Colombian patients with systemic and enteric outcomes. METHODOLOGY: A total of 125 strains of S. enterica belonging to different serovars were isolated from various clinical samples. Strains were identified and screened for the presence of various genes located in pathogenicity islands. The genes tested were selected according to the attributed pathogenic function and detected by PCR for the SPI-1 hilA and invA; for SPI-2 spiC and ttrC; for SPI-3 misL and mgtC; for SPI-4 orfL and SPI-4R; and for SPI-5 pipD and sopB. RESULTS: Salmonella pathogenicity islands 1 to 5 were detected in isolates from patients with systemic and gastrointestinal infection. All the systemic isolates possessed all the genes tested; in contrast, 16 isolates from stool samples lacked one or more sequences encoded by the SPI-3 and SPI-4 (p < 0.000001). CONCLUSIONS: These results describe the heterogeneous distribution of SPIs-encoded sequences within the genomes of Colombian clinical isolates, and reveal important differences among systemic and stool sample isolates.

Impaired synthesis and secretion of SopA in Salmonella Typhimurium dam mutants.

DNA adenine methylation regulates virulence gene expression in certain bacteria, including Salmonella Typhimurium. The aim of this study was to investigate the involvement of DNA adenine methylase (Dam) methylation in the expression and secretion of the SPI-1 effector protein SopA. For this purpose, SopA-FLAG-tagged wild-type and dam strains of Salmonella Typhimurium were constructed. The expression and secretion of SopA were determined in bacterial culture and in intracellular bacteria recovered from infected HEp-2 epithelial cells. Bacterial culture supernatants and pellets were used to investigate secreted proteins and cell-associated proteins, respectively. Western blot and quantitative reverse transcriptase PCR analysis showed that the dam mutant expresses lower levels of SopA than the wild-type strain. Interestingly, the strain lacking Dam synthesizes SopA under nonpermissive conditions (28 degrees C). In addition, SopA secretion was drastically impaired in the dam mutant. In vivo experiments showed that the intracellular Salmonella dam mutant synthesizes SopA although in lower amounts than the wild-type strain. Taken together, our results suggest that Dam methylation modulates the expression and secretion of SopA in Salmonella Typhimurium.