Detection of RpoS Gene in Escherichia Coli O157:H7 and non-O157 and Their Survival Pattern in Water Treatment Methods

This study aimed to detect presence of RpoS gene in Escherichia coli O157:H7 and non-O157 and investigate their survival pattern in different water treatment methods. A total of fifteen serologically and molecularly identified E. coli was selected from a previous work, out of which eight were Escherichia coli O157 and seven were E. coli non-O157. From among these, S30 and S89 identified isolates served as presentative E. coli O157:H7 and non-O157 respectively for survival studies. The water treatment methods used employed included: use of silver, lime, storage, acidification (low pH), high temperature and Moringa oleifera. Survival pattern of the test organisms under the influence of these methods were carried out using standard techniques. Molecular detection of stress response gene, RpoS, in the fifteen (15) test organisms was performed following manufacturer’s instruction. Results showed that for both test organisms, silver was bactericidal at high concentration while storage allows their survival up till 21 days though with a reduction in cfu. Both organisms showed low survival at pH 9 while E. coli O157:H7 and non-O157 could survive at pH 4 and 6 respectively. E. coli O157:H7 survived better than non-O157 at high concentration of lime. While both survived at low temperature, E. coli O157:H7 survive better at 60oC. Sunlight and chlorine showed mild and complete bactericidal action respectively with increased exposure time for both test organisms. Moringa oleifera was only effective at a reduced concentration on the two organisms. Detection of RpoS genes showed that only 66.7% carried the gene in them while 33.3% did not. Findings from this study show that the possession of stressed genes in bacteria causing waterborne disease could allow these organisms to survive water treatment methods adopted in many under developed countries or rural communities. This suggests a threat to health of these communities.

Mutagenesis of the rpoS gene involved in alteration of outer membrane composition

Background and Objectives: rpoS is a bacterial sigma factor of RNA polymerase which is involved in the expression of the genes which control regulons and play a critical role in survival against stresses. Few suitable vectors are available which could be maintained successfully in Flexibacter chinesis cells and could in particular be used as a suicide vector to make mutation in the rpoS gene. The aim of this study was to investigate if rpoS mutagenesis has impact on bacterial morphology in addition to cell division. Materials and Methods: A 0.603 kb BamHI-PstI fragment subclone of pICRPOS38Ω was cloned into linearized pLYLO3. The final construct, pLRPOS38 suicide vector, was introduced into Flexibacter chinesis. Then the cytoplasm of mutant strain and wild-type were investigated by transmission electron microscopy. Results: After successful subcloning of suicide vector into F. chinesis, based on TEM study, it was demonstrated that muta- tion in rpoS gene leads to decomposition of outer membrane of F. chinesis. Conclusion: A suitable vector to make suicide mutation in rpoS was constructed for F. chinesi.

Genotypic characterisation of the Erwinia genus by PCR-RFLP analysis of rpoS gene

Genotypic characterisation of the members of the genus Erwinia, based on the PCR-RFLP analysis of a fragment of the rpoS gene was done. PCR primers deduced from described rpoS gene sequences of E. carotovora allowed the amplification of about 880 bp DNA fragments from all tested Erwinia species. The rpoS fragments, amplified from 20 species of the studied Erwinia genus, were compared by RFLP analysis with 4 enzymes (AluI, Hin6I, HinfI, and Tru1I). Restriction analysis allowed drawing 63 common profiles of RFLP products for all tested Erwinia. From 1 to 3 specific RFLP profiles were identified among most of the species tested. However, in two cases: E. chrysanthemi and E. c. subsp. carotovora 15 and 20 specific RFLP groups were detected, respectively. High variability of genetic profiles of the E. chrysanthemi and E. c. subsp. carotovora can be explained by the wide spectrum of plants, which they infect. The results indicated that rpoS PCR-RFLP analysis is a useful tool for identification of species and subspecies belonging to the former Erwinia genus, as well as for differentiation of strains within E. c. subsp. carotovora and E. chrysanthemi.

Role of the rpoS gene in the survival of Salmonella enterica serovar Typhimurium in the marine environmentRôle du gène rpoS pour la survie de Salmonella enterica serovar Typhimurium dans l'environnement marin

The survival of Salmonella enterica serovar Typhimurium rpoS+ and rpoS− exposed to sunlight or incubated in the dark was studied in a seawater microcosm and a diffusion chamber. The total number of bacteria and physiologically active ones were estimated by direct counting using epifluorescence microscopy, while cultivable bacteria were enumerated by plating onto an agar culture medium. The results obtained showed that the bacteria exposed to sunlight, either in the microcosm or in the diffusion chamber, exhibited a fast decline of cultivable cells, whereas the epifluorescence enumeration revealed a conservation of physiological activity after a total loss of cultivability. In a transparent diffusion chamber, the evolution of cultivable and physiological active bacteria was characterized by a diurnal decrease and a nocturnal stabilization in the count levels, reflecting the effect of phototoxicity on the survival of Salmonella. The transformation and persistence of a physiological active state and cellular integrity in the diffusion chamber or the microcosm showed a clear dependence on the presence of the rpoS gene. Survival in the dark was also characterized by a decrease of cultivability; however, this was significantly inferior to that observed in bacteria exposed to sunlight. This loss of cultivability was accompanied by the preservation of a physiologically active state. The study of seasonal survival carried out in the transparent diffusion chamber was characterized by an enhanced survival of the rpoS+ strain in comparison with the rpoS– mutant. The highest survival rates were obtained in the autumn, thus highlighting the relevant effect of temperature and exposure to sunlight on the persistence of bacteria in seawater.