Antibiotic persistence in Salmonella enterica serovar Typhimurium : involvement of the CspA paralogues

Chronic infections are often attributed to bacterial biofilms. These biofilms are extremely tolerant to antimicrobial treatment due to the presence of dormant persister cells. Whilst a number of persister genes and pathways have been identified, it is likely that others remain. Investigating persistence of S. Typhimurium was therefore undertaken. A csp null mutant of Salmonella enterica sv. Typhimurium, lacking all six cold shock protein (CspA) paralogues was previously constructed (Hutchinson 2005). At 10°C, this strain is unable to divide, but remains viable for several weeks. However it remains capable of growth at 37°C and thus is conditionally dormant. Using this strain, the link between dormancy and persistence was investigated. Treatment of stationary phase planktonic cultures with fluoroquinolones revealed persister cells in SL1344. In contrast the csp null mutant was completely eliminated by treatment at 37°C; this could be prevented by cspC or cspE expression, implicating a role for cspA paralogues in persistence. Cold shock (10°C) substantially increased persister levels, although csp null cultures remained hypersensitive. Chloramphenicol pre-treatment also reduced elimination of the csp null mutant, linking translation with the persister phenotype. Mutations in 5 genes affecting chromosomal structure and function were investigated, 3 of which (hns, hfq, rpoS) were found to reduce persister levels, suggesting a possible role for DNA supercoiling in persistence. Plasmid topologies in the csp null mutant were highly supercoiled compared to SL1344, a phenotype prevented by cspC or cspE expression. Altered supercoiling is therefore proposed as a mechanism for fluoroquinolone sensitivity in the csp null mutant. Persister levels were also characterised in biofilms of SL1344 and the csp null mutant. In contrast to stationary phase planktonic cultures, the CspA paralogues did not appear to play a role in biofilm persistence under the experimental conditions tested. However, the study revealed a novel role for CspA paralogues in pellicle formation at the air-liquid interface. A plasmid library was used to identify chromosomal regions capable of rescuing the planktonic persister phenotype of the csp null mutant. One region which delayed fluoroquinolone elimination of the csp null mutant, contained components of the hpa gene cluster, replicated in 11 isolates. This locus is involved in hydroxyphenylacetate (HPA) catabolism, indicating a possible role of cellular metabolism in the persistence. Overall this study has revealed novel information about antibiotic persistence in S. Typhimurium and the involvement of the CspA paralogues. These results provide an important foundation for further investigations and contribute towards knowledge of the complex processes of dormancy, persistence and biofilm formation in bacteria.