Viable But Non-Culturable (VBNC) state in Salmonella Typhimurium

© 2019 Venkata Durga Naga Bhaskara Karthik PullelaSalmonella enterica serovar Typhimurium is a dangerous food-borne pathogen that causes severe diseases like gastroenteritis and septicaemia. It causes over 800,000 deaths p.a. globally. The majority of the cases are caused by contaminated food. Culturing is still a gold standard to detect bacteria in food, clinical and other samples and this approach assumes that those bacteria present in the samples, which are capable of causing disease, are culturable. There are numerous reports of bacteria entering the viable but non-culturable (VBNC) state, a state where bacteria exhibit loss of culturability under routine culturing conditions despite being alive, when placed under stress conditions. The VBNC state raises questions about the effectiveness of bacterial culturability as a tool for bacterial surveillance. The mechanism(s) of entry into, and maintenance of, the VBNC state is not very well understood. Bacteria can be ‘resuscitated’ from this state of lowered metabolic activity in the presence of certain ‘cues’ thus regaining the lost metabolic activity. To study the VBNC state in S. Typhimurium, a reliable and reproducible method was established to generate VBNC S. Typhimurium. The bacteria were subjected to an infection- and environment–relevant oxidative stress, using hydrogen peroxide. The non-culturability of the bacteria was established using routinely used media that support S. Typhimurium growth. Further, the viability of the ‘non-culturable’ population was studied using confocal microscopy by Live/Dead staining and flow cytometric analysis. Drug tolerance of the VBNC S. Typhimurium was revealed. Finally, the ultrastructure of the VBNC bacteria was compared with actively dividing bacteria using a transmission electron microscopy (TEM). After confirming and characterising the S. Typhimurium in the VBNC state, the transcriptome (by RNA-sequencing analysis) of the culturable and VBNC S. Typhimurium were compared. Gene knockouts were generated based on the RNA-seq data and the ability of the mutants to form VBNC cells was tested. The proteome of VBNC S. Typhimurium was analysed by mass-spectrometry. Unlike the large number of transcriptional changes, the proteome was relatively unaffected, suggesting that translation was reduced. Experiments were then conducted to study the ribosomal content, protein synthesis and ribosomal degradation in the VBNC S. Typhimurium and a possible mechanism of VBNC entry, based on ribosomal dissolution, was proposed based on these observations. Lastly, the pathogenicity of the VBNC S. Typhimurium was tested by using a highly sensitive murine intravenous infection model. VBNC S. Typhimurium were used to infect the mice and their growth phenotype was resuscitated in vivo. The infection with VBNC S. Typhimurium was not found to be lethal out to 20 days post-infection in C57BL/6 mice, or in two mice immunodeficient mice strains that were tested (RAG2 gamma-c chain double knockout mice and INF-gamma knockout mice) indicating a loss of virulence in the in vivo resuscitated VBNC bacteria. Unlike the attenuated infection seen in animals inoculated with VBNC, reisolated ‘VBNC‘ S. Typhimurium regained their virulence in the subsequent infections, causing mortality. This study describes a reproducible method to drive S. Typhimurium into non-culturability without complete loss of virulence and discusses various characteristics of VBNC S. Typhimurium. The transcriptomic and proteomic profiles of oxidative stress induced VBNC S. Typhimurium have been examined. A possible ribosome-related mechanism of entry into the VBNC state is proposed based on the transcriptome, proteome and mutagenesis studies. Finally, the virulence of the VBNC S. Typhimurium was tested in normal and immune-deficient mice. These observations help in gaining insights into the VBNC formation and virulence in S. Typhimurium