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

Reduction in DNA topoisomerase I level affects growth, phenotype and nucleoid architecture of Mycobacterium smegmatis

The steady-state negative supercoiling of eubacterial genomes is maintained by the action of DNA topoisomerases. Topoisomerase distribution varies in different species of mycobacteria. While Mycobacterium tuberculosis (Mtb) contains a single type I (Topol) and a single type II (Gyrase) enzyme, Mycobacterium smegmatis (Msm) and other members harbour additional relaxases. Topol is essential for Mtb survival. However, the necessity of Topol or other relaxases in Msm has not been investigated. To recognize the importance of Topol for growth, physiology and gene expression of Msm, we have developed a conditional knock-down strain of Topol in Msm. The Topol-depleted strain exhibited extremely slow growth and drastic changes in phenotypic characteristics. The cessation of growth indicates the essential requirement of the enzyme for the organism in spite of having additional DNA relaxation enzymes in the cell. Notably, the imbalance in Topol level led to the altered expression of topology modulatory proteins, resulting in a diffused nucleoid architecture. Proteomic and transcript analysis of the mutant indicated reduced expression of the genes involved in central metabolic pathways and core DNA transaction processes. RNA polymerase (RNAP) distribution on the transcription units was affected in the Topol-depleted cells, suggesting global alteration in transcription. The study thus highlights the essential requirement of Topol in the maintenance of cellular phenotype, growth characteristics and gene expression in mycobacteria. A decrease in Topol level led to altered RNAP occupancy and impaired transcription elongation, causing severe downstream effects

Autoregulation of topoisomerase I expression by supercoiling sensitive transcription

The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Es-cherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobac-terium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of suboptimal spacing between the -35 and -10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the -35 and -10 elements to facilitate the optimal transcription of topoI

A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide

The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, chromosomal genetic manipulation, and dense mutant library construction. Here we demonstrate that culturing clinical isolates in the presence of polymyxin B nonapeptide (PMBN) improves their transformation frequency via electroporation by up to 100-fold in a dose-dependent and reversible manner. The effect was observed for PMBN-binding uropathogenic Escherichia coli (UPEC) and Salmonella enterica strains but not naturally polymyxin resistant Proteus mirabilis. Using our PMBN electroporation method we show efficient delivery of large plasmid constructs into UPEC, which otherwise failed using a conventional electroporation protocol. Moreover, we show a fivefold increase in the yield of engineered mutant colonies obtained in S. enterica with the widely used lambda-Red recombineering method, when cells are cultured in the presence of PMBN. Lastly, we demonstrate that PMBN treatment can enhance the delivery of DNA-transposase complexes into UPEC and increase transposon mutant yield by eightfold when constructing Transposon Insertion Sequencing (TIS) libraries. Therefore, PMBN can be used as a powerful electropermeabilisation adjuvant to aid the delivery of DNA and DNA–protein complexes into clinically important bacteria.</p

Conditional silencing of topoisomerase I gene of Mycobacterium tuberculosis validates its essentiality for cell survival

Topoisomerases are an important class of enzymes for regulating the DNA transaction processes. Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens also posing serious challenges for therapeutic interventions. The organism contains only one type IA topoisomerase (Rv3646c), offering an opportunity to test its potential as a candidate drug target. To validate the essentiality of M.tuberculosis topoisomerase I (TopoI(Mt)) for bacterial growth and survival, we have generated a conditionally regulated strain of topoI in Mtb. The conditional knockdown mutant exhibited delayed growth on agar plate. In liquid culture, the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the M.tuberculosis growth and open up new avenues for targeting the enzyme

Characterization of bla CTX-M sequences of Indian origin and thirteen uropathogenic Escherichia coli isolates resistant to multiple antibiotics

Abstract Objectives ESBL-producing isolates of the Enterobacteriaceae occur throughout the world. The objectives of this study were to characterize uropathogenic Escherichia coli isolated at a tertiary care hospital in southern India, and shed light on bla CTX-M sequences of Indian origin. Results A cohort of 13 urinary isolates of E. coli (obtained from patients at the Sri Sathya Sai Institute of Higher Medical Sciences, Prasanthigram, Andhra Pradesh, India) were characterized and found to be resistant to multiple antibiotics, including extended-spectrum cephalosporins. All 13 isolates contained bla CTX-M-15, and many of them transferred this genotype to at least one laboratory strain of E. coli after conjugation. Analyses of bla CTX-M-15 sequences (n = 141) of Indian origin showed that > 85% of them were obtained from bacteria not associated with the urinary tract, and that E. coli isolates account for majority of all bla CTX-M-15-carrying bacteria reported from India. Other types of bla CTX-M appear to be rare in India, since only six such sequences were reported as of July 2015. The results indicate that ‘selection pressure’ exerted by extended-spectrum cephalosporins may have stabilized the bla CTX-M-15 genotype among E. coli in India. The rarity of other bla CTX-M suggests that they lack the survival advantage that bla CTX-M-15 may have

Transposon-Directed Insertion-Site Sequencing Reveals Glycolysis Gene gpmA as Part of the H2O2 Defense Mechanisms in Escherichia coli

Hydrogen peroxide (H2O2) is a common effector of defense mechanisms against pathogenic infections. However, bacterial factors involved in H2O2tolerance remain unclear. Here we used transposon-directed insertion-site sequencing (TraDIS), a technique allowing the screening of the whole genome, to identify genes implicated in H2O2tolerance inEscherichia coli. Our TraDIS analysis identified 10 mutants with fitness defect upon H2O2exposure, among which previously H2O2-associated genes (oxyR,dps,dksA,rpoS,hfqandpolA) and other genes with no known association with H2O2tolerance inE. coli(corA,rbsR,nhaAandgpmA). This is the first description of the impact ofgpmA, a gene involved in glycolysis, on the susceptibility ofE. colito H2O2. Indeed, confirmatory experiments showed that the deletion ofgpmAled to a specific hypersensitivity to H2O2comparable to the deletion of the major H2O2scavenger genekatG. This hypersensitivity was not due to an alteration of catalase function and was independent of the carbon source or the presence of oxygen. Transcription ofgpmAwas upregulated under H2O2exposure, highlighting its role under oxidative stress. In summary, our TraDIS approach identifiedgpmAas a member of the oxidative stress defense mechanism inE. coli