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High-density, strand-specific cDNA sequencing (ssRNA-seq) was used to analyze the transcriptome of Salmonella enterica serovar Typhi (S. Typhi). By mapping sequence data to the entire S. Typhi genome, we analyzed the transcriptome in a strand-specific manner and further defined transcribed regions encoded within prophages, pseudogenes, previously un-annotated, and 3'- or 5'-untranslated regions (UTR). An additional 40 novel candidate non-coding RNAs were identified beyond those previously annotated. Proteomic analysis was combined with transcriptome data to confirm and refine the annotation of a number of hpothetical genes. ssRNA-seq was also combined with microarray and proteome analysis to further define the S. Typhi OmpR regulon and identify novel OmpR regulated transcripts. Thus, ssRNA-seq provides a novel and powerful approach to the characterization of the bacterial transcriptome.

Background Although Mycoplasma genitalium is a common sexually transmitted pathogen causing clinically distinct diseases both in male and females, few genomes have been sequenced up to now, due mainly to its fastidious nature and slow growth. Hence, we lack a robust phylogenetic framework to provide insights into the population structure of the species. Currently our understanding of the nature and diversity of M. genitalium relies on molecular tests targeting specific genes or regions of the genome and knowledge is limited by a general under-testing internationally. This is set against a background of drug resistance whereby M. genitalium has developed resistance to mainly all therapeutic antimicrobials. Results We sequenced 28 genomes of Mycoplasma genitalium from temporally (1980–2010) and geographically (Europe, Japan, Australia) diverse sources. All the strain showed essentially the same genomic content without any accessory regions found. However, we identified extensive recombination across their genomes with a total of 25 regions showing heightened levels of SNP density. These regions include the MgPar loci, associated with host interactions, as well as other genes that could also be involved in this role. Using these data, we generated a robust phylogeny which shows that there are two main clades with differing degrees of genomic variability. SNPs found in region V of 23S rRNA and parC were consistent with azithromycin/erythromycin and fluoroquinolone resistances, respectively, and with their phenotypic MIC data. Conclusions The sequence data here generated is essential for designing rational approaches to type and track Mycoplasma genitalium as antibiotic resistance increases. It represents a first approach to its population genetics to better appreciate the role of this organism as a sexually transmitted pathogen.

Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn 5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Tra nsposon D irected I nsertion-site S equencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the “secondary resistome”. As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance ( dedA ) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5 μg/ml, 4-fold below the susceptibility breakpoint (S ≤ 2 μg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial “helper” drugs that restore the efficacy of existing antimicrobials.

Streptococcus pneumoniae of serotype 3 possess a mucoid capsule and cause disease associated with high mortality rates relative to other pneumococci. Phylogenetic analysis of a complete reference genome and 81 draft sequences from clonal complex 180, the predominant serotype 3 clone in much of the world, found most sampled isolates belonged to a clade affected by few diversifying recombinations. However, other isolates indicate significant genetic variation has accumulated over the clonal complex's entire history. Two closely related genomes, one from the blood and another from the cerebrospinal fluid, were obtained from a patient with meningitis. The pair differed in their behaviour in a mouse model of disease and in their susceptibility to antimicrobials, with at least some of these changes attributable to a mutation that up-regulated the patAB efflux pump. This indicates clinically important phenotypic variation can accumulate rapidly through small alterations to the genotype.

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

The Sfh protein is encoded by self-transmissible plasmids involved in human typhoid and is closely related to the global regulator H-NS. We have found that Sfh provides a stealth function that allows the plasmids to be transmitted to new bacterial hosts with minimal effects on their fitness. Introducing the plasmid without the sfh gene imposes a mild H-NS⁻ phenotype and a severe loss of fitness due to titration of the cellular pool of H-NS by the A+T-rich plasmid. This stealth strategy seems to be used widely to aid horizontal DNA transmission and has important implications for bacterial evolution.

Background Shigella flexneri is an important human pathogen that has to adapt to the anaerobic environment in the gastrointestinal tract to cause dysentery. To define the influence of anaerobiosis on the virulence of Shigella , we performed deep RNA sequencing to identify transcriptomic differences that are induced by anaerobiosis and modulated by the anaerobic Fumarate and Nitrate Reduction regulator, FNR. Results We found that 528 chromosomal genes were differentially expressed in response to anaerobic conditions; of these, 228 genes were also influenced by FNR. Genes that were up-regulated in anaerobic conditions are involved in carbon transport and metabolism ( e.g. ptsG, manX, murQ, cysP, cra ), DNA topology and regulation ( e.g. ygiP, stpA, hns ), host interactions ( e.g. yciD, nmpC, slyB, gapA, shf, msbB ) and survival within the gastrointestinal tract ( e.g. shiA, ospI, adiY, cysP ). Interestingly, there was a marked effect of available oxygen on genes involved in Type III secretion system (T3SS), which is required for host cell invasion and pathogenesis. These genes, located on the large Shigella virulence plasmid, were down regulated in anaerobiosis in an FNR-dependent manner. We also confirmed anaerobic induction of csrB and csrC small RNAs in an FNR-independent manner. Conclusions Anaerobiosis promotes survival and adaption strategies of Shigella , while modulating virulence plasmid genes involved in T3SS-mediated host cell invasion. The influence of FNR on this process is more extensive than previously appreciated, although aside from the virulence plasmid, this transcriptional regulator does not govern expression of genes on other horizontally acquired sequences on the chromosome such as pathogenicity islands.

Invasive Non-typhoidal Salmonella (iNTS) are an important cause of bacteraemia in children and HIV-infected adults in sub-Saharan Africa. Previous research has shown that iNTS strains exhibit a pattern of gene loss that resembles that of host adapted serovars such as Salmonella Typhi and Paratyphi A. Salmonella enterica serovar Bovismorbificans was a common serovar in Malawi between 1997 and 2004. We sequenced the genomes of 14 Malawian bacteraemia and four veterinary isolates from the UK, to identify genomic variations and signs of host adaptation in the Malawian strains. Whole genome phylogeny of invasive and veterinary S. Bovismorbificans isolates showed that the isolates are highly related, belonging to the most common international S. Bovismorbificans Sequence Type, ST142, in contrast to the findings for S. Typhimurium, where a distinct Sequence Type, ST313, is associated with invasive disease in sub-Saharan Africa. Although genome degradation through pseudogene formation was observed in ST142 isolates, there were no clear overlaps with the patterns of gene loss seen in iNTS ST313 isolates previously described from Malawi, and no clear distinction between S. Bovismorbificans isolates from Malawi and the UK. The only defining differences between S. Bovismorbificans bacteraemia and veterinary isolates were prophage-related regions and the carriage of a S. Bovismorbificans virulence plasmid (pVIRBov). iNTS S. Bovismorbificans isolates, unlike iNTS S. Typhiumrium isolates, are only distinguished from those circulating elsewhere by differences in the mobile genome. It is likely that these strains have entered a susceptible population and are able to take advantage of this niche. There are tentative signs of convergent evolution to a more human adapted iNTS variant. Considering its importance in causing disease in this region, S. Bovismorbificans may be at the beginning of this process, providing a reference against which to compare changes that may become fixed in future lineages in sub-Saharan Africa.

Bacterial DNA is maintained in a supercoiled state controlled by the action of topoisomerases. Alterations in supercoiling affect fundamental cellular processes, including transcription. Here, we show that substitution at position 87 of GyrA of Salmonella influences sensitivity to antibiotics, including nonquinolone drugs, alters global supercoiling, and results in an altered transcriptome with increased expression of stress response pathways. Decreased susceptibility to multiple antibiotics seen with a GyrA Asp87Gly mutant was not a result of increased efflux activity or reduced reactive-oxygen production. These data show that a frequently observed and clinically relevant substitution within GyrA results in altered expression of numerous genes, including those important in bacterial survival of stress, suggesting that GyrA mutants may have a selective advantage under specific conditions. Our findings help contextualize the high rate of quinolone resistance in pathogenic strains of bacteria and may partly explain why such mutant strains are evolutionarily successful. IMPORTANCE Fluoroquinolones are a powerful group of antibiotics that target bacterial enzymes involved in helping bacteria maintain the conformation of their chromosome. Mutations in the target enzymes allow bacteria to become resistant to these antibiotics, and fluoroquinolone resistance is common. We show here that these mutations also provide protection against a broad range of other antimicrobials by triggering a defensive stress response in the cell. This work suggests that fluoroquinolone resistance mutations may be beneficial under a range of conditions. Fluoroquinolones are a powerful group of antibiotics that target bacterial enzymes involved in helping bacteria maintain the conformation of their chromosome. Mutations in the target enzymes allow bacteria to become resistant to these antibiotics, and fluoroquinolone resistance is common. We show here that these mutations also provide protection against a broad range of other antimicrobials by triggering a defensive stress response in the cell. This work suggests that fluoroquinolone resistance mutations may be beneficial under a range of conditions.

Shigellosis or bacillary dysentery is endemic worldwide and is a significant cause of death in children less than five years of age in developing countries. There are no licensed Shigella vaccines and glycoconjugates are among the leading candidate vaccines against shigellosis today. We used whole genome sequence analysis (WGA) to find out whether immunization, with an investigational Shigella sonnei glycoconjugate, could induce selective pressure leading to changes in the genome of S. sonnei. An outbreak of culture-proven S. sonnei shigellosis which occurred immediately after vaccination in one of the cohorts of volunteers participating in a phase III trial of the vaccine in Israel created a unique condition in which the epidemic agent “co-existed” with the developing immune responses induced by the vaccine and natural infection among vaccinees who developed S. sonnei shigellosis. By comparing the whole genomes of S. sonnei isolated from vaccinees and from volunteers in the control group, we show at a very high sensitivity that a potent S. sonnei glycoconjugate that conferred 74% protective efficacy against the homologous disease did not induce changes in the genome of S. sonnei and in particular on the O-antigen gene cluster.