Rebekah N. Whitehead

Coordinated Regulation of the Neisseria gonorrhoeae-truncated Denitrification Pathway by the Nitric Oxide-sensitive Repressor, NsrR, and Nitrite-insensitive NarQ-NarP

Neisseria gonorrhoeae survives anaerobically by reducing nitrite to nitrous oxide catalyzed by the nitrite and nitric oxide reductases, AniA and NorB. PaniA is activated by FNR (regulator of fumarate and nitrate reduction), the two-component regulatory system NarQ-NarP, and induced by nitrite; PnorB is induced by NO independently of FNR by an uncharacterized mechanism. We report the results of microarray analysis, bioinformatic analysis, and chromatin immunoprecipitation, which revealed that only five genes with readily identified NarP-binding sites are differentially expressed in narP+ and narP strains. These include three genes implicated in the truncated gonococcal denitrification pathway: aniA, norB, and narQ. We also report that (i) nitrite induces aniA transcription in a narP mutant; (ii) nitrite induction involves indirect inactivation by nitric oxide of a gonococcal repressor, NsrR, identified from a multigenome bioinformatic study; (iii) in an nsrR mutant, aniA, norB, and dnrN (encoding a putative reactive nitrogen species response protein) were expressed constitutively in the absence of nitrite, suggesting that NsrR is the only NO-sensing transcription factor in N. gonorrhoeae; and (iv) NO rather than nitrite is the ligand to which NsrR responds. When expressed in Escherichia coli, gonococcal NarQ and chimaeras of E. coli and gonococcal NarQ are ligand-insensitive and constitutively active: a “locked-on” phenotype. We conclude that genes involved in the truncated denitrification pathway of N. gonorrhoeae are key components of the small NarQP regulon, that NarP indirectly regulates PnorB by stimulating NO production by AniA, and that NsrR plays a critical role in enabling gonococci to evade NO generated as a host defense mechanism.

Is the abundance of Faecalibacterium prausnitzii relevant to Crohn's disease?

Reports that bacteria within the Firmicutes phylum, especially the species Faecalibacterium prausnitzii, are less abundant in Crohns disease (CD) patients and supernatants from cultures of this bacterium are anti-inflammatory prompted the investigation of the possible correlations between the abundance of F. prausnitzii and the response to treatment in patients with gut diseases and healthy controls. In a randomized, double-blind trial, faeces were collected from healthy volunteers, and from patients with active CD, ulcerative colitis (UC) and irritable bowel syndrome before and after treatment. The levels of F. prausnitzii DNA in faecal suspensions were determined by PCR. Treatment by an elemental diet was effective, resulting in decreases in both the Harvey and Bradshaw index (P<0.001) and the concentrations of serum C-reactive protein (P<0.05). The total levels of F. prausnitzii in faecal samples from CD patients at presentation were lower than those in the other groups both before and after the treatment. There was no correlation between F. prausnitzii abundance and the severity of CD before treatment. Clinical improvement unexpectedly correlated with a significant decrease in the abundance of F. prausnitzii, especially the A2-165 subgroup (P<0.05). Our data suggest that a paucity of F. prausnitzii in the gastrointestinal microbial communities is likely to be a minor aetiological factor in CD: recovery following elemental diet is attributed to lower levels of gut flora.

Exposure of Salmonella enterica Serovar Typhimurium to High Level Biocide Challenge Can Select Multidrug Resistant Mutants in a Single Step

Background Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux. Methodology/Principal Findings Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide. Conclusions/Significance These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.

Analysis of volatile organic compounds of bacterial origin in chronic gastrointestinal diseases

Background: The aim of this study was to determine whether volatile organic compounds (VOCs) present in the headspace of feces could be used to diagnose or distinguish between chronic diseases of the gastrointestinal tract and apparently healthy volunteers. Methods: A total of 87 people were recruited, divided between 4 categories: healthy volunteers (n = 19), Crohn’s disease (n = 22), ulcerative colitis (n = 20), and irritable bowel syndrome (n = 26). They each supplied fecal samples before, and except for the healthy volunteers, after treatment. Fecal samples were incubated in a sample bag with added purified air at 40°C and headspace samples were taken and concentrated on thermal sorption tubes. Gas chromatography–mass spectrometry then desorbed and analyzed these. The concentrations of a selection of high-abundance compounds were determined and assessed for differences in concentration between the groups. Results: Crohns disease samples showed significant elevations in the concentrations of ester and alcohol derivates of short-chain fatty acids and indole compared with the other groups; indole and phenol were elevated in ulcerative colitis and irritable bowel syndrome but not at a statistically significant level. After treatment, the levels of many of the VOCs were significantly reduced and were more similar to those concentrations in healthy controls. Conclusions: The abundance of a number of VOCs in feces differs markedly between Crohns disease and other gastrointestinal conditions. Following treatment, the VOC profile is altered to more closely resemble that of healthy volunteers.

Parallel evolutionary pathways to antibiotic resistance selected by biocide exposure

Objectives Biocides are widely used to prevent infection. We aimed to determine whether exposure of Salmonella to various biocides could act as a driver of antibiotic resistance. Methods Salmonella enterica serovar Typhimurium was exposed to four biocides with differing modes of action. Antibiotic-resistant mutants were selected during exposure to all biocides and characterized phenotypically and genotypically to identify mechanisms of resistance. Results All biocides tested selected MDR mutants with decreased antibiotic susceptibility; these occurred randomly throughout the experiments. Mutations that resulted in de-repression of the multidrug efflux pump AcrAB-TolC were seen in MDR mutants. A novel mutation in rpoA was also selected and contributed to the MDR phenotype. Other mutants were highly resistant to both quinolone antibiotics and the biocide triclosan. Conclusions This study shows that exposure of bacteria to biocides can select for antibiotic-resistant mutants and this is mediated by clinically relevant mechanisms of resistance prevalent in human pathogens.

Diversity and distribution of sulphate‐reducing bacteria in human faeces from healthy subjects and patients with inflammatory bowel disease

The relative abundance of different groups of sulphate-reducing bacteria (SRB) in faecal DNA collected before and after therapy from patients suffering from Crohns disease (CD), irritable bowel syndrome (IBS) or ulcerative colitis (UC) has been compared with that from healthy controls. Growth tests revealed that SRB were not more abundant in samples from patients with CD before treatment than in the healthy control group. For most of the 128 samples available, these preliminary results were confirmed using degenerate PCR primers that amplify the dsrAB gene. However, some samples from patients with CD before treatment contained a growth inhibitor that was absent from IBS or UC samples. In-depth sequencing of PCR-generated dsrB fragments revealed that the diversity detected was surprisingly low, with only eight strains of SRB and the sulphite-reducing bacterium, Bilophila wadsworthia, detected above the 0.1% threshold. The proportion of the two major species detected, B. wadsworthia and Desulfovibrio piger, was as high as 93.5% of the total SRB population in the healthy control group and lower in all patient groups. Four previously undescribed species were found: it is impossible to predict whether they are sulphate or sulphite-reducing bacteria.

Clinically Relevant Mutant DNA Gyrase Alters Supercoiling, Changes the Transcriptome, and Confers Multidrug Resistance

ABSTRACT 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.

Prevalence of decreased susceptibility to triclosan in Salmonella enterica isolates from animals and humans and association with multiple drug resistance

Previous laboratory studies have implicated triclosan as a possible selective force driving resistance to multiple antibiotics and have identified a number of triclosan resistance mechanisms in Salmonella enterica. The aim of this work was to determine the prevalence of decreased susceptibility to triclosan in a panel of human and animal isolates of S. enterica and to identify the mechanisms of triclosan resistance in these strains. Over 400 animal and human isolates of non-typhoidal Salmonella were screened for decreased susceptibility to triclosan and a panel of antibiotics. The prevalence of decreased susceptibility to triclosan was ca. 4%. Of the isolates with decreased triclosan susceptibility, 56% were multidrug-resistant (MDR) compared with 12% of triclosan-sensitive isolates. MDR and triclosan-resistant strains showed increased efflux activity compared with strains with reduced susceptibility to triclosan alone. No high-level triclosan resistance was seen in this panel of isolates. A reservoir of strains with low-level decreased triclosan susceptibility is present in animals and humans. These isolates are MDR as a result of generic mechanisms of antimicrobial resistance and do not carry specific mutations within fabI.

Enteral feeding reduces metabolic activity of the intestinal microbiome in Crohn's disease: an observational study.

Background/Objectives:Enteral feeding will induce remission in as many as 80–90% of compliant patients with active Crohn’s disease (CD), but its method of action remains uncertain. This study was designed to examine its effects on the colonic microbiome.Methods/Subjects:Healthy volunteers and patients with CD followed a regimen confined to enteral feeds alone for 1 or 2 weeks, respectively. Chemicals excreted on breath or in faeces were characterised at the start and at the end of the feeding period by gas chromatography/mass spectrometry.Results:One week of feeding in healthy volunteers caused significant changes in stool colour and deterioration in breath odour, together with increased excretion of phenol and indoles on the breath. Feeding for 2 weeks in patients with CD produced significant improvements in symptoms and a decrease in the concentration of C-reactive protein. The faecal concentrations of microbial products, including short-chain fatty acids (SCFAs), and potentially toxic substances, including 1-propanol, 1-butanol and the methyl and ethyl esters of SCFAs, showed significant falls.Conclusions:A significant change occurs in the production of microbial metabolites after enteral feeding in both healthy volunteers and patients with CD. Many of those detected in CD are toxic and may feasibly lead to the immunological attack on the gut microbiota, which is characteristic of inflammatory bowel disease. The reduction in the production of such metabolites after enteral feeding may be the reason for its effectiveness in CD.

Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA.

Generic primers are available for detecting bacterial genes required for almost every reaction of the biological nitrogen cycle, the one notable exception being napA (gene for the molybdoprotein of the periplasmic nitrate reductase) encoding periplasmic nitrate reductases. Using an iterative approach, we report the first successful design of three forward oligonucleotide primers and one reverse primer that, in three separate PCRs, can amplify napA DNA from all five groups of Proteobacteria. All 140 napA sequences currently listed in the NCBI (National Center for Biotechnology Information) database are predicted to be amplified by one or more of these primer pairs. We demonstrate that two pairs of these primers also amplify PCR products of the predicted sizes from DNA isolated from human faeces, confirming their ability to direct the amplification of napA fragments from mixed populations. Analysis of the resulting amplicons by high-throughput sequencing will enable a good estimate to be made of both the range and relative abundance of nitrate-reducing bacteria in any community, subject only to any unavoidable bias inherent in a PCR approach to molecular characterization of a highly diverse target.