Irene M. Monahan

Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages Insights into the Phagosomal Environment

Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2–deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon γ– and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and β-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of σE-dependent, sodium dodecyl sulfate–regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogens cell envelope.

Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection

Summary Background Diagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection. Methods In this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12 420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative. Findings Clinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p<0·001), but not in group 2 compared with group 3 (p=0·4). A multivariate analysis accounting for potential confounders confirmed the mortality differences between groups 1 and 3 (OR 1·61, 95% CI 1·12–2·31). Multistage algorithms performed better than did standalone assays. Interpretation We noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection. Funding Department of Health and Health Protection Agency, UK.

Differential expression of mycobacterial proteins following phagocytosis by macrophages

Mycobacterium tuberculosis resides within the macrophages of the host, but the molecular and cellular mechanisms of survival are poorly understood. Recent evidence suggests that the attenuated vaccine strain Mycobacterium bovis BCG is both a deletion and regulatory mutant, yet retains both its immunoprotective and intra-macrophage survival potential. In an attempt to define M. bovis BCG genes expressed during interaction with macrophages, the patterns of protein synthesis were examined by both one- and two-dimensional gel electrophoresis of BCG while inside the human leukaemic macrophage cell line THP-1. This study demonstrated that BCG expresses proteins while resident inside macrophages that are not expressed during in vitro growth in culture media or under conditions of heat shock. Western blotting analysis revealed that some of the differentially expressed proteins are specifically recognized by human M. tuberculosis-infected sera. Proteome analysis by two-dimensional electrophoresis and MS identified six abundant proteins that showed increased expression inside macrophages: 16 kDa alpha-crystallin (HspX), GroEL-1 and GroEL-2, a 31.7 kDa hypothetical protein (Rv2623), InhA and elongation factor Tu (Tuf). Identification of proteins by such a strategy will help elucidate the molecular basis of the attenuation and the vaccine potential of BCG, and may provide antigens that distinguish infection with M. tuberculosis from vaccination with BCG.

Gene expression during host—pathogen interactions: Approaches to bacterial mRNA extraction and labelling for microarray analysis

Publisher Summary The expression of genes in response to the signals associated with environmental stimuli is a prerequisite for the survival of bacterial pathogens within the host and is, therefore, the underlying basis of infectious disease. With the increasing amount of information about gene complement of bacterial pathogens and the development of functional genomics technologies, such as microarrays, it is now possible to monitor bacterial gene expression at a whole genome level both in vitro and during host-pathogen interactions. Such considerations impact on the choice of an experimental approach to extracting bacterial RNA for microarray analysis from different models of infection, for example, axenic culture, cell monocultures infected with bacteria in vitro, or whole tissues from the infected animal models. This chapter presents the experiences and methods that address both the biological and technical issues involved in extracting biologically “meaningful” RNA from the pathogenic mycobacterium, Mycobacterium tuberculosis , for the purposes of studying whole-genome expression during host-pathogen interactions using microarray analysis.

Extraction of RNA from Intracellular Mycobacterium tuberculosis

Pathogenicity in Mycobacterium tuberculosis may be thought of as a multifactorial process with both pathogen and host-response effector molecules contributing to the process of infection, leading either to immunopathology and disease or control of infection and long-term persistence. Little is known about this at a genetic level, but it is becoming recognized that bacterial virulence constitutes the correct temporal and spatial regulation of many genes that may be necessary for a particular phase in infection in response to specific environmental cues.

Accurate detection of Neisseria gonorrhoeae ciprofloxacin susceptibility directly from genital and extragenital clinical samples: towards genotype-guided antimicrobial therapy

Introduction Increasing use of nucleic acid amplification tests (NAATs) as the primary means of diagnosing gonococcal infection has resulted in diminished availability of Neisseria gonorrhoeae antimicrobial susceptibility data. We conducted a prospective diagnostic assessment of a real-time PCR assay (NGSNP) enabling direct detection of gonococcal ciprofloxacin susceptibility from a range of clinical sample types. Methods NGSNP, designed to discriminate an SNP associated with ciprofloxacin resistance within the N. gonorrhoeae genome, was validated using a characterized panel of geographically diverse isolates (n = 90) and evaluated to predict ciprofloxacin susceptibility directly on N. gonorrhoeae-positive NAAT lysates derived from genital (n = 174) and non-genital (n = 116) samples (n = 290), from 222 culture-confirmed clinical episodes of gonococcal infection. Results NGSNP correctly genotyped all phenotypically susceptible (n = 49) and resistant (n = 41) panel isolates. Ciprofloxacin-resistant N. gonorrhoeae was responsible for infection in 29.7% (n = 66) of clinical episodes evaluated. Compared with phenotypic susceptibility testing, NGSNP demonstrated sensitivity and specificity of 95.8% (95% CI 91.5%–98.3%) and 100% (95% CI 94.7%–100%), respectively, for detecting ciprofloxacin-susceptible N. gonorrhoeae, with a positive predictive value of 100% (95% CI 97.7%–100%). Applied to urogenital (n = 164), rectal (n = 40) and pharyngeal samples alone (n = 30), positive predictive values were 100% (95% CI 96.8%–100%), 100% (95% CI 87.2%–100%) and 100% (95% CI 82.4%–100%), respectively. Conclusions Genotypic prediction of N. gonorrhoeae ciprofloxacin susceptibility directly from clinical samples was highly accurate and, in the absence of culture, will facilitate use of tailored therapy for gonococcal infection, sparing use of current empirical treatment regimens and enhancing acquisition of susceptibility data for surveillance.

The expression profile of Mycobacterium tuberculosis infecting the human monocytic cell line THP-1 using whole genome microarray analysis

The expression profile of Mycobacterium tuberculosis infecting the human monocytic cell line THP-1 using whole genome microarray analysis

Sexually active students’ acceptability of providing saline oral samples for future human papillomavirus testing

Dear Editor, Most (70%) 16–24 year olds report having oral sex, but many are unaware that this can lead to sexually transmitted infections (STIs) in the throat. In addition, we lack UK data on both the prevalence of oropharyngeal STIs in young people not attending healthcare facilities and on acceptable oral sampling techniques. We aimed to assess the acceptability of saline oral rinses in sexually-active, ethnically-diverse teenagers (50% black, 25% white and 25% other ethnic groups) attending six London further education colleges. From April to May 2017, “Test n Treat” trial participants were asked to complete a follow-up questionnaire and provide a self-taken vaginal swab (females) or urine sample (males) for rapid chlamydia/gonorrhoea testing and underwent on-site treatment on the same day, if required. They were then asked to read a brief information sheet and (if willing) to sign a consent form to provide a mouthwash sample for future STI research. They were asked to gargle 10 ml of normal saline for 15 s, swish it around their mouth for a further 15 s and spit it back into the container. At two sites, participants were also asked two questions:

P5.098 Clinical Prediction of Fluoroquinolone Susceptibility, Directly from Residual Routine NAAT Gonococcal-Positive Samples Using a gyrA SNP Detection Assay

Background Antimicrobial resistance in Neisseria gonorrhoeae (NG) poses significant challenges for empirical therapy, heightened by nucleic acid amplification tests (NAAT) use over culture for diagnosis. Molecular susceptibility testing, directly on NAAT-positive clinical samples may allow fluoroquinolone use, currently precluded because community resistance rates are > 5%. We determined whether a genotypic resistance test performed directly on NG NAAT positive routine clinical samples predicted susceptibility to ciprofloxacin with > 95% confidence. Methods A real time PCR SNP detection assay targeting the C > T SNP in codon 91 (S91P) of gyrA, commonly linked with other gyrA resistance associated SNPs, was used on 81 previously identified NG isolates, tested for ciprofloxacin MICs by E-test after sample blinding (Phase 1) and also (Phase 2) on 103 blinded clinical samples from 89 patients, positive by NAAT and culture (25 women, 64 men; 21 cervical, 3 vaginal, 1 urethral, 48 urine, 10 throat and 21 rectal). Results Phase 1 and Phase 2: 61/81(75%) and 68/103(66%) respectively were phenotypically susceptible to ciprofloxacin; 81/81 and 87/103 respectively of S91P assays worked. Phase 2 assay failure was not associated with sample site. Of Phase 2 assays that worked, predictive values for identifying ciprofloxacin susceptible gonorrhoea, using wild-type (S91P absence), and resistant gonorrhoea, using S91P presence, on clinical samples from: genital sites was 100%(95% CI: 91%–100%)/86%(67%–95%) respectively; non-genital sites was 93%(68%–99%)/92%(65%–99%) respectively; overall was 98%(90%–100%)/88%(73%–95%) respectively. Among all 89 patients, assay use would have identified 47(53%) as treatable with ciprofloxacin, one incorrectly (predictive value of assay for susceptibility 97.9%(88.9%–99.6%). However, nearly 80% of men and 60% of women received treatment for gonorrhoea before these results would have been available. Conclusions Direct genotypic testing of routine gonococcal NAAT-positive genital samples allows for safe use of ciprofloxacin in gonorrhoea but may have limited impact, particularly for men until available at the point of care.