Ronan Doyle

Regulatory B cells are induced by gut microbiota-driven interleukin-1β and interleukin-6 production

Regulatory B cells (Breg cells) differentiate in response to inflammation and subsequently restrain excessive immune responses via the release of interleukin-10 (IL-10). However, the precise inflammatory signals governing their differentiation remain to be elucidated. Here we show that the gut microbiota promotes the differentiation of Breg cells in the spleen as well as in the mesenteric lymph nodes. Perturbation of the gut microbiome imposed either by antibiotic treatment or by changes in the sterility of housing conditions reduces the number and function of Breg cells. Following the induction of arthritis, IL-1β and IL-6 are produced only in conventionally housed mice and both cytokines directly promote Breg cell differentiation and IL-10 production. Mice lacking IL-6 receptor (IL-6R) or IL-1 receptor 1 (IL-1R1) specifically on B cells have a reduced number of IL-10–producing B cells and develop exacerbated arthritis compared to control animals. Thus, in response to inflammatory signals induced by both the gut flora and arthritis, Breg cells increase in number and restrain excessive inflammation.

Term and preterm labour are associated with distinct microbial community structures in placental membranes which are independent of mode of delivery.

Infection is considered a possible trigger for preterm labour, supported by evidence showing the presence of bacteria in the placenta and placental membranes from preterm births. In this study, 16S rDNA pyrosequencing was used to identify bacteria in placental membranes. Caesarean sections and vaginal deliveries at term were found to harbour common genera. Mycoplasma hominis, Aerococcus christensenii, Gardnerella vaginalis and Fusobacterium nucleatum were either only present in preterm membranes or in greater abundance than at term. These data support previous studies that used either targeted qPCR or broad-range 16S rDNA PCR and cloning but not a recent microbiome analysis of placental tissue using high-throughput sequencing.

Bacterial communities found in placental tissues are associated with severe chorioamnionitis and adverse birth outcomes

Preterm birth is a major cause of neonatal mortality and morbidity worldwide. Bacterial infection and the subsequent inflammatory response are recognised as an important cause of preterm birth. It is hypothesised that these organisms ascend the cervical canal, colonise placental tissues, cause chorioamnionitis and in severe cases infect amniotic fluid and the foetus. However, the presence of bacteria within the intrauterine cavity does not always precede chorioamnionitis or preterm birth. Whereas previous studies observing the types of bacteria present have been limited in size and the specificity of a few predetermined organisms, in this study we characterised bacteria found in placental tissues from a cohort of 1391 women in rural Malawi using 16S ribosomal RNA gene sequencing. We found that specific bacteria found concurrently on placental tissues associate with chorioamnionitis and delivery of a smaller newborn. Severe chorioamnionitis was associated with a distinct difference in community members, a higher bacterial load and lower species richness. Furthermore, Sneathia sanguinengens and Peptostreptococcus anaerobius found in both matched participant vaginal and placental samples were associated with a lower newborn length-for-age Z-score. This is the largest study to date to examine the placental microbiome and its impact of birth outcomes. Our results provide data on the role of the vaginal microbiome as a source of placental infection as well as the possibility of therapeutic interventions against targeted organisms during pregnancy.

Rapid identification of a Mycobacterium tuberculosis full genetic drug resistance profile through whole genome sequencing directly from sputum

INTRODUCTION Resistance to second-line tuberculosis drugs is common, but slow to diagnose with phenotypic drug sensitivity testing. Rapid molecular tests speed up diagnosis, but can only detect limited mutations. Whole genome sequencing (WGS) of culture isolates can generate a complete genetic drug resistance profile, but is delayed by the initial culture step. In the case presented here, successful WGS directly from sputum was achieved using targeted enrichment. CASE REPORT A 29-year-old Nigerian woman was diagnosed with tuberculosis. Xpert MTB/RIF and Hain line probe assays identified rpoB and inhA mutations consistent with rifampicin and intermediate isoniazid resistance, and a further possible mutation conferring fluoroquinolone resistance. WGS directly from sputum identified a further inhA mutation consistent with high-level isoniazid resistance and confirmed the absence of fluoroquinolone resistance. Isoniazid was stopped, and the patient has completed 18 months of a fluoroquinolone-based regimen without relapse. DISCUSSION Compared to rapid molecular tests (which can only examine a limited number of mutations) and WGS of culture isolates (which requires a culture step), WGS directly from sputum can quickly generate a complete genetic drug resistance profile. In this case, WGS altered the clinical management of drug-resistant tuberculosis and demonstrated potential for guiding individualized drug treatment where second-line drug resistance is common.

Distinguishing the Signals of Gingivitis and Periodontitis in Supragingival Plaque: a Cross-Sectional Cohort Study in Malawi

ABSTRACT Periodontal disease ranges from gingival inflammation (gingivitis) to the inflammation and loss of tooth-supporting tissues (periodontitis). Previous research has focused mainly on subgingival plaque, but supragingival plaque composition is also known to be associated with disease. Quantitative modeling of bacterial abundances across the natural range of periodontal severities can distinguish which features of disease are associated with particular changes in composition. We assessed a cross-sectional cohort of 962 Malawian women for periodontal disease and used 16S rRNA gene amplicon sequencing (V5 to V7 region) to characterize the bacterial compositions of supragingival plaque samples. Associations between bacterial relative abundances and gingivitis/periodontitis were investigated by using negative binomial models, adjusting for epidemiological factors. We also examined bacterial cooccurrence networks to assess community structure. The main differences in supragingival plaque compositions were associated more with gingivitis than periodontitis, including higher bacterial diversity and a greater abundance of particular species. However, even after controlling for gingivitis, the presence of subgingival periodontitis was associated with an altered supragingival plaque. A small number of species were associated with periodontitis but not gingivitis, including members of Prevotella, Treponema, and Selenomonas, supporting a more complex disease model than a linear progression following gingivitis. Cooccurrence networks of periodontitis-associated taxa clustered according to periodontitis across all gingivitis severities. Species including Filifactor alocis and Fusobacterium nucleatum were central to this network, which supports their role in the coaggregation of periodontal biofilms during disease progression. Our findings confirm that periodontitis cannot be considered simply an advanced stage of gingivitis even when only considering supragingival plaque. IMPORTANCE Periodontal disease is a major public health problem associated with oral bacteria. While earlier studies focused on a small number of periodontal pathogens, it is now accepted that the whole bacterial community may be important. However, previous high-throughput marker gene sequencing studies of supragingival plaque have largely focused on high-income populations with good oral hygiene without including a range of periodontal disease severities. Our study includes a large number of low-income participants with poor oral hygiene and a wide range of severities, and we were therefore able to quantitatively model bacterial abundances as functions of both gingivitis and periodontitis. A signal associated with periodontitis remains after controlling for gingivitis severity, which supports the concept that, even when only considering supragingival plaque, periodontitis is not simply an advanced stage of gingivitis. This suggests the future possibility of diagnosing periodontitis based on bacterial occurrences in supragingival plaque.

Direct whole genome sequencing of sputum accurately identifies drug resistant Mycobacterium tuberculosis faster than MGIT culture sequencing

The current methods available to diagnose antimicrobial-resistant Mycobacterium tuberculosis infections require a positive culture or only test a limited number of resistance-associated mutations. A rapid accurate identification of antimicrobial resistance enables the prompt initiation of effective treatment. ABSTRACT The current methods available to diagnose antimicrobial-resistant Mycobacterium tuberculosis infections require a positive culture or only test a limited number of resistance-associated mutations. A rapid accurate identification of antimicrobial resistance enables the prompt initiation of effective treatment. Here, we determine the utility of whole-genome sequencing (WGS) of M. tuberculosis directly from routinely obtained diagnostic sputum samples to provide a comprehensive resistance profile compared to that from mycobacterial growth indicator tube (MGIT) WGS. We sequenced M. tuberculosis from 43 sputum samples by targeted DNA enrichment using the Agilent SureSelectXT kit, and 43 MGIT positive samples from each participant. Thirty two (74%) sputum samples and 43 (100%) MGIT samples generated whole genomes. The times to antimicrobial resistance profiles and concordance were compared with Xpert MTB/RIF and phenotypic resistance testing from cultures of the same samples. Antibiotic susceptibility could be predicted from WGS of sputum within 5 days of sample receipt and up to 24 days earlier than WGS from MGIT culture and up to 31 days earlier than phenotypic testing. Direct sputum results could be reduced to 3 days with faster hybridization and if only regions encoding drug resistance are sequenced. We show that direct sputum sequencing has the potential to provide comprehensive resistance detection significantly faster than MGIT whole-genome sequencing or phenotypic testing of resistance from cultures in a clinical setting. This improved turnaround time enables prompt appropriate treatment with associated patient and health service benefits. Improvements in sample preparation are necessary to ensure comparable sensitivities and complete resistance profile predictions in all cases.

Enrichment of clinically relevant organisms in spontaneous preterm delivered placenta and reagent contamination across all clinical groups in a large UK pregnancy cohort

Preterm birth is associated with both psychological and physical disabilities and is the leading cause of infant morbidity and mortality worldwide. Infection is known to be an important cause of spontaneous preterm birth, and recent research has implicated variation in the “placental microbiome” in the risk of preterm birth. Consistent with data from previous studies, the abundances of certain clinically relevant species differed between spontaneous preterm- and nonspontaneous preterm- or term-delivered placentas. These results support the view that a proportion of spontaneous preterm births have an intrauterine-infection component. However, an additional observation from this study was that a substantial proportion of sequenced reads were contaminating reads rather than DNA from endogenous, clinically relevant species. This observation warrants caution in the interpretation of sequencing outputs from low-biomass samples such as the placenta. ABSTRACT In this study, differences in the placental microbiota from term and preterm deliveries in a large pregnancy cohort in the United Kingdom were studied by using 16S-targeted amplicon sequencing. The impacts of contamination from DNA extraction, PCR reagents, and the delivery itself were also examined. A total of 400 placental samples from 256 singleton pregnancies were analyzed, and differences between spontaneous preterm-, nonspontaneous preterm-, and term-delivered placentas were investigated. DNA from recently delivered placentas was extracted, and screening for bacterial DNA was carried out by using targeted sequencing of the 16S rRNA gene on the Illumina MiSeq platform. Sequenced reads were analyzed for the presence of contaminating operational taxonomic units (OTUs) identified via sequencing of negative extraction and PCR-blank samples. Differential abundances and between-sample (beta) diversity metrics were then compared. A large proportion of the reads sequenced from the extracted placental samples mapped to OTUs that were also found for negative extractions. Striking differences in the compositions of samples were also observed, according to whether the placenta was delivered abdominally or vaginally, providing strong circumstantial evidence for delivery contamination as an important contributor to observed microbial profiles. When OTU- and genus-level abundances were compared between the groups of interest, a number of organisms were enriched in the spontaneous preterm-delivery cohort, including organisms that have been associated previously with adverse pregnancy outcomes, specifically Mycoplasma spp. and Ureaplasma spp. However, analyses of the overall community structure did not reveal convincing evidence for the existence of a reproducible “preterm placental microbiome.” IMPORTANCE Preterm birth is associated with both psychological and physical disabilities and is the leading cause of infant morbidity and mortality worldwide. Infection is known to be an important cause of spontaneous preterm birth, and recent research has implicated variation in the “placental microbiome” in the risk of preterm birth. Consistent with data from previous studies, the abundances of certain clinically relevant species differed between spontaneous preterm- and nonspontaneous preterm- or term-delivered placentas. These results support the view that a proportion of spontaneous preterm births have an intrauterine-infection component. However, an additional observation from this study was that a substantial proportion of sequenced reads were contaminating reads rather than DNA from endogenous, clinically relevant species. This observation warrants caution in the interpretation of sequencing outputs from low-biomass samples such as the placenta.

Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture

Background Repeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity. We compared M. tuberculosis whole genome sequences generated from 33 paired clinical samples using two methods. In one method DNA was extracted directly from sputum then enriched with custom-designed SureSelect (Agilent) oligonucleotide baits and in the other it was extracted from mycobacterial growth indicator tube (MGIT) culture. Results DNA directly sequenced from sputum showed significantly more within-sample diversity than that from MGIT culture (median 5.0 vs 4.5 heterozygous alleles per sample, p=0.04). Resistance associated variants present as HAs occurred in four patients, and in two cases may provide a genotypic explanation for phenotypic resistance. Conclusions Culture-free M. tuberculosis whole genome sequencing detects more within-sample diversity than a leading culture-based method and may allow detection of mycobacteria that are not actively replicating.

Children with cystic fibrosis are infected with multiple subpopulations of Mycobacterium abscessus with different antimicrobial resistance profiles

Background Children with cystic fibrosis (CF) can develop life-threatening infections of Mycobacterium abscessus. These present a significant clinical challenge, particularly when the strains involved are resistant to antibiotics. Recent evidence of within-patient subclones of M. abscessus in adults with CF suggests the possibility that within-patient diversity may be relevant for the treatment of pediatric CF patients. Methods We performed whole genome sequencing (WGS) on 32 isolates of M. abscessus from multiple body sites for two patients with CF undergoing treatment at Great Ormond Street Hospital, UK, in 2015. Results We found evidence of extensive diversity within patients over time. Clustering analysis of single nucleotide variants (SNVs) revealed that each patient harboured multiple subpopulations, which were differentially abundant between sputum, lung samples, chest wounds, and pleural fluid. Sputum isolates did not reflect overall within-patient diversity, including failing to detect subclones with mutations previously associated with macrolide resistance (rrl 2058/2059). Some variants were present at intermediate frequencies before lung transplant. The time of transplant coincided with extensive variation, suggesting that this event is particularly disruptive for the microbial community, but transplant did not clear the M. abscessus infection and both patients died as a result of this infection. Conclusions Isolates of M. abscessus from sputum do not always reflect the entire diversity present within the patient, which can include subclones with differing AMR profiles. Awareness of this phenotypic variability, with sampling of multiple body sites in conjunction with WGS, may be necessary to ensure the best treatment for this vulnerable patient group. Key point summary Children with cystic fibrosis undergoing lung transplant harbour multiple subpopulations of M. abscessus. Subpopulations can have different antimicrobial resistance genotypes. Sputum isolates do not reflect the genetic diversity within a patient.

Co-causation of reduced newborn size by maternal undernutrition, infections, and inflammation.

Abstract More than 20 million babies are born with low birthweight annually. Small newborns have an increased risk for mortality, growth failure, and other adverse outcomes. Numerous antenatal risk factors for small newborn size have been identified, but individual interventions addressing them have not markedly improved the health outcomes of interest. We tested a hypothesis that in low‐income settings, newborn size is influenced jointly by multiple maternal exposures and characterized pathways associating these exposures with newborn size. This was a prospective cohort study of pregnant women and their offspring nested in an intervention trial in rural Malawi. We collected information on maternal and placental characteristics and used regression analyses, structural equation modelling, and random forest models to build pathway maps for direct and indirect associations between these characteristics and newborn weight‐for‐age Z‐score and length‐for‐age Z‐score. We used multiple imputation to infer values for any missing data. Among 1,179 pregnant women and their babies, newborn weight‐for‐age Z‐score was directly predicted by maternal primiparity, body mass index, and plasma alpha‐1‐acid glycoprotein concentration before 20 weeks of gestation, gestational weight gain, duration of pregnancy, placental weight, and newborn length‐for‐age Z‐score (p < .05). The latter 5 variables were interconnected and were predicted by several more distal determinants. In low‐income conditions like rural Malawi, maternal infections, inflammation, nutrition, and certain constitutional factors jointly influence newborn size. Because of this complex network, comprehensive interventions that concurrently address multiple adverse exposures are more likely to increase mean newborn size than focused interventions targeting only maternal nutrition or specific infections.