RESULTS:

Crohn disease (CD) is a chronic inflammatory condition primarily affecting the gastro-intestinal tract and is characterized by reduced bacterial diversity. The exact cause of disease is unknown; however evidence suggests that several components including microbiota may contribute to the underlying pathology and disease development. Perturbation of the host–microbe commensal relationship is considered the main driving force of tissue destruction and pathological changes seen in CD. Several putative bacterial pathogens including species from Mycobacterium Campylobacter and Helicobacter are postulated in the aetiology of CD. However to date no strong evidence supports a single bacterium contributing overall to CD pathogenesis. Alternatively dysbiosis or bacterial imbalance is more widely accepted as a leading factor in the disrupted host–immune system cross-talk resulting in subsequent intestinal inflammation. Depletion of symbiont microbes including Firmicutes Bifidobacterium and Clostridia in conjunction with an increase in pathobiont microbes from Bacteroidetes and Enterobacteria is a striking feature observed in CD. No single factor has been identified as driving this dysbiosis although diet antibiotic exposure and possible early life events in presence of underlying genetic susceptibility may contribute. The aim of this review is to highlight the current accumulating literature on the proposed role of bacteria in the pathogenesis of CD.

Purpose. To date molecular methods that circumvent the limitations of traditional culture methods have not been used to describe the vaginal microflora in India. Here we compared culture and culture-independent molecular methods in characterizing the vaginal microbiota in Indian women.

Methodology. Culture methods involved traditional cultivation on Rogosa and sheep blood agar whereas culture-independent methods bypassed a culturing step by performing broadrange 16S rDNA PCR on DNA isolated directly from vaginal swabs.

Results. A total of 13 women were included in the study of which five were characterized as healthy two were bacterial vaginosis intermediate and six were bacterial vaginosis positive according to Nugent scoring. Lactobacillus jensenii was detected most frequently when using culture methods. On the other hand Lactobacillus iners which was not detected by culture methods was the most common Lactobacillus sp. detected using cultivation-independent methods.

Conclusion. We found little overlap between the species found using cultivation-dependent and cultivation-independent methods. Rather culture-dependent and culture-independent methods were found to be complementary in describing the vaginal microflora among South Indian women. Culture-independent methods were found to be superior in detecting clinically relevant vaginal flora.

A few studies have highlighted the importance of the respiratory microbiome in modulating the frequency and outcome of viral respiratory infections. However there are insufficient data on the use of microbial signatures as prognostic biomarkers to predict respiratory disease outcomes. In this study we aimed to evaluate whether specific bacterial community compositions in the nasopharynx of children at the time of hospitalization are associated with different influenza clinical outcomes. We utilized retrospective nasopharyngeal (NP) samples (n=36) collected at the time of hospital arrival from children who were infected with influenza virus and had been symptomatic for less than 2 days. Based on their clinical course children were classified into two groups: patients with mild influenza and patients with severe respiratory or neurological complications. We implemented custom 16S rRNA gene sequencing metagenomic sequencing and computational analysis workflows to classify the bacteria present in NP specimens at the species level. We found that increased bacterial diversity in the nasopharynx of children was strongly associated with influenza severity. In addition patients with severe influenza had decreased relative abundance of Staphylococcus aureus and increased abundance of Prevotella (including P. melaninogenica) Streptobacillus Porphyromonas Granulicatella (including G. elegans) Veillonella (including V. dispar) Fusobacterium and Haemophilus in their nasopharynx. This pilot study provides proof-of-concept data for the use of microbial signatures as prognostic biomarkers of influenza outcomes. Further large prospective cohort studies are needed to refine and validate the performance of such microbial signatures in clinical settings.

Introduction. Prosthetic joint infections (PJIs) are challenging to treat therapeutically because the infectious agents often are resistant to antibiotics and capable of abundant growth in surface-attached biofilms. Though infection rates are low ca. 1–2 % the overall increase in the sheer number of joint replacement surgeries results in an increase in patients at risk.

Aims. This study investigates the consensus of microbial species comprising PJI ecology which is currently lacking.

Methodology. In this study PJI populations from seven patients were analysed using combined culturing and whole-genome shotgun sequencing (WGSS) to establish population profiles and compare WGSS and culture methods for detection and identification of the PJI microbiome.

Results. WGSS detected strains when culture did not notably dormant culture-resistant and rare microbes. The CosmosID algorithm was used to predict micro-organisms present in the PJI and discriminate contaminants. However culturing indicated the presence of microbes falling below the WGSS algorithm threshold. In these instances microbes cultured are believed to be minor species. The two strategies were combined to build a population profile.

Conclusions. Variability between and among PJIs showed that most infections were distinct and unique. Comparative analysis of populations revealed PJIs to form clusters that were related to but separate from vaginal skin and gut microbiomes. Fungi and protists were detected by WGSS but the role of fungi is just beginning to be understood and for protists it is unknown. These micro-organisms and their novel and strain-specific microbial interactions remain to be determined in current clinical tests.

Purpose. While some micro-organisms such as Staphylococcus aureus are clearly implicated in causing tissue damage in diabetic foot ulcers (DFUs) our knowledge of the contribution of the entire microbiome to clinical outcomes is limited. We profiled the microbiome of a longitudinal sample series of 28 people with diabetes and DFUs of the heel in an attempt to better characterize the relationship between healing infection and the microbiome.

Methodology. In total 237 samples were analysed from 28 DFUs collected at fortnightly intervals for 6 months or until healing. Microbiome profiles were generated by 16S rRNA gene sequence analysis supplemented by targeted nanopore sequencing.

Result/Key findings. DFUs which failed to heal during the study period (20/28 71.4 %) were more likely to be persistently colonized with a heterogeneous community of micro-organisms including anaerobes and Enterobacteriaceae (log-likelihood ratio 9.56 P=0.008). During clinically apparent infection a reduction in the diversity of micro-organisms in a DFU was often observed due to expansion of one or two taxa with recovery in diversity at resolution. Modelling of the predicted species interactions in a single DFU with high diversity indicated that networks of metabolic interactions may exist that contribute to the formation of stable communities.

Conclusion. Longitudinal profiling is an essential tool for improving our understanding of the microbiology of chronic wounds as community dynamics associated with clinical events can only be identified by examining changes over multiple time points. The development of complex communities particularly involving Enterobacteriaceae and strict anaerobes may be contributing to poor outcomes in DFUs and requires further investigation.

A strain of an obligately anaerobic Gram-stain-negative rod-shaped bacterium is described by phenotypical biochemical and genotypical characterization. This strain A2879T was isolated from an abscess swab of a patient sampled during routine care at hospital. Phylogenetic analyses (full-length 16S rRNA gene and whole-genome sequence) revealed the strain to belong to the genus Prevotella but to be distant from recognized species with the closest relationship to Prevotella veroralis . Unambiguous identification also proved possible by MALDI-TOF MS. The genomic DNA G+C content was 41.5 mol%. Strain A2879T was moderately saccharolytic and proteolytic. The most abundant cellular long-chain fatty acids were anteiso-C15 : 0 and iso-C15 : 0. In view of these data strain A2879T is considered to represent a novel species within the genus Prevotella for which the name Prevotella vespertina sp. nov. is proposed. The type strain is A2879T (=DSM 108027T=CCOS 1233T=CCUG72808T). As this strain has been isolated from a clinical sample it is considered relevant for human medicine and health in general and in particular for the fields of clinical microbiology and infectious diseases. This description will enable routine and research laboratories alike to easily identify the novel taxon allowing its role in the context of human health and disease or microbiota to be further elucidated.

Unique morphologies can enable bacteria to survive in their native environment. Furthermore many bacteria change their cell shape to adapt to different environmental conditions. For instance some bacteria increase their surface area under carbon or nitrogen starvation. Bacteriodes thetaiotaomicron is an abundant human gut species; it efficiently degrades a number of carbohydrates and also supports the growth of other bacteria by breaking down complex polysaccharides. The gut provides a variable environment as nutrient availability is subject to the diet and health of the host yet how gut bacteria adapt and change their morphologies under different nutrient conditions has not been studied. Here for the first time we report an elongated B. thetaiotaomicron morphology under sugar-limited conditions using live-cell imaging; this elongated morphology is enhanced in the presence of sodium bicarbonate. Similarly we also observed that sodium bicarbonate produces an elongated-length phenotype in another Gram-negative gut bacterium Escherichia coli . The increase in cell length might provide an adaptive advantage for cells to survive under nutrient-limited conditions.

In the mid-1960s the microbial aetiology of periodontal diseases was introduced based on classical experimental gingivitis studies . Since then numerous studies have addressed the fundamental role that oral microbiota plays in the initiation and progression of periodontal diseases. Recent advances in laboratory identification techniques have contributed to a better understanding of the complexity of the oral microbiome in both health and disease. Modern culture-independent methods such as human oral microbial identification microarray and next-generation sequencing have been used to identify a wide variety of microbial taxa residing in the gingival sulcus and the periodontal pocket. The first theory of the ‘non-specific plaque’ hypothesis gave rise to the ‘ecological plaque’ hypothesis and more recently to the ‘polymicrobial synergy and dysbiosis hypothesis’. Periodontitis is now considered to be a multimicrobial inflammatory disease in which the various bacterial species within the dental biofilm are in a dysbiotic state and this imbalance favours the establishment of chronic inflammatory conditions and ultimately the destruction of tooth-supporting tissues. Apart from the known putative periodontal pathogens the whole biofilm community is now considered to play a role in the establishment of inflammation and the initiation and progression of periodontitis in a susceptible host. Treatment is unlikely to eliminate putative pathogens but when it is thoroughly performed it has the potential to establish a healthy ecosystem by altering the microbial community in numbers and composition and also contribute to the maturation of the host immune response.

Antarctic sea-ice forms a complex and dynamic system that drives many ecological processes in the Southern Ocean. Sea-ice microalgae and their associated microbial communities are understood to influence nutrient flow and allocation in marine polar environments. Sea-ice microalgae and their microbiota can have high seasonal and regional (>1000 km2) compositional and abundance variation driven by factors modulating their growth symbiotic interactions and function. In contrast our knowledge of small-scale variation in these communities is limited. Understanding variation across multiple scales and its potential drivers is critical for informing on how multiple stressors impact sea-ice communities and the functions they provide. Here we characterized bacterial communities associated with sea-ice microalgae and the potential drivers that influence their variation across a range of spatial scales (metres to >10 kms) in a previously understudied area in Commonwealth Bay East Antarctica where anomalous events have substantially and rapidly expanded local sea-ice coverage. We found a higher abundance and different composition of bacterial communities living in sea-ice microalgae closer to the shore compared to those further from the coast. Variation in community structure increased linearly with distance between samples. Ice thickness and depth to the seabed were found to be poor predictors of these communities. Further research on the small-scale environmental drivers influencing these communities is needed to fully understand how large-scale regional events can affect local function and ecosystem processes.

In metagenomic analyses of microbiomes one of the first steps is usually the taxonomic classification of reads by comparison to a database of previously taxonomically classified genomes. While different studies comparing metagenomic taxonomic classification methods have determined that different tools are ‘best’ there are two tools that have been used the most to-date: Kraken (k-mer-based classification against a user-constructed database) and MetaPhlAn (classification by alignment to clade-specific marker genes) the latest versions of which are Kraken2 and MetaPhlAn 3 respectively. We found large discrepancies in both the proportion of reads that were classified as well as the number of species that were identified when we used both Kraken2 and MetaPhlAn 3 to classify reads within metagenomes from human-associated or environmental datasets. We then investigated which of these tools would give classifications closest to the real composition of metagenomic samples using a range of simulated and mock samples and examined the combined impact of tool–parameter–database choice on the taxonomic classifications given. This revealed that there may not be a one-size-fits-all ‘best’ choice. While Kraken2 can achieve better overall performance with higher precision recall and F1 scores as well as alpha- and beta-diversity measures closer to the known composition than MetaPhlAn 3 the computational resources required for this may be prohibitive for many researchers and the default database and parameters should not be used. We therefore conclude that the best tool–parameter–database choice for a particular application depends on the scientific question of interest which performance metric is most important for this question and the limit of available computational resources.