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Refereed/Peer-reviewed Hotel quarantine for international travelers has been used to prevent coronavirus disease spread into Australia. A quarantine hotel–associated community outbreak was detected in South Australia. Real-time genomic sequencing enabled rapid confirmation tracking the outbreak to a recently returned traveler and linked 2 cases of infection in travelers at the same facility.

Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501YV2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected., Wellcome Open Research, 6

Background.Campylobacter curvusis a Gram-negative bacteria associated with periodontal disease in humans. Cases of extra-oral manifestations of infection are rare with only six reported cases of extra-oral infection including this report that have been identified in the current literature. Molecular methods are generally used to identifyC. curvuswhile optimal antibiotic choice and duration to treat extra-oral infections for this pathogen is unknown.Case presentation.A 63-year-old male with a background history of alcoholic pancreatitis presented with fever and malaise who was found to have radiological intra-abdominal collections. Drainage of these collections identifiedC. curvusvia matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry with high probability and identification further confirmed by whole-genome sequencing. Antibiotic susceptibility testing to erythromycin and ciprofloxacin ofC. curvuswas performed using E-test diffusion methods along with investigation for the presence of resistance genes. The patient was treated with intravenous piperacillin-tazobactam followed by ciprofloxacin for 4 weeks total with good clinical recovery.Conclusions.Extra-oral manifestations with the pathogenC. curvusare rare with few cases described in the literature. There is minimal data on susceptibility patterns, optimal antibiotic treatment and duration. Treatment of extraintestinalC. curvusinfections in humans should encompass both adequate source control and antibiotic therapy.

Pseudomonas aeruginosa is an opportunistic pathogen displaying high intrinsic antimicrobial resistance and the ability to thrive in different ecological environments. In this study, the ability of P. aeruginosa to develop simultaneous resistance to multiple antibiotics and disinfectants in different natural niches were investigated using strains collected from clinical samples, veterinary samples,and wastewater. The correlation between biocide and antimicrobial resistance was determined by employing principal component analysis. Molecular mechanisms linking biocide and antimicrobial resistance were interrogated by determining gene expression using RT-q PCR and identifying a potential genetic determinant for co- and cross-resistance using whole-genome sequencing.A subpopulation of P. aeruginosa isolates belonging to three sequence types was resistant against the common preservative benzalkonium chloride and showed cross-resistance to fluoroquinolones,cephalosporins, aminoglycosides, and multidrug resistance. Of these, the epidemiological high-risk ST235 clone was the most abundant. The over expression of the MexAB-OprM drug efflux pump resulting from amino acid mutations in regulators MexR, NalC, or NalD was the major contributing factor for cross-resistance that could be reversed by an efflux pump inhibitor. This is the first comparison of antibiotic-biocide cross-resistance in samples isolated from different ecological niches and serves as a confirmation of laboratory-based studies on biocide adapted isolates. The isolates from wastewater had a higher incidence of multidrug resistance and biocide-antibiotic cross-resistance than those from clinical and veterinary settings. Refereed/Peer-reviewed

Poor diet and lifestyle exposures are implicated in substantial global increases in non-communicable disease burden in low-income, remote, and Indigenous communities. This observational study investigated the contribution of the fecal microbiome to influence host physiology in two Indigenous communities in the Torres Strait Islands: Mer, a remote island where a traditional diet predominates, and Waiben a more accessible island with greater access to takeaway food and alcohol. Counterintuitively, disease markers were more pronounced in Mer residents. However, island-specific differences in disease risk were explained, in part, by microbiome traits. The absence of Alistipes onderdonkii, for example, significantly (p=0.014) moderated island-specific patterns of systolic blood pressure in multivariate-adjusted models. We also report mediatory relationships between traits of the fecal metagenome, disease markers, and risk exposures. Understanding how intestinal microbiome traits influence response to disease risk exposures is critical for the development of strategies that mitigate the growing burden of cardiometabolic disease in these communities.

Chronic disruption of the intestinal microbiota in adult cystic fibrosis (CF) patients is associated with local and systemic inflammation, and has been linked to the risk of serious comorbidities. Supplementation with high amylose maize starch (HAMS) might provide clinical benefit by promoting commensal bacteria and the biosynthesis of immunomodulatory metabolites. However, whether the disrupted CF gut microbiota has the capacity to utilise these substrates is not known. We combined metagenomic sequencing, in vitro fermentation, amplicon sequencing, and metabolomics to define the characteristics of the faecal microbiota in adult CF patients and assess HAMS fermentation capacity. Compared to healthy controls, the faecal metagenome of adult CF patients had reduced bacterial diversity and prevalence of commensal fermentative clades. In vitro fermentation models seeded with CF faecal slurries exhibited reduced acetate levels compared to healthy control reactions, but comparable levels of butyrate and propionate. While the commensal genus Faecalibacterium was strongly associated with short chain fatty acid (SCFA) production by healthy microbiota, it was displaced in this role by Clostridium sensu stricto 1 in the microbiota of CF patients. A subset of CF reactions exhibited enterococcal overgrowth, resulting in lactate accumulation and reduced SCFA biosynthesis. The addition of healthy microbiota to CF faecal slurries failed to displace predominant CF taxa, or substantially influence metabolite biosynthesis. Despite significant microbiota disruption, the adult CF gut microbiota retains the capacity to exploit HAMS. Our findings highlight the potential for taxa associated with the altered CF gut microbiotato mediate prebiotic effects in microbial systems subject to ongoing perturbation, irrespective of the depletion of common commensal clades.

Recent molecular-based studies have suggested that a diverse middle ear microbiome in adults and children can exist in the absence of disease. These studies have been largely unsupported by culture and feature species that commonly contaminate low-biomass sequencing data. While 16S rRNA gene amplicon sequencing has proven to be a highly informative technique in many clinical contexts, it is susceptible to spurious signal arising from sequencing reagent contaminants where sample biomass is low. Combining culture-based and molecular techniques, we undertook a detailed investigation of the evidence for bacterial colonization of the healthy middle ear. In finding no evidence of viable bacterial cells in middle ear samples, our study further underlines the importance of careful consideration of amplicon sequence data derived from very-low-biomass contexts and the value of analytical approaches that combine culture and molecular techniques., Otitis media (OM) is a cluster of diseases of the middle ear that commonly result from bacterial infection. OM subtypes in which the tympanic membrane is intact (acute otitis media and otitis media with effusion) are presumed to result from pathogen translocation through the eustachian tube. Recent molecular-based studies have suggested that a diverse middle ear microbiome exists in the absence of disease. These have been largely unsupported by culture and feature species that commonly contaminate low-biomass sequencing data. Combining culture-based and molecular techniques, we undertook a detailed investigation of the evidence for bacterial colonization of the healthy middle ear. Middle ear (ME), nasopharynx (NP), and external ear canal (EC) swabs were collected from a total of 25 adult patients undergoing cochlear implant, stapedotomy, or translabyrinthine vestibular schwannoma resection. Diagnostic culture, microscopy, quantitative PCR, and 16S rRNA gene amplicon sequencing were used to assess sample bacterial content. EC and NP microbiota were consistent with previous reports. In contrast, bacterial levels in ME samples were not significantly above those in unused control swabs. Commonly detected taxa were among recognized sequencing contaminants (Methylobacterium, Pseudomonas, and Acinetobacter). Linear regression of dominant ME taxa confirmed a negative relationship between relative abundance and bacterial load, consistent with contamination. No bacteria were detected by microscopy or diagnostic culture in any middle ear sample. Our findings cast substantial doubt on previous reports identifying a healthy middle ear microbiome using 16S amplicon sequencing. IMPORTANCE Recent molecular-based studies have suggested that a diverse middle ear microbiome in adults and children can exist in the absence of disease. These studies have been largely unsupported by culture and feature species that commonly contaminate low-biomass sequencing data. While 16S rRNA gene amplicon sequencing has proven to be a highly informative technique in many clinical contexts, it is susceptible to spurious signal arising from sequencing reagent contaminants where sample biomass is low. Combining culture-based and molecular techniques, we undertook a detailed investigation of the evidence for bacterial colonization of the healthy middle ear. In finding no evidence of viable bacterial cells in middle ear samples, our study further underlines the importance of careful consideration of amplicon sequence data derived from very-low-biomass contexts and the value of analytical approaches that combine culture and molecular techniques.

Recent demonstrations that long-term macrolide therapy can prevent exacerbations in chronic airways diseases have led to a dramatic increase in their use. However, little is known about the wider, potentially adverse impacts of these treatments. Substantial disruption of the upper airway commensal microbiota might reduce its contribution to host defense and local immune regulation, while increases in macrolide resistance carriage would represent a serious public health concern. Using samples from a randomized controlled trial, we show that low-dose erythromycin given over 48 weeks influences the composition of the oropharyngeal commensal microbiota. We report that macrolide therapy is associated with significant changes in the relative abundances of members of the Actinomyces genus and with significant increases in the carriage of transmissible macrolide resistance. Determining the clinical significance of these changes, relative to treatment benefit, now represents a research priority., Long-term macrolide therapy reduces rates of pulmonary exacerbation in bronchiectasis. However, little is known about the potential for macrolide therapy to alter the composition and function of the oropharyngeal commensal microbiota or to increase the carriage of transmissible antimicrobial resistance. We assessed the effect of long-term erythromycin on oropharyngeal microbiota composition and the carriage of transmissible macrolide resistance genes in 84 adults with bronchiectasis, enrolled in the Bronchiectasis and Low-dose Erythromycin Study (BLESS) 48-week placebo-controlled trial of twice-daily erythromycin ethylsuccinate (400 mg). Oropharyngeal microbiota composition and macrolide resistance gene carriage were determined by 16S rRNA gene amplicon sequencing and quantitative PCR, respectively. Long-term erythromycin treatment was associated with a significant increase in the relative abundance of oropharyngeal Haemophilus parainfluenzae (P = 0.041) and with significant decreases in the relative abundances of Streptococcus pseudopneumoniae (P = 0.024) and Actinomyces odontolyticus (P = 0.027). Validation of the sequencing results by quantitative PCR confirmed a significant decrease in the abundance of Actinomyces spp. (P = 0.046). Erythromycin treatment did not result in a significant increase in the number of subjects who carried erm(A), erm(B), erm(C), erm(F), mef(A/E), and msrA macrolide resistance genes. However, the abundance of erm(B) and mef(A/E) gene copies within carriers who had received erythromycin increased significantly (P < 0.05). Our findings indicate that changes in oropharyngeal microbiota composition resulting from long-term erythromycin treatment are modest and are limited to a discrete group of taxa. Associated increases in levels of transmissible antibiotic resistance genes within the oropharyngeal microbiota highlight the potential for this microbial system to act as a reservoir for resistance. IMPORTANCE Recent demonstrations that long-term macrolide therapy can prevent exacerbations in chronic airways diseases have led to a dramatic increase in their use. However, little is known about the wider, potentially adverse impacts of these treatments. Substantial disruption of the upper airway commensal microbiota might reduce its contribution to host defense and local immune regulation, while increases in macrolide resistance carriage would represent a serious public health concern. Using samples from a randomized controlled trial, we show that low-dose erythromycin given over 48 weeks influences the composition of the oropharyngeal commensal microbiota. We report that macrolide therapy is associated with significant changes in the relative abundances of members of the Actinomyces genus and with significant increases in the carriage of transmissible macrolide resistance. Determining the clinical significance of these changes, relative to treatment benefit, now represents a research priority.

Antibody-mediated responses play a critical role in vaccine-mediated immunity. However, for reasons that are poorly understood, these responses are highly variable between individuals. Using a mouse model, we report that antibiotic-driven intestinal dysbiosis, specifically in early life, leads to significantly impaired antibody responses to five different adjuvanted and live vaccines. Restoration of the commensal microbiota following antibiotic exposure rescues these impaired responses. In contrast, antibiotic-treated adult mice do not exhibit impaired antibody responses to vaccination. Interestingly, in contrast to impaired antibody responses, immunized mice exposed to early-life antibiotics display significantly enhanced T cell cytokine recall responses upon ex vivo restimulation with the vaccine antigen. Our results demonstrate that, in mice, antibioticdriven dysregulation of the gut microbiota in early life can modulate immune responses to vaccines that are routinely administered to infants worldwide. Refereed/Peer-reviewed

Enterobacter cloacae is a common member of the gut microbiota in healthy individuals. However, it is also an opportunistic pathogen, capable of causing bacteremia. We report the draft genomes of two Enterobacter cloacae subspecies cloacae strains isolated from hematology patients with bacteremia. Both isolates carry genes encoding extended-spectrum β-lactamases.

Inbred mice are used to investigate many aspects of human physiology, including susceptibility to disease and response to therapies. Despite increasing evidence that the composition and function of the murine intestinal microbiota can substantially influence a broad range of experimental outcomes, relatively little is known about microbiome dynamics within experimental mouse populations. We investigated changes in the intestinal microbiome between C57BL/6J mice spanning six generations (assessed at generations 1, 2, 3, and 6), following their introduction to a stringently controlled facility. Fecal microbiota composition and function were assessed by 16S rRNA gene amplicon sequencing and liquid chromatography mass spectrometry, respectively. Significant divergence of the intestinal microbiota between founder and second generation mice, as well as continuing inter-generational variance, was observed. Bacterial taxa whose relative abundance changed significantly through time included Akkermansia, Turicibacter, and Bifidobacterium (p < 0.05), all of which are recognized as having the potential to substantially influence host physiology. Shifts in microbiota composition were mirrored by corresponding differences in the fecal metabolome (r = 0.57, p = 0.0001), with notable differences in levels of tryptophan pathway metabolites and amino acids, including glutamine, glutamate and aspartate. We related the magnitude of changes in the intestinal microbiota and metabolome characteristics during acclimation to those observed between populations housed in separate facilities, which differed in regards to husbandry, barrier conditions and dietary intake. The microbiome variance reported here has implications for experimental reproducibility, and as a consequence, experimental design and the interpretation of research outcomes across wide range of contexts.

Culture-based detection of nontuberculous Mycobacteria (NTM) in respiratory samples is time consuming and can be subject to overgrowth by nonmycobacterial bacteria. We describe a single-reaction TaqMan quantitative PCR assay for the direct detection of NTM species in clinical samples that is specific, sensitive, and robust.

Sequencing-based studies of the human faecal microbiota are increasingly common. Appropriate storage of sample material is essential to avoid the introduction of post-collection bias in microbial community composition. Rapid freezing to −80 °C is commonly considered to be best-practice. However, this is not feasible in many studies, particularly those involving sample collection in participants’ homes. We determined the extent to which a range of stabilisation and storage strategies maintained the composition of faecal microbial community structure relative to freezing to −80 °C. Refrigeration at 4 °C, storage at ambient temperature and the use of several common preservative buffers (RNAlater, OMNIgene.GUT, Tris-EDTA) were assessed relative to freezing. Following 72 hours of storage, faecal microbial composition was assessed by 16 S rRNA amplicon sequencing. Refrigeration was associated with no significant alteration in faecal microbiota diversity or composition. However, samples stored using other conditions showed substantial divergence compared to −80 °C control samples. Aside from refrigeration, the use of OMNIgene.GUT resulted in the least alteration, while the greatest change was seen in samples stored in Tris-EDTA buffer. The commercially available OMNIgene.GUT kit may provide an important alternative where refrigeration and cold chain transportation is not available.

Background The rapid expansion of 16S rRNA gene sequencing in challenging clinical contexts has resulted in a growing body of literature of variable quality. To a large extent, this is due to a failure to address spurious signal that is characteristic of samples with low levels of bacteria and high levels of non-bacterial DNA. We have developed a workflow based on the paired-end read Illumina MiSeq-based approach, which enables significant improvement in data quality, post-sequencing. We demonstrate the efficacy of this methodology through its application to paediatric upper-respiratory samples from several anatomical sites. Results A workflow for processing sequence data was developed based on commonly available tools. Data generated from different sample types showed a marked variation in levels of non-bacterial signal and ‘contaminant’ bacterial reads. Significant differences in the ability of reference databases to accurately assign identity to operational taxonomic units (OTU) were observed. Three OTU-picking strategies were trialled as follows: de novo, open-reference and closed-reference, with open-reference performing substantially better. Relative abundance of OTUs identified as potential reagent contamination showed a strong inverse correlation with amplicon concentration allowing their objective removal. The removal of the spurious signal showed the greatest improvement in sample types typically containing low levels of bacteria and high levels of human DNA. A substantial impact of pre-filtering data and spurious signal removal was demonstrated by principal coordinate and co-occurrence analysis. For example, analysis of taxon co-occurrence in adenoid swab and middle ear fluid samples indicated that failure to remove the spurious signal resulted in the inclusion of six out of eleven bacterial genera that accounted for 80% of similarity between the sample types. Conclusions The application of the presented workflow to a set of challenging clinical samples demonstrates its utility in removing the spurious signal from the dataset, allowing clinical insight to be derived from what would otherwise be highly misleading output. While other approaches could potentially achieve similar improvements, the methodology employed here represents an accessible means to exclude the signal from contamination and other artefacts. Electronic supplementary material The online version of this article (doi:10.1186/s40168-015-0083-8) contains supplementary material, which is available to authorized users.