Method for culturing Candidatus Ornithobacterium hominis.

Candidatus Ornithobacterium hominis has been detected in nasopharyngeal microbiota sequence data from around the world. This report provides the first description of culture conditions for isolating this bacterium. The availability of an easily reproducible culture method is expected to facilitate deeper understanding of the clinical significance of this species

Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.

BACKGROUND: The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. RESULTS: In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. CONCLUSIONS: These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised

'Candidatus Ornithobacterium hominis': insights gained from draft genomes obtained from nasopharyngeal swabs.

'Candidatus Ornithobacterium hominis' represents a new member of the Flavobacteriaceae detected in 16S rRNA gene surveys of people from South-East Asia, Africa and Australia. It frequently colonizes the infant nasopharynx at high proportional abundance, and we demonstrate its presence in 42 % of nasopharyngeal swabs from 12-month-old children in the Maela refugee camp in Thailand. The species, a Gram-negative bacillus, has not yet been cultured, but the cells can be identified in mixed samples by fluorescent hybridization. Here, we report seven genomes assembled from metagenomic data, two to improved draft standard. The genomes are approximately 1.9 Mb, sharing 62 % average amino acid identity with the only other member of the genus, the bird pathogen Ornithobacterium rhinotracheale. The draft genomes encode multiple antibiotic-resistance genes, competition factors, Flavobacterium johnsoniae-like gliding motility genes and a homologue of the Pasteurella multocida mitogenic toxin. Intra- and inter-host genome comparison suggests that colonization with this bacterium is both persistent and strain exclusive

Freshwater monitoring by nanopore sequencing.

While traditional microbiological freshwater tests focus on the detection of specific bacterial indicator species, including pathogens, direct tracing of all aquatic DNA through metagenomics poses a profound alternative. Yet, in situ metagenomic water surveys face substantial challenges in cost and logistics. Here, we present a simple, fast, cost-effective and remotely accessible freshwater diagnostics workflow centred around the portable nanopore sequencing technology. Using defined compositions and spatiotemporal microbiota from surface water of an example river in Cambridge (UK), we provide optimised experimental and bioinformatics guidelines, including a benchmark with twelve taxonomic classification tools for nanopore sequences. We find that nanopore metagenomics can depict the hydrological core microbiome and fine temporal gradients in line with complementary physicochemical measurements. In a public health context, these data feature relevant sewage signals and pathogen maps at species level resolution. We anticipate that this framework will gather momentum for new environmental monitoring initiatives using portable devices

Freshwater monitoring by nanopore sequencing.

While traditional microbiological freshwater tests focus on the detection of specific bacterial indicator species, including pathogens, direct tracing of all aquatic DNA through metagenomics poses a profound alternative. Yet, in situ metagenomic water surveys face substantial challenges in cost and logistics. Here, we present a simple, fast, cost-effective and remotely accessible freshwater diagnostics workflow centred around the portable nanopore sequencing technology. Using defined compositions and spatiotemporal microbiota from surface water of an example river in Cambridge (UK), we provide optimised experimental and bioinformatics guidelines, including a benchmark with twelve taxonomic classification tools for nanopore sequences. We find that nanopore metagenomics can depict the hydrological core microbiome and fine temporal gradients in line with complementary physicochemical measurements. In a public health context, these data feature relevant sewage signals and pathogen maps at species level resolution. We anticipate that this framework will gather momentum for new environmental monitoring initiatives using portable devices

Pneumococcal within-host diversity during colonization, transmission and treatment

Characterizing the genetic diversity of pathogens within the host promises to greatly improve surveillance and reconstruction of transmission chains. For bacteria, it also informs our understanding of inter-strain competition and how this shapes the distribution of resistant and sensitive bacteria. Here we study the genetic diversity of Streptococcus pneumoniae within 468 infants and 145 of their mothers by deep sequencing whole pneumococcal populations from 3,761 longitudinal nasopharyngeal samples. We demonstrate that deep sequencing has unsurpassed sensitivity for detecting multiple colonization, doubling the rate at which highly invasive serotype 1 bacteria were detected in carriage compared with gold-standard methods. The greater resolution identified an elevated rate of transmission from mothers to their children in the first year of the child's life. Comprehensive treatment data demonstrated that infants were at an elevated risk of both the acquisition and persistent colonization of a multidrug-resistant bacterium following antimicrobial treatment. Some alleles were enriched after antimicrobial treatment, suggesting that they aided persistence, but generally purifying selection dominated within-host evolution. Rates of co-colonization imply that in the absence of treatment, susceptible lineages outcompeted resistant lineages within the host. These results demonstrate the many benefits of deep sequencing for the genomic surveillance of bacterial pathogens. Longitudinal population deep sequencing of Streptococcus pneumoniae sampled from infants and their mothers improves our understanding of the dynamics of colonization, transmission, inter-strain competition and the impact of antibiotic treatment.Peer reviewe

A longitudinal study of the infant nasopharyngeal microbiota: The effects of age, illness and antibiotic use in a cohort of South East Asian children.

A longitudinal study was undertaken in infants living in the Maela refugee camp on the Thailand-Myanmar border between 2007 and 2010. Nasopharyngeal swabs were collected monthly, from birth to 24 months of age, with additional swabs taken if the infant was diagnosed with pneumonia according to WHO clinical criteria. At the time of collection, swabs were cultured for Streptococcus pneumoniae and multiple serotype carriage was assessed. The bacterial 16S rRNA gene profiles of 544 swabs from 21 infants were analysed to see how the microbiota changes with age, respiratory infection, antibiotic consumption and pneumococcal acquisition. The nasopharyngeal microbiota is a somewhat homogenous community compared to that of other body sites. In this cohort it is dominated by five taxa: Moraxella, Streptococcus, Haemophilus, Corynebacterium and an uncharacterized Flavobacteriaceae taxon of 93% nucleotide similarity to Ornithobacterium. Infant age correlates with certain changes in the microbiota across the cohort: Staphylococcus and Corynebacterium are associated with the first few months of life while Moraxella and the uncharacterised Flavobacteriaceae increase in proportional abundance with age. Respiratory illness and antibiotic use often coincide with an unpredictable perturbation of the microbiota that differs from infant to infant and in different illness episodes. The previously described interaction between Dolosigranulum and Streptococcus was observed in these data. Monthly sampling demonstrates that the nasopharyngeal microbiota is in flux throughout the first two years of life, and that in this refugee camp population the pool of potential bacterial colonisers may be limited

Patent Human Infections with the Whipworm, Trichuris trichiura, Are Not Associated with Alterations in the Faecal Microbiota

Background: The soil-transmitted helminth (STH), Trichuris trichiura colonises the human large intestine where it may modify inflammatory responses, an effect possibly mediated through alterations in the intestinal microbiota. We hypothesised that patent T. trichiura infections would be associated with altered faecal microbiota and that anthelmintic treatment would induce a microbiota resembling more closely that observed in uninfected individuals. Materials and Methods: School children in Ecuador were screened for STH infections and allocated to 3 groups: uninfected, T. trichiura only, and mixed infections with T. trichiura and Ascaris lumbricoides. A sample of uninfected children and those with T. trichiura infections only were given anthelmintic treatment. Bacterial community profiles in faecal samples were studied by 454 pyrosequencing of 16 S rRNA genes. Results: Microbiota analyses of faeces were done for 97 children: 30 were uninfected, 17 were infected with T. trichiura, and 50 with T. trichiura and A. lumbricoides. Post-treatment samples were analyzed for 14 children initially infected with T. trichiura alone and for 21 uninfected children. Treatment resulted in 100% cure of STH infections. Comparisons of the microbiota at different taxonomic levels showed no statistically significant differences in composition between uninfected children and those with T. trichiura infections. We observed a decreased proportional abundance of a few bacterial genera from the Clostridia class of Firmicutes and a reduced bacterial diversity among children with mixed infections compared to the other two groups, indicating a possible specific effect of A. lumbricoides infection. Anthelmintic treatment of children with T. trichiura did not alter faecal microbiota composition. Discussion: Our data indicate that patent human infections with T. trichiura may have no effect on faecal microbiota but that A. lumbricoides colonisation might be associated with a disturbed microbiota. Our results also catalogue the microbiota of rural Ecuadorians and indicate differences with individuals from more urban industrialised societies

Comprehensive identification of single nucleotide polymorphisms associated with beta-lactam resistance within pneumococcal mosaic genes.

Traditional genetic association studies are very difficult in bacteria, as the generally limited recombination leads to large linked haplotype blocks, confounding the identification of causative variants. Beta-lactam antibiotic resistance in Streptococcus pneumoniae arises readily as the bacteria can quickly incorporate DNA fragments encompassing variants that make the transformed strains resistant. However, the causative mutations themselves are embedded within larger recombined blocks, and previous studies have only analysed a limited number of isolates, leading to the description of "mosaic genes" as being responsible for resistance. By comparing a large number of genomes of beta-lactam susceptible and non-susceptible strains, the high frequency of recombination should break up these haplotype blocks and allow the use of genetic association approaches to identify individual causative variants. Here, we performed a genome-wide association study to identify single nucleotide polymorphisms (SNPs) and indels that could confer beta-lactam non-susceptibility using 3,085 Thai and 616 USA pneumococcal isolates as independent datasets for the variant discovery. The large sample sizes allowed us to narrow the source of beta-lactam non-susceptibility from long recombinant fragments down to much smaller loci comprised of discrete or linked SNPs. While some loci appear to be universal resistance determinants, contributing equally to non-susceptibility for at least two classes of beta-lactam antibiotics, some play a larger role in resistance to particular antibiotics. All of the identified loci have a highly non-uniform distribution in the populations. They are enriched not only in vaccine-targeted, but also non-vaccine-targeted lineages, which may raise clinical concerns. Identification of single nucleotide polymorphisms underlying resistance will be essential for future use of genome sequencing to predict antibiotic sensitivity in clinical microbiology

Freshwater monitoring by nanopore sequencing

Many water-dwelling bacteria can cause severe diseases such as cholera, typhoid or leptospirosis. One way to prevent outbreaks is to test water sources to find out which species of microbes they contain, and at which levels. Traditionally, this involves taking a water sample, followed by growing a few species of ‘indicator bacteria’ that help to estimate whether the water is safe. An alternative technique, called metagenomics, has been available since the mid-2000s. It consists in reviewing (or ‘sequencing’) the genetic information of most of the bacteria present in the water, which allows scientists to spot harmful species. Both methods, however, require well-equipped laboratories with highly trained staff, making them challenging to use in remote areas. The MinION is a pocket-sized device that – when paired with a laptop or mobile phone – can sequence genetic information ‘on the go’. It has already been harnessed during Ebola, Zika or SARS-CoV-2 epidemics to track the genetic information of viruses in patients and environmental samples. However, it is still difficult to use the MinION and other sequencers to monitor bacteria in water sources, partly because the genetic information of the microbes is highly fragmented during DNA extraction. To address this challenge, Urban, Holzer et al. set out to optimise hardware and software protocols so the MinION could be used to detect bacterial species present in rivers. The tests focussed on the River Cam in Cambridge, UK, a waterway which faces regular public health problems: local rowers and swimmers often contract waterborne infections, sometimes leading to river closures. For six months, Urban, Holzer et al. used the MinION to map out the bacteria present across nine river sites, assessing the diversity of species and the presence of disease-causing microbes in the water. In particular, the results showed that optimising the protocols made it possible to tell the difference between closely related species – an important feature since harmful and inoffensive bacteria can sometimes be genetically close. The data also revealed that the levels of harmful bacteria were highest downstream of urban river sections, near a water treatment plant and river barge moorings. Together, these findings demonstrate that optimising MinION protocols can turn this device into a useful tool to easily monitor water quality. Around the world, climate change, rising urbanisation and the intensification of agriculture all threaten water quality. In fact, access to clean water is one of the United Nations sustainable development goals for 2030. Using the guidelines developed by Urban, Holzer et al., communities could harness the MinION to monitor water quality in remote areas, offering a cost-effective, portable DNA analysis tool to protect populations against deadly diseases