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. 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. 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.

In 2003 the World Health Organization (WHO) convened a working group and published a set of standard methods for studies measuring nasopharyngeal carriage of Streptococcus pneumoniae (the pneumococcus). The working group recently reconvened under the auspices of the WHO and updated the consensus standard methods. These methods describe the collection, transport and storage of nasopharyngeal samples, as well as provide recommendations for the identification and serotyping of pneumococci using culture and non-culture based approaches. We outline the consensus position of the working group, the evidence supporting this position, areas worthy of future research, and the epidemiological role of carriage studies. Adherence to these methods will reduce variability in the conduct of pneumococcal carriage studies undertaken in the context of pneumococcal vaccine trials, implementation studies, and epidemiology studies more generally so variability in methodology does not confound the interpretation of study findings.

Stephen Bentley, Paul Turner and colleagues report whole-genome sequencing of 3,085 pneumococci collected from a densely sampled pneumococcal carriage cohort in a Thai refugee camp over a 3-year period. They provide a high-resolution analysis of natural pneumococcal evolution and bacterial recombination, identifying lineage-specific variation in recombination. Evasion of clinical interventions by Streptococcus pneumoniae occurs through selection of non-susceptible genomic variants. We report whole-genome sequencing of 3,085 pneumococcal carriage isolates from a 2.4-km2 refugee camp. This sequencing provides unprecedented resolution of the process of recombination and its impact on population evolution. Genomic recombination hotspots show remarkable consistency between lineages, indicating common selective pressures acting at certain loci, particularly those associated with antibiotic resistance. Temporal changes in antibiotic consumption are reflected in changes in recombination trends, demonstrating rapid spread of resistance when selective pressure is high. The highest frequencies of receipt and donation of recombined DNA fragments were observed in non-encapsulated lineages, implying that this largely overlooked pneumococcal group, which is beyond the reach of current vaccines, may have a major role in genetic exchange and the adaptation of the species as a whole. These findings advance understanding of pneumococcal population dynamics and provide information for the design of future intervention strategies.

Escherichia coli sequence type 131 (ST131) has emerged globally as the most predominant extraintestinal pathogenic lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined. This study of the largest worldwide collection ( n = 215) of sequenced ST131 E. coli isolates to date demonstrates that the clonal expansion of two previously recognized antimicrobial-resistant clades, C1/ H 30R and C2/ H 30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/ H 30Rx clade driven by the acquisition of a bla _CTX-M-15 -containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids and chromosomal integration of bla _CTX-M within subclusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed among ST131, the bla _{CTX-M-14/14-like} group. The complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages of E. coli . These data also highlight the global imperative to reduce specific antibiotic selection pressures and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages. IMPORTANCE Escherichia coli , perennially a major bacterial pathogen, is becoming increasingly difficult to manage due to emerging resistance to all preferred antimicrobials. Resistance is concentrated within specific E. coli lineages, such as sequence type 131 (ST131). Clarification of the genetic basis for clonally associated resistance is key to devising intervention strategies. We used high-resolution genomic analysis of a large global collection of ST131 isolates to define the evolutionary history of extended-spectrum beta-lactamase production in ST131. We documented diverse contributory genetic processes, including stable chromosomal integrations of resistance genes, persistence and evolution of mobile resistance elements within sublineages, and sporadic acquisition of different resistance elements. Both global distribution and regional segregation were evident. The diversity of resistance element acquisition and propagation within ST131 indicates a need for control and surveillance strategies that target both bacterial strains and mobile genetic elements.

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. Streptococcus pneumoniae is carried asymptomatically in the nasopharyngeal tract. However, it is capable of causing multiple diseases, including pneumonia, bacteraemia and meningitis, which are common causes of morbidity and mortality in young children. Antibiotic treatment has become more difficult, especially that involving the group of beta-lactam antibiotics where resistance has developed rapidly. The organism is known to be highly recombinogenic, and this allows variants conferring beta-lactam resistance to be readily introduced into the genome. Identification of the specific genetic determinants of beta-lactam resistance is essential to understand both the mechanism of resistance and the spread of resistant variants in the pneumococcal population. Here, we performed a genome-wide association study on 3,701 isolates collected from two different locations and identified candidate variants that may explain beta-lactam resistance as well as discriminating potential genetic hitchhiking variants from potential causative variants. We report 51 loci, containing 301 SNPs, that are associated with beta-lactam non-susceptibility. 71 out of 301 polymorphic changes result in amino acid alterations, 28 of which have been reported previously. Understanding the determinants of resistance at the single nucleotide level will be important for the future use of sequence data to predict resistance in the clinical setting.