Identifying large-scale recombination and capsular switching events in Streptococcus agalactiae strains causing disease in adults in the UK between 2014 and 2015

Cases of invasive group B streptococcal infection in the adult UK population have steadily increased over recent years, with the most common serotypes being V, III and Ia, but less is known of the genetic background of these strains. We have carried out in-depth analysis of the whole-genome sequences of 193 clinically important group B Streptococcus (GBS) isolates (184 were from invasive infection, 8 were from non-invasive infection and 1 had no information on isolation site) isolated from adults and submitted to the National Reference Laboratory at the UK Health Security Agency between January 2014 and December 2015. We have determined that capsular serotypes III (26.9%), Ia (26.4%) and V (15.0%) were most commonly identified, with slight differences in gender and age distribution. Most isolates (n=182) grouped to five clonal complexes (CCs), CC1, CC8/CC10, CC17, CC19 and CC23, with common associations between specific serotypes and virulence genes. Additionally, we have identified large recombination events mediating potential capsular switching events between sequence type (ST)1 serotype V and serotypes Ib (n=2 isolates), II (n=2 isolates) and VI (n=2 isolates); between ST19 serotype III and serotype V (n=5 isolates); and between CC17 serotype III and serotype IV (n=1 isolate). The high genetic diversity of disease-causing isolates and multiple recombination events reported in this study highlight the need for routine surveillance of the circulating disease-causing GBS strains. This information is crucial to better understand the global spread of GBS serotypes and genotypes, and will form the baseline information for any future GBS vaccine research in the UK and worldwide

Ten simple rules for the sharing of bacterial genotype—Phenotype data on antimicrobial resistance

The increasing availability of high-throughput sequencing (frequently termed next-generation sequencing (NGS)) data has created opportunities to gain deeper insights into the mechanisms of a number of diseases and is already impacting many areas of medicine and public health. The area of infectious diseases stands somewhat apart from other human diseases insofar as the relevant genomic data comes from the microbes rather than their human hosts. A particular concern about the threat of antimicrobial resistance (AMR) has driven the collection and reporting of large-scale datasets containing information from microbial genomes together with antimicrobial susceptibility test (AST) results. Unfortunately, the lack of clear standards or guiding principles for the reporting of such data is hampering the field's advancement. We therefore present our recommendations for the publication and sharing of genotype and phenotype data on AMR, in the form of 10 simple rules. The adoption of these recommendations will enhance AMR data interoperability and help enable its large-scale analyses using computational biology tools, including mathematical modelling and machine learning. We hope that these rules can shed light on often overlooked but nonetheless very necessary aspects of AMR data sharing and enhance the field's ability to address the problems of understanding AMR mechanisms, tracking their emergence and spread in populations, and predicting microbial susceptibility to antimicrobials for diagnostic purposes

Genetic diversity of Streptococcus pneumoniae causing meningitis and sepsis in Singapore during the first year of PCV7 implementation.

Streptococcus pneumoniae is a major cause of sepsis, meningitis and respiratory disease worldwide. Pneumococcal conjugate vaccines (PCVs) have now been implemented in many countries worldwide, including Singapore. To evaluate the effectiveness of these vaccines, pneumococcal surveillance studies are required. Detailed and unified pneumococcal epidemiology data are currently scarce in South East Asia. Thus, we present data on invasive pneumococcal (IPD) isolates from Singapore that could assist in evaluating the effectiveness of pneumococcal vaccine in Singapore. One hundred and fifty-nine invasive pneumococcal disease isolates were received by the National Public Health Laboratory in Singapore between June 2009 and August 2010. Isolates were characterized using serotyping and multilocus sequence typing. Twenty-four different serotypes were found, the most common of which were 19A, 3, 7F, 23F, 6B, 14, 8 and 19F (in rank order). One hundred and two sequence types were observed, of which 38 were novel due to new alleles or new combinations of already existing alleles. Based on the Simpson's Index of Diversity, serotypes 3, 6B and 19A were the most genetically diverse. Novel sequence types were more prevalent among conjugate vaccine serotypes 3, 19F and 23F and non-conjugate vaccine serotype 8, serogroup 15 and in non-typable isolates. We have demonstrated considerable genetic diversity among invasive pneumococci before and during the widespread use of conjugate vaccines in Singapore. Approximately half of all novel IPD clones identified in this study were non-conjugate vaccine serotypes. Although PCVs would target the most common serotypes, the high genetic diversity in non-vaccine serotypes would require further surveillance studies

Rapid detection of mobilized colistin resistance using a nucleic acid based lab-on-a-chip diagnostic system

The increasing prevalence of antimicrobial resistance is a serious threat to global public health. One of the most concerning trends is the rapid spread of Carbapenemase-Producing Organisms (CPO), where colistin has become the last-resort antibiotic treatment. The emergence of colistin resistance, including the spread of mobilized colistin resistance (mcr) genes, raises the possibility of untreatable bacterial infections and motivates the development of improved diagnostics for the detection of colistin-resistant organisms. This work demonstrates a rapid response for detecting the most recently reported mcr gene, mcr−9, using a portable and affordable lab-on-a-chip (LoC) platform, offering a promising alternative to conventional laboratory-based instruments such as real-time PCR (qPCR). The platform combines semiconductor technology, for non-optical real-time DNA sensing, with a smartphone application for data acquisition, visualization and cloud connectivity. This technology is enabled by using loop-mediated isothermal amplification (LAMP) as the chemistry for targeted DNA detection, by virtue of its high sensitivity, specificity, yield, and manageable temperature requirements. Here, we have developed the first LAMP assay for mcr−9 - showing high sensitivity (down to 100 genomic copies/reaction) and high specificity (no cross-reactivity with other mcr variants). This assay is demonstrated through supporting a hospital investigation where we analyzed nucleic acids extracted from 128 carbapenemase-producing bacteria isolated from clinical and screening samples and found that 41 carried mcr−9 (validated using whole genome sequencing). Average positive detection times were 6.58 ± 0.42 min when performing the experiments on a conventional qPCR instrument (n = 41). For validating the translation of the LAMP assay onto a LoC platform, a subset of the samples were tested (n = 20), showing average detection times of 6.83 ± 0.92 min for positive isolates (n = 14). All experiments detected mcr−9 in under 10 min, and both platforms showed no statistically significant difference (p-value > 0.05). When sample preparation and throughput capabilities are integrated within this LoC platform, the adoption of this technology for the rapid detection and surveillance of antimicrobial resistance genes will decrease the turnaround time for DNA detection and resistotyping, improving diagnostic capabilities, patient outcomes, and the management of infectious diseases

Frequency of transmission, asymptomatic shedding, and airborne spread of Streptococcus pyogenes in schoolchildren exposed to scarlet fever: a prospective, longitudinal, multicohort, molecular epidemiological, contact-tracing study in England, UK

Background: Despite recommendations regarding prompt treatment of cases and enhanced hygiene measures, scarlet fever outbreaks increased in England between 2014 and 2018. We aimed to assess the effects of standard interventions on transmission of Streptococcus pyogenes to classroom contacts, households, and classroom environments to inform future guidance. Methods: We did a prospective, longitudinal, multicohort, molecular epidemiological, contact-tracing study in six settings across five schools in Greater London, UK. Schools and nurseries were eligible to participate if they had reported two cases of scarlet fever within 10 days of each other among children aged 2–8 years from the same class, with the most recent case arising in the preceding 48 h. We cultured throat swabs from children with scarlet fever, classroom contacts, and household contacts at four timepoints. We also cultured hand swabs and cough plates from all cases in years 1 and 2 of the study, and from classroom contacts in year 2. Surface swabs from toys and other fomites in classrooms were cultured in year 1, and settle plates from classrooms were collected in year 2. Any sample with S pyogenes detected was recorded as positive and underwent emm genotyping and genome sequencing to compare with the outbreak strain. Findings: Six classes, comprising 12 cases of scarlet fever, 17 household contacts, and 278 classroom contacts were recruited between March 1 and May 31, 2018 (year 1), and between March 1 and May 31, 2019 (year 2). Asymptomatic throat carriage of the outbreak strains increased from 11 (10%) of 115 swabbed children in week 1, to 34 (27%) of 126 in week 2, to 26 (24%) of 108 in week 3, and then five (14%) of 35 in week 4. Compared with carriage of outbreak S pyogenes strains, colonisation with non-outbreak and non-genotyped S pyogenes strains occurred in two (2%) of 115 swabbed children in week 1, five (4%) of 126 in week 2, six (6%) of 108 in week 3, and in none of the 35 children in week 4 (median carriage for entire study 2·8% [IQR 0·0–6·6]). Genome sequencing showed clonality of outbreak isolates within each of six classes, confirming that recent transmission accounted for high carriage. When transmissibility was tested, one (9%) of 11 asymptomatic carriers of emm4 and five (36%) of 14 asymptomatic carriers of emm3.93 had a positive cough plate. The outbreak strain was identified in only one (2%) of 60 surface swabs taken from three classrooms; however, in the two classrooms with settle plates placed in elevated locations, two (17%) of 12 and six (50%) of 12 settle plates yielded the outbreak strain. Interpretation: Transmission of S pyogenes in schools is intense and might occur before or despite reported treatment of cases, underlining a need for rapid case management. Despite guideline adherence, heavy shedding of S pyogenes by few classroom contacts might perpetuate outbreaks, and airborne transmission has a plausible role in its spread. These findings highlight the need for research to improve understanding and to assess effectiveness of interventions to reduce airborne transmission of S pyogenes. Funding: Action Medical Research, UK Research Innovation, and National Institute for Health Research

Coupling Machine Learning and High Throughput Multiplex Digital PCR Enables Accurate Detection of Carbapenem-Resistant Genes in Clinical Isolates

Rapid and accurate identification of patients colonised with carbapenemase-producing organisms (CPOs) is essential to adopt prompt prevention measures to reduce the risk of transmission. Recent studies have demonstrated the ability to combine machine learning (ML) algorithms with real-time digital PCR (dPCR) instruments to increase classification accuracy of multiplex PCR assays when using synthetic DNA templates. We sought to determine if this novel methodology could be applied to improve identification of the five major carbapenem-resistant genes in clinical CPO-isolates, which would represent a leap forward in the use of PCR-based data-driven diagnostics for clinical applications. We collected 253 clinical isolates (including 221 CPO-positive samples) and developed a novel 5-plex PCR assay for detection of blaIMP, blaKPC, blaNDM, blaOXA-48, and blaVIM. Combining the recently reported ML method "Amplification and Melting Curve Analysis" (AMCA) with the abovementioned multiplex assay, we assessed the performance of the AMCA methodology in detecting these genes. The improved classification accuracy of AMCA relies on the usage of real-time data from a single-fluorescent channel and benefits from the kinetic/thermodynamic information encoded in the thousands of amplification events produced by high throughput real-time dPCR. The 5-plex showed a lower limit of detection of 10 DNA copies per reaction for each primer set and no cross-reactivity with other carbapenemase genes. The AMCA classifier demonstrated excellent predictive performance with 99.6% (CI 97.8-99.9%) accuracy (only one misclassified sample out of the 253, with a total of 160,041 positive amplification events), which represents a 7.9% increase (p-value <0.05) compared to conventional melting curve analysis. This work demonstrates the use of the AMCA method to increase the throughput and performance of state-of-the-art molecular diagnostic platforms, without hardware modifications and additional costs, thus potentially providing substantial clinical utility on screening patients for CPO carriage

Hospital outbreak of carbapenem-resistant Enterobacterales associated with a bla OXA-48 plasmid carried mostly by Escherichia coli ST399

A hospital outbreak of carbapenem-resistant Enterobacterales was detected by routine surveillance. Whole genome sequencing and subsequent analysis revealed a conserved promiscuous bla OXA-48 carrying plasmid as the defining factor within this outbreak. Four different species of Enterobacterales were involved in the outbreak. Escherichia coli ST399 accounted for 35 of all the 55 isolates. Comparative genomics analysis using publicly available E. coli ST399 genomes showed that the outbreak E. coli ST399 isolates formed a unique clade. We developed a mathematical model of pOXA-48-like plasmid transmission between host lineages and used it to estimate its conjugation rate, giving a lower bound of 0.23 conjugation events per lineage per year. Our analysis suggests that co-evolution between the pOXA-48-like plasmid and E. coli ST399 could have played a role in the outbreak. This is the first study to report carbapenem-resistant E. coli ST399 carrying blaOXA-48 as the main cause of a plasmid-borne outbreak within a hospital setting. Our findings suggest complementary roles for both plasmid conjugation and clonal expansion in the emergence of this outbreak

Emergence of dominant toxigenic M1T1 Streptococcus pyogenes clone during increased scarlet fever activity in England: a population-based molecular epidemiological study.

BACKGROUND: Since 2014, England has seen increased scarlet fever activity unprecedented in modern times. In 2016, England's scarlet fever seasonal rise coincided with an unexpected elevation in invasive Streptococcus pyogenes infections. We describe the molecular epidemiological investigation of these events. METHODS: We analysed changes in S pyogenes emm genotypes, and notifications of scarlet fever and invasive disease in 2014-16 using regional (northwest London) and national (England and Wales) data. Genomes of 135 non-invasive and 552 invasive emm1 isolates from 2009-16 were analysed and compared with 2800 global emm1 sequences. Transcript and protein expression of streptococcal pyrogenic exotoxin A (SpeA; also known as scarlet fever or erythrogenic toxin A) in sequenced, non-invasive emm1 isolates was quantified by real-time PCR and western blot analyses. FINDINGS: Coincident with national increases in scarlet fever and invasive disease notifications, emm1 S pyogenes upper respiratory tract isolates increased significantly in northwest London in the March to May period, from five (5%) of 96 isolates in 2014, to 28 (19%) of 147 isolates in 2015 (p=0·0021 vs 2014 values), to 47 (33%) of 144 in 2016 (p=0·0080 vs 2015 values). Similarly, invasive emm1 isolates collected nationally in the same period increased from 183 (31%) of 587 in 2015 to 267 (42%) of 637 in 2016 (p<0·0001). Sequences of emm1 isolates from 2009-16 showed emergence of a new emm1 lineage (designated M1UK)-with overlap of pharyngitis, scarlet fever, and invasive M1UK strains-which could be genotypically distinguished from pandemic emm1 isolates (M1global) by 27 single-nucleotide polymorphisms. Median SpeA protein concentration in supernatant was nine-times higher among M1UK isolates (190·2 ng/mL [IQR 168·9-200·4]; n=10) than M1global isolates (20·9 ng/mL [0·0-27·3]; n=10; p<0·0001). M1UK expanded nationally to represent 252 (84%) of all 299 emm1 genomes in 2016. Phylogenetic analysis of published datasets identified single M1UK isolates in Denmark and the USA. INTERPRETATION: A dominant new emm1 S pyogenes lineage characterised by increased SpeA production has emerged during increased S pyogenes activity in England. The expanded reservoir of M1UK and recognised invasive potential of emm1 S pyogenes provide plausible explanation for the increased incidence of invasive disease, and rationale for global surveillance. FUNDING: UK Medical Research Council, UK National Institute for Health Research, Wellcome Trust, Rosetrees Trust, Stoneygate Trust

Exploiting genomics for antimicrobial resistance surveillance at One Health interfaces.

The intersection of human, animal, and ecosystem health at One Health interfaces is recognised as being of key importance in the evolution and spread of antimicrobial resistance (AMR) and represents an important, and yet rarely realised opportunity to undertake vital AMR surveillance. A working group of international experts in pathogen genomics, AMR, and One Health convened to take part in a workshop series and online consultation focused on the opportunities and challenges facing genomic AMR surveillance in a range of settings. Here we outline the working group's discussion of the potential utility, advantages of, and barriers to, the implementation of genomic AMR surveillance at One Health interfaces and propose a series of recommendations for addressing these challenges. Embedding AMR surveillance at One Health interfaces will require the development of clear beneficial use cases, especially in low-income and middle-income countries. Evidence of directionality, risks to human and animal health, and potential trade implications were also identified by the working group as key issues. Addressing these challenges will be vital to enable genomic surveillance technology to reach its full potential for assessing the risk of transmission of AMR between the environment, animals, and humans at One Health interfaces.

Genomics for antimicrobial resistance surveillance to support infection prevention and control in health-care facilities

Integration of genomic technologies into routine antimicrobial resistance (AMR) surveillance in health-care facilities has the potential to generate rapid, actionable information for patient management and inform infection prevention and control measures in near real time. However, substantial challenges limit the implementation of genomics for AMR surveillance in clinical settings. Through a workshop series and online consultation, international experts from across the AMR and pathogen genomics fields convened to review the evidence base underpinning the use of genomics for AMR surveillance in a range of settings. Here, we summarise the identified challenges and potential benefits of genomic AMR surveillance in health-care settings, and outline the recommendations of the working group to realise this potential. These recommendations include the definition of viable and cost-effective use cases for genomic AMR surveillance, strengthening training competencies (particularly in bioinformatics), and building capacity at local, national, and regional levels using hub and spoke models