Search

Background Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global health challenge. Limitations to AMR surveillance reporting, alongside reduction in culture-based susceptibility testing, has resulted in a need for rapid diagnostics and strain detection. We investigated Nanopore sequencing time, and depth, to accurately identify closely related N. gonorrhoeae isolates, compared to Illumina sequencing. Methods N. gonorrhoeae strains collected from a London Sexual Health clinic were cultured and sequenced with MiSeq and MinION sequencing platforms. Accuracy was determined by comparing variant calls at 68 nucleotide positions (37 resistance-associated markers). Accuracy at varying MinION sequencing depths was determined through retrospective time-stamped read analysis. Results Of 22 MinION-MiSeq pairs reaching sufficient sequencing depth, agreement of variant call positions passing quality control criteria was 185/185 (100%, 95%CI: 98.0-100.0), 502/503 (99.8%, CI:98.9-99.9) and 564/565 (99.8%, CI:99.0-100.0) at 10x, 30x and 40x MinION depth, respectively. Isolates identified as closely related by MiSeq, within one yearly evolutionary distance of ≤5 single nucleotide polymorphisms, were accurately identified via MinION. Conclusion Nanopore sequencing shows utility as a rapid surveillance tool, identifying closely related N. gonorrhoeae strains, with just 10x sequencing depth, taking a median time of 29 minutes. This highlights its potential for tracking local transmission and AMR markers.

Background WGS has significant potential to help tackle the major public health problem of TB. The Republic of Korea has the third highest rates of TB of all Organisation for Economic Cooperation and Development countries but there has been very limited use of WGS in TB to date. Objectives A retrospective comparison of Mycobacterium tuberculosis (MTB) clinical isolates from 2015 to 2017 from two centres in the Republic of Korea using WGS to compare phenotypic drug susceptibility testing (pDST) and WGS drug susceptibility predictions (WGS-DSP). Methods Fifty-seven MTB isolates had DNA extracted and were sequenced using the Illumina HiSeq platform. The WGS analysis was performed using bwa mem, bcftools and IQ-Tree; resistance markers were identified using TB profiler. Phenotypic susceptibilities were carried out at the Supranational TB reference laboratory (Korean Institute of Tuberculosis). Results For first-line antituberculous drugs concordance for rifampicin, isoniazid, pyrazinamide and ethambutol was 98.25%, 92.98%, 87.72% and 85.96%, respectively. The sensitivity of WGS-DSP compared with pDST for rifampicin, isoniazid, pyrazinamide and ethambutol was 97.30%, 92.11%, 78.95% and 95.65%, respectively. The specificity for these first-line antituberculous drugs was 100%, 94.74%, 92.11% and 79.41%, respectively. The sensitivity and specificity for second-line drugs ranged from 66.67% to 100%, and from 82.98% to 100%, respectively. Conclusions This study confirms the potential role for WGS in drug susceptibility prediction, which would reduce turnaround times. However, further larger studies are needed to ensure current databases of drug resistance mutations are reflective of the TB present in the Republic of Korea.

COG-UK is supported by funding from the MRC part of UK Research & Innovation, the National Institute of Health Research (Grant code MC_PC_19027), and Genome Research Limited, operating as the Welcome Sanger Institute. Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine. Publisher PDF

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.

ObjectivesA lactobacilli-dominated vaginal microbiome may protect against pelvic inflammatory disease (PID), but one dominated byGardnerellaspecies might increase susceptibility. Not all lactobacilli are equally protective. Recent research suggests that D(−) isomer lactic acid producing lactobacilli (Lactobacillus crispatus, Lactobacillus jenseniiandLactobacillus gasseri) may protect against infection withChlamydia trachomatis, an important cause of PID.Lactobacillus iners,which produces L(+) isomer lactic acid, may be less protective. We investigated the microbiome in stored vaginal samples from participants who did or did not develop PID during the prevention of pelvic infection (POPI) chlamydia screening trial.MethodsLong-read 16S rRNA gene nanopore sequencing was used on baseline vaginal samples (one per participant) from all 37 women who subsequently developed clinically diagnosed PID during 12-month follow-up, and 111 frequency matched controls who did not, matched on four possible risk factors for PID: age ResultsSamples from 106 women (median age 19 years, 40% black ethnicity, 22% chlamydia positive, 54% reporting multiple partners) were suitable for analysis. Three main taxonomic clusters were identified dominated byL. iners, L. crispatusandGardnerella vaginalis. There was no association between a more diverse,G. vaginalisdominated microbiome and subsequent PID, although increased Shannon diversity was associated with black ethnicity (p=0.002) and bacterial vaginosis (diagnosed by Gram stain pConclusionsIn the first-ever community-based prospective study of PID, there was no clear association between the vaginal microbiome and subsequent development of PID. Future studies using serial samples may identify vaginal microbial communities that may predispose to PID.

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.

Concentration dependency of phenotypic and genotypic isoniazid-rifampicin resistance emergence was investigated to obtain a mechanistic understanding on how anti-mycobacterial drugs facilitate the emergence of bacterial populations that survive throughout treatment. Using static kill curve experiments, observing two evolution cycles, it was demonstrated that rifampicin resistance was the result of non-specific mechanisms and not associated with accumulation of drug resistance encoding SNPs. Whereas, part of isoniazid resistance could be accounted for by accumulation of specific SNPs, which was concentration dependent. Using a Hollow Fibre Infection Model it was demonstrated that emergence of genotypic resistance only occurs when antibiotic levels fall below MIC although MICs are typically maintained following clinical dosing provided that adherence to the regimen is good. This study showed that disentangling and quantifying concentration dependent emergence of resistance provides improved rational for drug and dose selection although further work on understanding underlying mechanisms is needed to improve the drug development pipeline.One Sentence SummaryDisentangling and quantifying concentration dependent emergence of resistance will contribute to better informed drug and dose selection for anti-mycobacterial combination therapy.

Background\ud The bedaquiline–pretomanid–linezolid regimen has been reported to have 90% efficacy against highly drug-resistant tuberculosis, but the incidence of adverse events with 1200 mg of linezolid daily has been high. The appropriate dose of linezolid and duration of treatment with this agent to minimize toxic effects while maintaining efficacy against highly drug-resistant tuberculosis are unclear.\ud \ud Methods\ud We enrolled participants with extensively drug-resistant (XDR) tuberculosis (i.e., resistant to rifampin, a fluoroquinolone, and an aminoglycoside), pre-XDR tuberculosis (i.e., resistant to rifampin and to either a fluoroquinolone or an aminoglycoside), or rifampin-resistant tuberculosis that was not responsive to treatment or for which a second-line regimen had been discontinued because of side effects. We randomly assigned the participants to receive bedaquiline for 26 weeks (200 mg daily for 8 weeks, then 100 mg daily for 18 weeks), pretomanid (200 mg daily for 26 weeks), and daily linezolid at a dose of 1200 mg for 26 weeks or 9 weeks or 600 mg for 26 weeks or 9 weeks. The primary end point in the modified intention-to-treat population was the incidence of an unfavorable outcome, defined as treatment failure or disease relapse (clinical or bacteriologic) at 26 weeks after completion of treatment. Safety was also evaluated.\ud \ud Results\ud A total of 181 participants were enrolled, 88% of whom had XDR or pre-XDR tuberculosis. Among participants who received bedaquiline–pretomanid–linezolid with linezolid at a dose of 1200 mg for 26 weeks or 9 weeks or 600 mg for 26 weeks or 9 weeks, 93%, 89%, 91%, and 84%, respectively, had a favorable outcome; peripheral neuropathy occurred in 38%, 24%, 24%, and 13%, respectively; myelosuppression occurred in 22%, 15%, 2%, and 7%, respectively; and the linezolid dose was modified (i.e., interrupted, reduced, or discontinued) in 51%, 30%, 13%, and 13%, respectively. Optic neuropathy developed in 4 participants (9%) who had received linezolid at a dose of 1200 mg for 26 weeks; all the cases resolved. Six of the seven unfavorable microbiologic outcomes through 78 weeks of follow-up occurred in participants assigned to the 9-week linezolid groups.\ud \ud Conclusions\ud A total of 84 to 93% of the participants across all four bedaquiline–pretomanid–linezolid treatment groups had a favorable outcome. The overall risk–benefit ratio favored the group that received the three-drug regimen with linezolid at a dose of 600 mg for 26 weeks, with a lower incidence of adverse events reported and fewer linezolid dose modifications. (Funded by the TB Alliance and others; ZeNix ClinicalTrials.gov number, NCT03086486. opens in new tab.)

Concentration dependency of phenotypic and genotypic isoniazid-rifampicin resistance emergence was investigated to obtain a mechanistic understanding on how anti-mycobacterial drugs facilitate the emergence of bacterial populations that survive throughout treatment. Using static kill curve experiments, observing two evolution cycles, it was demonstrated that rifampicin resistance was the result of non-specific mechanisms and not associated with accumulation of drug resistance encoding SNPs. Whereas, part of isoniazid resistance could be accounted for by accumulation of specific SNPs, which was concentration dependent. Using a Hollow Fibre Infection Model it was demonstrated that emergence of resistance did not occur at concentration–time profiles mimicking the granuloma. This study showed that disentangling and quantifying concentration dependent emergence of resistance provides an improved rational for drug and dose selection although further work to understand the underlying mechanisms is needed to improve the drug development pipeline.

Aims Idiopathic left ventricular hypertrophy (LVH) is defined as LVH in the absence of myocyte disarray or secondary causes. It is unclear whether idiopathic LVH represents the phenotypic spectrum of hypertrophic cardiomyopathy (HCM) or whether it is a unique disease entity. We aimed to ascertain the prevalence of HCM in first-degree relatives of decedents from sudden death with idiopathic LVH at autopsy. Decedents also underwent molecular autopsy to identify the presence of pathogenic variants in genes implicated in HCM. Methods and results Families of 46 decedents with idiopathic LVH (125 first-degree relatives) were investigated with electrocardiogram, echocardiogram exercise tolerance test, cardiovascular magnetic resonance imaging, 24-h Holter, and ajmaline provocation test. Next-generation sequencing molecular autopsy was performed in 14 (30%) cases. Decedents with idiopathic LVH were aged 33 ± 14 years and 40 (87%) were male. Fourteen families (30%) comprising 16 individuals were diagnosed with cardiac disease, including Brugada syndrome (n = 8), long QT syndrome (n = 3), cardiomyopathy (n = 2), and Wolff–Parkinson–White syndrome (n = 1). None of the family members were diagnosed with HCM. Molecular autopsy did not identify any pathogenic or likely pathogenic variants in genes encoding sarcomeric proteins. Two decedents had pathogenic variants associated with long QT syndrome, which were confirmed in relatives with the clinical phenotype. One decedent had a pathogenic variant associated with Danon disease in the absence of any histopathological findings of the condition or clinical phenotype in the family. Conclusion Idiopathic LVH appears to be a distinct disease entity from HCM and is associated with fatal arrhythmias in individuals with primary arrhythmia syndromes. Family screening in relatives of decedents with idiopathic LVH should be comprehensive and encompass the broader spectrum of inherited cardiac conditions, including channelopathies.

Background The Public Health Alliance for Genomic Epidemiology (PHA4GE) (https://pha4ge.org) is a global coalition that is actively working to establish consensus standards, document and share best practices, improve the availability of critical bioinformatics tools and resources, and advocate for greater openness, interoperability, accessibility, and reproducibility in public health microbial bioinformatics. In the face of the current pandemic, PHA4GE has identified a need for a fit-for-purpose, open-source SARS-CoV-2 contextual data standard. Results As such, we have developed a SARS-CoV-2 contextual data specification package based on harmonizable, publicly available community standards. The specification can be implemented via a collection template, as well as an array of protocols and tools to support both the harmonization and submission of sequence data and contextual information to public biorepositories. Conclusions Well-structured, rich contextual data add value, promote reuse, and enable aggregation and integration of disparate datasets. Adoption of the proposed standard and practices will better enable interoperability between datasets and systems, improve the consistency and utility of generated data, and ultimately facilitate novel insights and discoveries in SARS-CoV-2 and COVID-19. The package is now supported by the NCBI’s BioSample database.

Background: Acquired long QT syndrome (aLQTS) is a serious unpredictable adverse drug reaction. Pharmacogenomic markers may predict risk. Methods: Among 153 aLQTS patients (mean age 58 years [range, 14–88], 98.7% White, 85.6% symptomatic), computational methods identified proteins interacting most significantly with 216 QT-prolonging drugs. All cases underwent sequencing of 31 candidate genes arising from this analysis or associating with congenital LQTS. Variants were filtered using a minor allele frequency Results: In 25.5% of cases, at least one rare variant was identified: 22.2% of cases carried a rare variant in a gene associated with congenital LQTS, and in 4% of cases that variant was known to be pathogenic or likely pathogenic for congenital LQTS; 7.8% cases carried a cytochrome-P450 (CYP) gene variant. Of 12 identified CYP variants, 11 (92%) were in an enzyme known to metabolize at least one culprit drug to which the subject had been exposed. Drug-drug interactions that affected culprit drug metabolism were found in 19% of cases. More than one congenital LQTS variant, CYP gene variant, or drug interaction was present in 7.8% of cases. Gene-burden analyses of the primary cohort compared to control exomes (n=452), and an independent replication aLQTS exome sequencing cohort (n=67) and drug-tolerant controls (n=148) demonstrated an increased burden of rare (minor allele frequency Conclusions: Rare susceptibility variants in CYP genes are emerging as potentially important pharmacogenomic risk markers for aLQTS and could form part of personalized medicine approaches in the future.

It is widely recognized that pathogens can be transmitted across the placenta from mother to foetus. Recent re-evaluation of metagenomic studies indicates that the placenta has no unique microbiome of commensal bacteria. However, viral transmission across the placenta, including transmission of DNA viruses such as the human herpesviruses, is possible. A fuller understanding of which DNA virus sequence can be found in the placenta is required. We employed a metagenomic analysis to identify viral DNA sequences in placental metagenomes from full-term births (20 births), pre-term births (13 births), births from pregnancies associated with antenatal infections (12 births) or pre-term births with antenatal infections (three births). Our analysis found only a small number of DNA sequences corresponding to the genomes of human herpesviruses in four of the 48 metagenomes analysed. Therefore, our data suggest that DNA virus infection of the placenta is rare and support the concept that the placenta is largely free of pathogen infection.

We identify amino acid variants within dominant SARS-CoV-2 T-cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T-cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T-cell responsiveness was seen due to Q213K in the A*01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215, due to P13L, P13S and P13T in the B*27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17, and due to T362I and P365S in the A*03:01/A*11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T-cell lines unable to recognise variant epitopes have diverse T-cell receptor repertoires. These data demonstrate the potential for T-cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T-cell as well as humoral immunity., Graphical Abstract

Objectives To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. Methods The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. Results We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. Conclusions In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage B.1.1.7 has been associated with an increased rate of transmission and disease severity among subjects testing positive in the community. Its impact on hospitalised patients is less well documented.MethodsWe collected viral sequences and clinical data of patients admitted with SARS-CoV-2 and hospital-onset COVID-19 infections (HOCIs), sampled 16/11/2020 - 10/01/2021, from eight hospitals participating in the COG-UK-HOCI study. Associations between the variant and the outcomes of all-cause mortality and intensive therapy unit (ITU) admission were evaluated using mixed effects Cox models adjusted by age, sex, comorbidities, care home residence, pregnancy and ethnicity.ResultsSequences were obtained from 2341 inpatients (HOCI cases = 786) and analysis of clinical outcomes was carried out in 2147 inpatients with all data available. The hazard ratio (HR) for mortality of B.1.1.7 compared to other lineages was 1.01 (95% CI 0.79-1.28, P=0.94) and for ITU admission was 1.01 (95% CI 0.75-1.37, P=0.96). Analysis of sex-specific effects of B.1.1.7 identified increased risk of mortality (HR 1.30, 95% CI 0.95-1.78) and ITU admission (HR 1.82, 95% CI 1.15-2.90) in females infected with the variant but not males (mortality HR 0.82, 95% CI 0.61-1.10; ITU HR 0.74, 95% CI 0.52-1.04).ConclusionsIn common with smaller studies of patients hospitalised with SARS-CoV-2 we did not find an overall increase in mortality or ITU admission associated with B.1.1.7 compared to other lineages. However, women with B.1.1.7 may be at an increased risk of admission to intensive care and at modestly increased risk of mortality.

Mortality in neonates with Gram-negative bloodstream infections has remained unacceptably high. Very few data are available on the impact of resistance profiles, virulence factors, appropriateness of empirical treatment and clinical characteristics on patients’ mortality. A survival analysis to investigate 28-day mortality probability and predictors was performed including (I) infants &lt, 90 days (II) with an available Enterobacterales blood isolate with (III) clinical, treatment and 28-day outcome data. Eighty-seven patients were included. Overall, 299 virulence genes were identified among all the pathogens. Escherichia&nbsp, coli had significantly more virulence genes identified compared with other species. A strong positive correlation between the number of resistance and virulence genes carried by each isolate was found. The cumulative probability of death obtained by the Kaplan-Meier survival analysis was 19.5%. In the descriptive analysis, early age at onset, gestational age at onset, culture positive for E. coli and number of classes of virulence genes carried by each isolate were significantly associated with mortality. By Cox multivariate regression, none of the investigated variables was significant. This pilot study has demonstrated the feasibility of investigating the association between neonatal sepsis mortality and the causative Enterobacterales isolates virulome. This relationship needs further exploration in larger studies, ideally including host immunopathological response, in order to develop a tailor-made therapeutic strategy.

Background\ud The gut microbiota potentially plays an important role in the immunologic education of the host during early infancy.\ud \ud Objective\ud We sought to determine how the infant gut microbiota evolve during infancy, particularly in relation to hygiene-related environmental factors, atopic disorders, and a randomized introduction of allergenic solids.\ud \ud Methods\ud A total of 1303 exclusively breast-fed infants were enrolled in a dietary randomized controlled trial (Enquiring About Tolerance study) from 3 months of age. In this nested longitudinal study, fecal samples were collected at baseline, with additional sampling of selected cases and controls at 6 and 12 months to study the evolution of their gut microbiota, using 16S ribosomal RNA gene-targeted amplicon sequencing.\ud \ud Results\ud In the 288 baseline samples from exclusively breast-fed infant at 3 months, the gut microbiota was highly heterogeneous, forming 3 distinct clusters: Bifidobacterium-rich, Bacteroides-rich, and Escherichia/Shigella-rich. Mode of delivery was the major discriminating factor. Increased Clostridium sensu stricto relative abundance at 3 months was associated with presence of atopic dermatitis on examination at age 3 and 12 months. From the selected cases and controls with longitudinal samples (n = 70), transition to Bacteroides-rich communities and influx of adult-specific microbes were observed during the first year of life. The introduction of allergenic solids promoted a significant increase in Shannon diversity and representation of specific microbes, such as genera belonging to Prevotellaceae and Proteobacteria (eg, Escherichia/Shigella), as compared with infants recommended to exclusively breast-feed.\ud \ud Conclusions\ud Specific gut microbiota characteristics of samples from 3-month-old breast-fed infants were associated with cesarean birth, and greater Clostridium sensu stricto abundance was associated with atopic dermatitis. The randomized introduction of allergenic solids from age 3 months alongside breast-feeding was associated with differential dynamics of maturation of the gut microbial communities.

Background: Recently emerging SARS-CoV-2 variants have been associated with an increased rate of transmission within the community. Little is known about the impact their increased infectivity has on transmission within hospitals. Methods: We collected viral sequences and epidemiological data of patients with community and healthcare associated SARS-CoV-2 infections, sampled from 16th November 2020 to 10th January 2021, from nine hospitals participating in the COG-UK HOCI study. Outbreaks were identified using ward information, lineage and pairwise genetic differences between viral sequences. Findings: Mixed effects logistic regression analysis of 4184 sequences showed healthcare-acquired infections were no more likely to be identified as the Alpha variant than community acquired infections. Nosocomial outbreaks were investigated based on overlapping ward stay and SARS-CoV-2 genome sequence similarity. There was no significant difference in the number of patients involved in outbreaks caused by the Alpha variant compared to outbreaks caused by other lineages. Interpretation: Notwithstanding evidence from community studies that the Alpha variant is more transmissible, we find no evidence to support it causing more nosocomial transmission than previous lineages. This suggests that the stringent infection prevention measures already in place in UK hospitals contained the spread of the Alpha variant as effectively as other less transmissible lineages, providing reassurance of their efficacy against emerging variants of concern. Funding Information: COG-UK HOCI funded by COG-UK consortium. The COG-UK consortium is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute. Declaration of Interests: None to declare. Ethics Approval Statement: Ethical approval for the HOCI study was provided by REC 20/EE/0118.

The Public Health Alliance for Genomic Epidemiology (PHA4GE) (https://pha4ge.org) is a global coalition that is actively working to establish consensus standards, document and share best practices, improve the availability of critical bioinformatic tools and resources, and advocate for greater openness, interoperability, accessibility and reproducibility in public health microbial bioinformatics. In the face of the current pandemic, PHA4GE has identified a clear and present need for a fit-for-purpose, open source SARS-CoV-2 contextual data standard. As such, we have developed an extension to the INSDC pathogen package, providing a SARS-CoV-2 contextual data specification based on harmonisable, publicly available, community standards. The specification is implementable via a collection template, as well as an array of protocols and tools to support the harmonisation and submission of sequence data and contextual information to public repositories. Well-structured, rich contextual data adds value, promotes reuse, and enables aggregation and integration of disparate data sets. Adoption of the proposed standard and practices will better enable interoperability between datasets and systems, improve the consistency and utility of generated data, and ultimately facilitate novel insights and discoveries in SARS-CoV-2 and COVID-19.

para-Aminosalicylic acid (PAS) remains one of the drugs of last resort for the treatment of tuberculosis, but its mechanism of action is still not completely understood. The main aim of this project was to identify new potential mechanisms of action and resistance to PAS by performing whole-genome sequencing on PAS-resistant laboratory mutants. A new variant in the folC gene was identified, as well as some other mutations that require further study.

Mycobacterium tuberculosis is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the essential protein kinase B (PknB), that control transitions between growth states. Here, we establish that depletion of PknB in replicating M. tuberculosis results in transcriptional adaptations that implicate the DNA-binding protein Lsr2 in coordinating these changes. We show that Lsr2 is phosphorylated by PknB, and that phosphorylation of Lsr2 at threonine 112 is important for M. tuberculosis growth and survival under hypoxic conditions. Fluorescence anisotropy and electrophoretic mobility shift assays demonstrate that phosphorylation reduces Lsr2 binding to DNA, and ChIP-sequencing confirms increased DNA binding of a phosphoablative (T112A) Lsr2 mutant in M. tuberculosis. Altered expression of target genes in T112A Lsr2 compared to wild type Lsr2 M. tuberculosis offers further evidence that phosphorylation mediates expression of the Lsr2 regulon. Structural studies reveal increased dynamics of the Lsr2 DNA binding domain from a T112D phosphomimetic Lsr2 mutant, providing a molecular basis for decreased DNA binding by phosphorylated Lsr2. Our findings suggest that, the essential protein kinase, PknB controls M. tuberculosis growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

Summary Objectives We describe the first published cluster of extensively drug resistant Tuberculosis (XDR-TB) in the UK and show how early whole genome sequencing (WGS) of Mtb can assist in case management and contact investigations. Methods We describe the contact tracing investigation undertaken after the presentation of an adult with XDR-TB. Active cases were treated with an XDR-TB drug regimen and contacts underwent a programme of follow-up for 2 years. All isolates of Mycobacterium tuberculosis ( Mtb ) were assessed early using whole genome sequencing (WGS) as well as routine drug susceptibility testing (DST). Results Thirty-three contacts were screened. In the first year one confirmed and one probable case were identified through contact tracing. A further possible case was identified through epidemiological links. Two confirmed cases were identified through WGS 2 years later. Twenty-five (80%) contacts without evidence of tuberculosis were adherent to 1 year of follow-up and 14 (45%) were adherent to 2 years of follow-up. WGS of Mtb was used to guide drug choices, rapidly identify transmission events, and alter public health management. Conclusion WGS of Mtb enabled rapid effective individualized treatment and facilitated public health interventions by early identification of transmission events.

Background Para-aminosalicylic acid (PAS) was one of the first antibiotics to be used against tuberculosis (TB) and it is still one of the last remaining drugs available to treat extensively drug-resistant (XDR) disease. Despite being on the market for decades, the mechanism of action of PAS is not completely understood yet. Sixteen new compounds against Mycobacterium tuberculosis were created in the laboratory as salicylate analogues (based on their chemical structures) and their antimycobacterial activity had never been tested before. The main aim of this project was to test the activity of these new analogues and to understand their mechanism of action (including PAS). Methods The compounds were tested using three different methods (spot culture, resazurin, and MGIT system). Additionally, resistant mutants were created against PAS and the most promising analogue; whole-genome sequencing (WGS) was performed to understand their mechanism of action. Results One compound in particular, AD25a, showed the lowest critical concentration (0.04 µg/mL) among the salicylate analogues. The WGS analysis identified a total of 28 single nucleotide polymorphisms (SNPs) in the AD25a-resistant mutants and 40 SNPs in the PAS-resistant mutants (when compared with the reference strain H37Rv). The SNPs identified in the AD25a and PAS-resistant mutants did not overlap. The genes rrs, rrl and folC were mostly involved in the PAS-resistant mutants. Conclusion The complete difference in the mutation profiles suggests that AD25a has a mechanism of action different to that of PAS, despite AD25a being synthesized as a salicylate analogue. WGS analysis of PAS-resistant mutants has also provided some interesting results. In particular, all our PAS mutants showed mutations in the rrs and rrl genes (16S and 23S RNA genes, respectively). These mutations should affect the ribosomes and the overall synthesis of proteins. This highlights a new potential mechanism of resistance for PAS that has never been observed before. Disclosures All authors: No reported disclosures.

Emerging infectious diseases 22(7), 1262-1264 (2016). doi:10.3201/eid2207.151511, Published by CDC, Atlanta, Ga.

Nigeria has an emerging problem with multidrug-resistant tuberculosis (MDR-TB). Whole-genome sequencing was used to understand the epidemiology of tuberculosis and genetics of multi-drug resistance among patients from two tertiary referral centers in Southwest Nigeria. In line with previous molecular epidemiology studies, most isolates of Mycobacterium tuberculosis from this dataset belonged to the Cameroon clade within the Euro-American lineage. Phylogenetic analysis showed this clade was undergoing clonal expansion in this region, and suggests that it was involved in community transmission of sensitive and multidrug-resistant tuberculosis. Five patients enrolled for retreatment were infected with pre-extensively drug resistant (pre-XDR) due to fluoroquinolone resistance in isolates from the Cameroon clade. In all five cases resistance was conferred through a mutation in the gyrA gene. In some patients, genomic changes occurred in bacterial isolates during the course of treatment that potentially led to decreased drug susceptibility. We conclude that inter-patient transmission of resistant isolates, principally from the Cameroon clade, contributes to the spread of MDR-TB in this setting, underscoring the urgent need to curb the spread of multi-drug resistance in this region.

Introduction Next generation sequencing can accurately predict antibiotic susceptibility in Neisseria gonorrhoeae (NG) allowing preservation of first-line treatments in the face of widespread antimicrobial resistance (AMR). The rapid nature of novel hand-held nanopore sequencing (NPS) gives promise for utility at the point of care. We evaluated time to result post DNA extraction, and accuracy of MinION (Oxford Nanopore Technologies) NPS to predict phenotypic antimicrobial susceptibility (PAMS) of NG to ciprofloxacin and azithromycin. Methods One-directional (1-D) NPS using bar-coded DNA library preparations from 48 NG isolates, prospectively collected from a London clinic, were run on NPS flow cells (3 per R9.0 flow cell) and illumina MiSeq as a comparator. NPS raw sequences were transferred to the cloud for base-calling, alignment, and variant calling using standard tools. Results Mean time for 1-D library preparation was roughly 1 hour; NPS and alignment took 30X per isolate. Of 48 samples, PAMS to ciprofloxacin, and azithromycin was 74% and 87% respectively. Accuracy of NPS-based genotypic susceptibility, defined as absence of any known AMR-associated SNP’s, to predict PAMS for ciprofloxacin and azithromycin, was 34/34 (100%; 95% CI 89.8%–100%) and 35/40 (87.5%; 95% CI 73.9%–94.5%) respectively. Accuracies improved significantly for azithromycin when only high quality reads were included, and with Illumina sequencing. 30 of the 34 isolates susceptible to azithromycin were also susceptible to ciprofloxacin, and 3 of 6 isolates resistant to azithromycin were also resistant to Ciprofloxacin. Conclusion NPS accurately predicted ciprofloxacin PAMS but was less accurate for azithromycin. With new iterations of the technology, imminent rapid barcoded library preparation (10 min) and rapid DNA extraction from clinical samples, NPS may allow accurate ceftriaxone-adjunctive treatment combinations, for a substantial proportion of patients.

Background RIFAQUIN was a tuberculosis chemotherapy trial in southern Africa including regimens with high-dose rifapentine with moxifloxacin. Here, the application of whole-genome sequencing (WGS) is evaluated within RIFAQUIN for identifying new infections in treated patients as either relapses or reinfections. WGS is further compared with mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) typing. This is the first report of WGS being used to evaluate new infections in a completed clinical trial for which all treatment and epidemiological data are available for analysis. Methods DNA from 36 paired samples of Mycobacterium tuberculosis cultured from patients before and after treatment was typed using 24-loci MIRU-VNTR, in silico spoligotyping and WGS. Following WGS, the sequences were mapped against the reference strain H37Rv, the single-nucleotide polymorphism (SNP) differences between pairs were identified, and a phylogenetic reconstruction was performed. Results WGS indicated that 32 of the paired samples had a very low number of SNP differences (0–5; likely relapses). One pair had an intermediate number of SNP differences, and was likely the result of a mixed infection with a pre-treatment minor genotype that was highly related to the post-treatment genotype; this was reclassified as a relapse, in contrast to the MIRU-VNTR result. The remaining three pairs had very high SNP differences (>750; likely reinfections). Conclusions WGS and MIRU-VNTR both similarly differentiated relapses and reinfections, but WGS provided significant extra information. The low proportion of reinfections seen suggests that in standard chemotherapy trials with up to 24 months of follow-up, typing the strains brings little benefit to an analysis of the trial outcome in terms of differentiating relapse and reinfection. However, there is a benefit to using WGS as compared to MIRU-VNTR in terms of the additional genotype information obtained, in particular for defining the presence of mixed infections and the potential to identify known and novel drug-resistance markers. Electronic supplementary material The online version of this article (doi:10.1186/s12916-017-0834-4) contains supplementary material, which is available to authorized users.

Background The largest outbreak of isoniazid-resistant (INH-R) Mycobacterium tuberculosis in Western Europe is centred in North London, with over 400 cases diagnosed since 1995. In the current study, we evaluated the genetic variation in a subset of clinical samples from the outbreak with the hypothesis that these isolates have unique biological characteristics that have served to prolong the outbreak. Methods Fitness assays, mutation rate estimation, and whole-genome sequencing were performed to test for selective advantage and compensatory mutations. Results This detailed analysis of the genetic variation of these INH-R samples suggests that this outbreak consists of successful, closely related, circulating strains with heterogeneous resistance profiles and little or no associated fitness cost or impact on their mutation rate. Conclusions Specific deletions and SNPs could be a peculiar feature of these INH-R M. tuberculosis isolates, and could potentially explain their persistence over the years.

Drug-resistant tuberculosis (TB) remains a major challenge to global health and to healthcare in the UK. In 2014, a total of 6,520 cases of TB were recorded in England, of which 1.4 % were multidrug-resistant TB (MDR-TB). Extensively drug-resistant TB (XDR-TB) occurs at a much lower rate, but the impact on the patient and hospital is severe. Current diagnostic methods such as drug susceptibility testing and targeted molecular tests are slow to return or examine only a limited number of target regions, respectively. Faster, more comprehensive diagnostics will enable earlier use of the most appropriate drug regimen, thus improving patient outcomes and reducing overall healthcare costs. Whole genome sequencing (WGS) has been shown to provide a rapid and comprehensive view of the genotype of the organism, and thus enable reliable prediction of the drug susceptibility phenotype within a clinically relevant timeframe. In addition, it provides the highest resolution when investigating transmission events in possible outbreak scenarios. However, robust software and database tools need to be developed for the full potential to be realized in this specialized area of medicine.

The Helicobacter pylori protein HP0958 is essential for flagellum biogenesis. It has been shown that HP0958 stabilizes the σ 54 factor RpoN. The aim of this study was to further investigate the role of HP0958 in flagellum production in H. pylori . Global transcript analysis identified a number of flagellar genes that were differentially expressed in an HP0958 mutant strain. Among these, the transcription of the major flagellin gene flaA was upregulated twofold, suggesting that HP0958 was a negative regulator of the flaA gene. However, the production of the FlaA protein was significantly reduced in the HP0958 mutant, and this was not due to the decreased stability of the FlaA protein. RNA stability analysis and binding assays indicated that HP0958 binds and destabilizes flaA mRNA. The HP0958 mutant was successfully complemented, confirming that the mutant phenotype described was due to the lack of HP0958. We conclude that HP0958 is a posttranscriptional regulator that modulates the amount of the flaA message available for translation in H. pylori .

Whole-genome sequencing (WGS) was carried out on 87 isolates of sequence type 111 (ST-111) of Pseudomonas aeruginosa collected between 2005 and 2014 from 65 patients and 12 environmental isolates from 24 hospital laboratories across the United Kingdom on an Illumina HiSeq instrument. Most isolates (73) carried VIM-2, but others carried IMP-1 or IMP-13 (5) or NDM-1 (1); one isolate had VIM-2 and IMP-18, and 7 carried no metallo-beta-lactamase (MBL) gene. Single nucleotide polymorphism analysis divided the isolates into distinct clusters; the NDM-1 isolate was an outlier, and the IMP isolates and 6/7 MBL-negative isolates clustered separately from the main set of 73 VIM-2 isolates. Within the VIM-2 set, there were at least 3 distinct clusters, including a tightly clustered set of isolates from 3 hospital laboratories consistent with an outbreak from a single introduction that was quickly brought under control and a much broader set dominated by isolates from a long-running outbreak in a London hospital likely seeded from an environmental source, requiring different control measures; isolates from 7 other hospital laboratories in London and southeast England were also included. Bayesian evolutionary analysis indicated that all the isolates shared a common ancestor dating back ∼50 years (1960s), with the main VIM-2 set separating approximately 20 to 30 years ago. Accessory gene profiling revealed blocks of genes associated with particular clusters, with some having high similarity (≥95%) to bacteriophage genes. WGS of widely found international lineages such as ST-111 provides the necessary resolution to inform epidemiological investigations and intervention policies.

Objectives MRSA is a major antimicrobial resistance (AMR) pathogen. The reservoir of infecting isolates is colonization, which is the site of evolutionary selection. The aim was to identify if AMRs in colonizing MRSA populations diversified and potential mechanisms of resistance gene transfer in vivo. Methods Nasal swabs from 38 MRSA carriers admitted to hospital were plated and 20 individual colonies from each patient tested for phenotypic antibiotic susceptibility and genetically for lineage, carriage of four prophages and three plasmid families. Free bacteriophages were detected in swabs as well as their capacity for transducing resistance genes. Results Nine (24%) patients carried phenotypic AMR variants and 24 (63%) carried prophage and plasmid variants. If a single colony was selected for testing, the probability of detecting all AMR in that patient was 87%. Sixty-four different AMR and mobile genetic element (MGE) profiles were detected, mostly in the MRSA CC22 background (where CC stands for clonal complex), with up to 8 profiles per patient. Nearly half of the patients carried detectable free bacteriophages and phages successfully transduced resistance genes between laboratory and patient isolates in vitro. WGS showed MRSA core genomes were stable, while AMR and MGEs varied. Conclusions ‘Clouds’ of MRSA variants that have acquired or lost AMR and MGEs are common in nasal colonizing populations and bacteriophages may play an important role in gene transfer. Accurate estimation of AMR and genetic variability has implications for diagnostics, epidemiology, antimicrobial stewardship and understanding the evolutionary selection of AMR in colonizing populations.

Clostridium difficile is the most frequent cause of nosocomial diarrhea worldwide, and recent reports suggested the emergence of a hypervirulent strain in North America and Europe. In this study, we applied comparative phylogenomics (whole-genome comparisons using DNA microarrays combined with Bayesian phylogenies) to model the phylogeny of C. difficile , including 75 diverse isolates comprising hypervirulent, toxin-variable, and animal strains. The analysis identified four distinct statistically supported clusters comprising a hypervirulent clade, a toxin A − B + clade, and two clades with human and animal isolates. Genetic differences among clades revealed several genetic islands relating to virulence and niche adaptation, including antibiotic resistance, motility, adhesion, and enteric metabolism. Only 19.7% of genes were shared by all strains, confirming that this enteric species readily undergoes genetic exchange. This study has provided insight into the possible origins of C. difficile and its evolution that may have implications in disease control strategies.

Staphylococcus aureus is a persistent resident of the human nose in 20% of the population and intermittently carried by another 60% (16). Most carriers harbor a single strain (2). S. aureus is a common cause of minor skin and wound infections, but only rarely causes severe community-acquired invasive infections such as bacteremia, endocarditis, and osteomyelitis. In contrast, S. aureus is the most common cause of hospital-acquired infection, which often occurs in association with breaches of the skin and mucous membranes in the immunocompromised host (14). Hundreds of S. aureus virulence factors and putative virulence genes have been described, including those involved in adherence to human tissue, evasion of the immune response, toxin secretion, and regulation of virulence gene expression (29). Specific toxins have also been described that play a pivotal role in toxin-mediated disease such as toxic shock syndrome (toxic shock syndrome toxin-1, encoded by tst; enterotoxins B and C, encoded by seb, sec) (5), scalded skin syndrome (exfoliative toxins A and B, encoded by eta, etb) (19), food poisoning (enterotoxin A, encoded by sea) (5), and more recently hemolytic pneumonia and skin and soft tissue infection (Panton Valentine leukocidin [PVL], encoded by the lukS-PV and lukF-PV genes) (20). Many of these genes are variably present as a result of being carried on mobile genetic elements (MGE) (22). However, critical to the development of targeted or preventive strategies is the elucidation of which if any of these genes are important in invasive infection. An important isolate collection associated with community-acquired invasive disease or carriage by healthy donors in the Oxford, United Kingdom, region (7) has been examined using multilocus sequence typing (MLST) (6), in which fragments of seven housekeeping genes were amplified and sequenced. Unique alleles at the seven loci were given an allelic number, and the allelic profile (string of seven integers) was used to define sequence type (ST) for each isolate. Isolates with an identical profile were considered to be clonal, and those with at least five of seven matching genes were considered to belong to the same clonal cluster (CC). Isolates clustered into 10 major CCs, none of which were associated with invasive disease (7). This argued against the presence of virulent genotypes but did not exclude the possibility that one or more variable genes were overrepresented in the invasive-isolate group. This possibility was examined during a study that defined the presence or absence of 33 putative virulence genes in this isolate collection using PCR (27). Seven genes were found to be present more commonly in invasive isolates, including eta and those encoding fibronectin binding protein A (fnbA), collagen binding protein (cna), serine-aspartate repeat containing protein E (sdrE), staphylococcal enterotoxin J (sej), gamma-hemolysin (hlg), and intracellular adhesin (ica). This suggested there were differences between isolates that did not correlate with lineage. It also suggested that some isolates are potentially more virulent than others. Each S. aureus isolate is thought to carry hundreds of variable genes including many putative virulence determinants. The first S. aureus comparative-genomics studies using a microarray (covering 92% of the genes found in the S. aureus COL genome) estimated that 22% of the S. aureus genome was variable (8). Many of the variable genes are known or putative virulence and resistance genes carried on MGE, and these elements are likely to transfer horizontally among staphylococci (see reference 22 for a review). The accumulation of such MGE may result in the emergence of “superbugs” that are increasingly resistant and virulent (22). The whole-genome sequencing of seven isolates of S. aureus (1, 10, 12, 18; www.genome.ou.edu/staph.html) has allowed us to design, print, and validate a multistrain PCR product S. aureus microarray carrying PCR products for every gene identified from these projects, probably the most comprehensive microarray of its kind (33). Here we describe the use of the seven-strain S. aureus microarray to investigate the Oxford collection of community-acquired S. aureus isolates. The aims were to identify which regions of the S. aureus genome vary, investigate gene distribution in a typical S. aureus population, and perform a comprehensive search for differences between invasive and carriage isolates.

Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from thePlasmodium yoelii yoeliigenomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protectiveP. y. yoeliigenes can then be identified in the genomic databases ofPlasmodium falciparumandPlasmodium vivaxand investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192P. y. yoeliiexons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encodingP. y. yoeliicircumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs inP. vivaxandP. knowlesibut notP. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.

Recent genomic-based studies have identified 13 two-component signal transduction systems (TCS) in Streptococcus pneumoniae. Bacterial TCSs are important for regulating expression of bacterial genes, including those which are important to the virulence of pathogenic bacteria. We have used virulence assays together with microarray analysis to investigate the importance of pneumococcal TCS04 in the virulence and gene regulation of this pathogen. Deletion mutants of the response regulator of TCS04, rr04, were examined in three independent pneumococcal strains representing three different pneumococcal serotypes. Analysis of the virulence of the three strains enabled us to identify a serotype-specific attenuation of virulence due to deletion of rr04. Microarray comparison of the transcriptional profiles of the wild-type strains with the rr04 mutants allowed us to determine which transcriptional changes were occurring in the rr04 mutants. Virulence-associated changes were demonstrated in the attenuated strain with significant downregulation of a previously determined virulence locus, psaB, psaC and psaA.

The recent explosion in genomic sequencing has made available a wealth of data that can now be analyzed to identify protein antigens, potential targets for vaccine development. Here we present, in the context ofPlasmodium falciparum, a strategy that rapidly identifies target antigens from large and complex genomes. Sixteen antigenic proteins recognized by volunteers immunized with radiation-attenuatedP. falciparumsporozoites, but not by mock immunized controls, were identified. Several of these were more antigenic than previously identified and well characterizedP. falciparum-derived protein antigens. The data suggest that immune responses toPlasmodiumare dispersed on a relatively large number of parasite antigens. These studies have implications for our understanding of immunodominance and breadth of responses to complex pathogens.

Staphylococcus aureus is a commensal and major pathogen of humans and animals. Comparative genomics of S. aureus populations suggests that colonization of different host species is associated with carriage of mobile genetic elements (MGE), particularly bacteriophages and plasmids capable of encoding virulence, resistance, and immune evasion pathways. Antimicrobial-resistant S. aureus of livestock are a potential zoonotic threat to human health if they adapt to colonize humans efficiently. We utilized the technique of experimental evolution and co-colonized gnotobiotic piglets with both human- and pig-associated variants of the lineage clonal complex 398, and investigated growth and genetic changes over 16 days using whole genome sequencing. The human isolate survived co-colonization on piglets more efficiently than in vitro. During co-colonization, transfer of MGE from the pig to the human isolate was detected within 4 h. Extensive and repeated transfer of two bacteriophages and three plasmids resulted in colonization with isolates carrying a wide variety of mobilomes. Whole genome sequencing of progeny bacteria revealed no acquisition of core genome polymorphisms, highlighting the importance of MGE. Staphylococcus aureus bacteriophage recombination and integration into novel sites was detected experimentally for the first time. During colonization, clones coexisted and diversified rather than a single variant dominating. Unexpectedly, each piglet carried unique populations of bacterial variants, suggesting limited transmission of bacteria between piglets once colonized. Our data show that horizontal gene transfer occurs at very high frequency in vivo and significantly higher than that detectable in vitro.