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BACKGROUND: Patients with prolonged hospitalisation have a significant risk of carriage of and subsequent infection with extended spectrum β-lactamase (ESBL)-producing and carbapenemase-producing Klebsiella pneumoniae. However, the distinctive roles of the community and hospital environments in the transmission of ESBL-producing or carbapenemase-producing K pneumoniae remain elusive. We aimed to investigate the prevalence and transmission of K pneumoniae within and between the two tertiary hospitals in Hanoi, Viet Nam, using whole-genome sequencing. METHODS: We did a prospective cohort study of 69 patients in intensive care units (ICUs) from two hospitals in Hanoi, Viet Nam. Patients were included if they were aged 18 years or older, admitted for longer than the mean length of stay in their ICU, and cultured K pneumoniae from their clinical samples. Longitudinally collected samples from patients (collected weekly) and the ICU environment (collected monthly) were cultured on selective media, and whole-genome sequences from K pneumoniae colonies analysed. We did phylogenetic analyses and correlated phenotypic antimicrobial susceptibility testing with genotypic features of K pneumoniae isolates. We constructed transmission networks of patient samples, relating ICU admission times and locations with genetic similarity of infecting K pneumoniae. FINDINGS: Between June 1, 2017, and Jan 31, 2018, 69 patients were in the ICUs and eligible for inclusion, and a total of 357 K pneumoniae isolates were cultured and successfully sequenced. 228 (64%) of K pneumoniae isolates carried between two and four different ESBL-encoding and carbapenemase-encoding genes, with 164 (46%) isolates carrying genes encoding both, with high minimum inhibitory concentrations. We found a novel co-occurrence of blaKPC-2 and blaNDM-1 in 46·6% of samples from the globally successful ST15 lineage. Despite being physically and clinically separated, the two hospitals shared closely related strains carrying the same array of antimicrobial resistance genes. INTERPRETATION: These results highlight the high prevalence of ESBL-positive carbapenem-resistant K pneumoniae in ICUs in Viet Nam. Through studying K pneumoniae ST15 in detail, we showed how important resistance genes are contained within these strains that are carried broadly by patients entering the two hospitals directly or through referral. FUNDING: Medical Research Council Newton Fund, Ministry of Science and Technology, Wellcome Trust, Academy of Medical Sciences, Health Foundation, and National Institute for Health and Care Research Cambridge Biomedical Research Centre.

Introductory paragraph Infections caused by carbapenemase-producing enterobacteria (CPE) are a major concern in clinical settings worldwide. Two fundamentally different processes shape the epidemiology of CPE in hospitals: the dissemination of CPE clones from patient to patient (between-patient transfer), and the transfer of carbapenemase-encoding plasmids between enterobacteria in the gut microbiota of individual patients (within-patient transfer). The relative contribution of each process to the overall dissemination of carbapenem resistance in hospitals remains poorly understood. Here, we used mechanistic models combining epidemiological data from more than 9,000 patients with whole genome sequence information from 250 enterobacteria clones to characterise the dissemination routes of a pOXA-48-like carbapenemase-encoding plasmid in a hospital setting over a two-year period. Our results revealed frequent between-patient transmission of high-risk pOXA-48-carrying clones, mostly of Klebsiella pneumoniae and sporadically Escherichia coli. The results also identified pOXA-48 dissemination hotspots within the hospital, such as specific wards and individual rooms within wards. Using high-resolution plasmid sequence analysis, we uncovered the pervasive within-patient transfer of pOXA-48, suggesting that horizontal plasmid transfer occurs in the gut of virtually every colonised patient. The complex and multifaceted epidemiological scenario exposed by this study provides insights for the development of intervention strategies to control the in-hospital spread of CPE.

The burden of antimicrobial resistance is a major threat to global health; however, prospective clinical outcome data from Africa are scarce. In Malawi, third-generation cephalosporins are the antibiotics of choice in patients admitted to hospital despite a rapid proliferation of resistance to these drugs. We aimed to quantify the effect of resistance to third-generation cephalosporins on mortality and length of hospital stay among patients with bloodstream infections.We did a prospective cohort study of patients admitted to Queen Elizabeth Central Hospital in Blantyre, Malawi. Patients of all ages who had positive blood cultures for Enterobacterales were included, with the exception of those from the genus Salmonella, and were followed up for 180 days. We characterised blood culture isolates using whole-genome sequencing and used Cox regression models to estimate the effect of resistance to third-generation cephalosporins on length of hospital stay, in-hospital mortality, and survival.Between Jan 31, 2018, and Jan 13, 2020, we recruited 326 patients, from whom 220 (68%) of 326 isolates were resistant to third-generation cephalosporins. The case fatality proportion was 45% (99 of 220) in patients with bloodstream infections that were resistant to third-generation cephalosporins, and 34% (36 of 106) in patients with bloodstream infections that were sensitive to third-generation cephalosporins. Resistance to third-generation cephalosporins was associated with an increased probability of in-hospital mortality (hazard ratio [HR] 1·44, 95% CI 1·02-2·04), longer hospital stays (1·5 days, 1·0-2·0) and decreased probability of discharge alive (HR 0·31, 0·22-0·45). Whole-genome sequencing showed a high diversity of sequence types of both Escherichia coli and Klebsiella pneumoniae. Although isolates associated with death were distributed across clades, we identified three E coli clades (ST410, ST617, and ST648) that were isolated from 14 patients who all died.Resistance to third-generation cephalosporins is associated with increased mortality and longer hospital stays in patients with bloodstream infections in Malawi. These data show the urgent need for allocation of resources towards antimicrobial resistance mitigation strategies in Africa.Wellcome Trust and Wellcome Asia and Africa Programme.

In sub-Saharan Africa (sSA), there is high morbidity and mortality from severe bacterial infection and this is compounded by antimicrobial resistance, in particular, resistance to 3rd-generation cephalosporins. This resistance is typically mediated by extended-spectrum beta lactamases (ESBLs). To interrupt ESBL transmission it will be important to investigate how human behaviour, water, sanitation, and hygiene (WASH) practices, environmental contamination, and antibiotic usage in both urban and rural settings interact to contribute to transmission of ESBL E. coli and ESBL K. pneumoniae between humans, animals, and the environment. Here we present the protocol for the Drivers of Resistance in Uganda and Malawi (DRUM) Consortium, in which we will collect demographic, geospatial, clinical, animal husbandry and WASH data from a total of 400 households in Uganda and Malawi. Longitudinal human, animal and environmental sampling at each household will be used to isolate ESBL E. coli and ESBL K. pneumoniae. This will be complimented by a Risks, Attitudes, Norms, Abilities and Self-Regulation (RANAS) survey and structured observations to understand the contextual and psychosocial drivers of regional WASH practices. Bacterial isolates and plate sweeps will be further characterised using a mixture of short-,long-read and metagenomic whole-genome sequencing. These datasets will be integrated into agent-based models to describe the transmission of EBSL resistance in Uganda and Malawi and allow us to inform the design of interventions for interrupting transmission of ESBL-bacteria.

Background Antimicrobial resistance (AMR) poses a major threat to human health around the world. Previous publications have estimated the effect of AMR on incidence, deaths, hospital length of stay, and health-care costs for specific pathogen–drug combinations in select locations. To our knowledge, this study presents the most comprehensive estimates of AMR burden to date. Methods We estimated deaths and disability-adjusted life-years (DALYs) attributable to and associated with bacterial AMR for 23 pathogens and 88 pathogen–drug combinations in 204 countries and territories in 2019. We obtained data from systematic literature reviews, hospital systems, surveillance systems, and other sources, covering 471 million individual records or isolates and 7585 study-location-years. We used predictive statistical modelling to produce estimates of AMR burden for all locations, including for locations with no data. Our approach can be divided into five broad components: number of deaths where infection played a role, proportion of infectious deaths attributable to a given infectious syndrome, proportion of infectious syndrome deaths attributable to a given pathogen, the percentage of a given pathogen resistant to an antibiotic of interest, and the excess risk of death or duration of an infection associated with this resistance. Using these components, we estimated disease burden based on two counterfactuals: deaths attributable to AMR (based on an alternative scenario in which all drug-resistant infections were replaced by drug-susceptible infections), and deaths associated with AMR (based on an alternative scenario in which all drug-resistant infections were replaced by no infection). We generated 95% uncertainty intervals (UIs) for final estimates as the 25th and 975th ordered values across 1000 posterior draws, and models were cross-validated for out-of-sample predictive validity. We present final estimates aggregated to the global and regional level. Findings On the basis of our predictive statistical models, there were an estimated 4·95 million (3·62–6·57) deaths associated with bacterial AMR in 2019, including 1·27 million (95% UI 0·911–1·71) deaths attributable to bacterial AMR. At the regional level, we estimated the all-age death rate attributable to resistance to be highest in western sub-Saharan Africa, at 27·3 deaths per 100 000 (20·9–35·3), and lowest in Australasia, at 6·5 deaths (4·3–9·4) per 100 000. Lower respiratory infections accounted for more than 1·5 million deaths associated with resistance in 2019, making it the most burdensome infectious syndrome. The six leading pathogens for deaths associated with resistance (Escherichia coli, followed by Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) were responsible for 929 000 (660 000–1 270 000) deaths attributable to AMR and 3·57 million (2·62–4·78) deaths associated with AMR in 2019. One pathogen–drug combination, meticillin-resistant S aureus, caused more than 100 000 deaths attributable to AMR in 2019, while six more each caused 50 000–100 000 deaths: multidrug-resistant excluding extensively drug-resistant tuberculosis, third-generation cephalosporin-resistant E coli, carbapenem-resistant A baumannii, fluoroquinolone-resistant E coli, carbapenem-resistant K pneumoniae, and third-generation cephalosporin-resistant K pneumoniae. Interpretation To our knowledge, this study provides the first comprehensive assessment of the global burden of AMR, as well as an evaluation of the availability of data. AMR is a leading cause of death around the world, with the highest burdens in low-resource settings. Understanding the burden of AMR and the leading pathogen–drug combinations contributing to it is crucial to making informed and location-specific policy decisions, particularly about infection prevention and control programmes, access to essential antibiotics, and research and development of new vaccines and antibiotics. There are serious data gaps in many low-income settings, emphasising the need to expand microbiology laboratory capacity and data collection systems to improve our understanding of this important human health threat. Funding Bill & Melinda Gates Foundation, Wellcome Trust, and Department of Health and Social Care using UK aid funding managed by the Fleming Fund.

A special-care neonatal unit from a large public hospital in Malawi was noted as having more frequent, difficult-to-treat infections, and a suspected outbreak of multi-drug-resistant Klebsiella pneumoniae was investigated using genomic characterisation. All K. pneumoniae bloodstream infections (BSIs) from patients in the neonatal ward (n=62), and a subset of K. pneumoniae BSI isolates (n=38) from other paediatric wards in the hospital, collected over a 4 year period were studied. After whole genome sequencing, the strain sequence types (STs), plasmid types, virulence and resistance genes were identified. One ST340 clone, part of clonal complex 258 (CC258) and an ST that drives hospital outbreaks worldwide, harbouring numerous resistance genes and plasmids, was implicated as the likely cause of the outbreak. This study contributes molecular information necessary for tracking and characterizing this important hospital pathogen in sub-Saharan Africa.

BackgroundExtended spectrum beta-lactamases-producing (ESBL-P) and/or carbapenem-resistant (CR) Klebsiella pneumoniae have severely restricted available treatment options in healthcare settings in Vietnam. Understanding the diversity and transmission mechanisms of ESBL- and carbapenemase-encoding K. pneumoniae is important in both hospital and community settings for patient management.MethodsWe conducted a 6-month prospective cohort study of 69 Intensive care unit (ICU) patients from two hospitals in Hanoi, Vietnam. Longitudinally collected samples from patients and the ICU environment were cultured on selective media, and 357 K. pneumoniae colonies were whole genome sequenced. We performed phylogenetic analyses, and correlated phenotypic antimicrobial susceptibility testing with genotypic features of K. pneumoniae isolates. We constructed transmission networks of patient samples, relating ICU admission times and locations with genetic similarity of infecting K. pneumoniae.FindingsDespite being geographically and clinically separated, the two hospitals shared closely related strains carrying the same array of antimicrobial resistance genes. Many patients carried the same resistant K. pneumoniae clone from admission to discharge. 45.9% of total isolates carried both ESBL- and carbapenemase-encoding genes, with high minimum inhibitory concentrations. We found a novel co-occurrence of blaKPC-2 and blaNDM-1 in 46. 6% of samples from the globally successful ST15 lineage.InterpretationThese results highlight the high prevalence of ESBL-positive carbapenem-resistant K. pneumoniae in Vietnamese ICUs. Through studying K. pneumoniae ST15 in detail, we illustrated how important resistance genes are coalescing in stains carried broadly by patients entering the two hospitals directly or through referral.FundingThis study was supported by the Medical Research Council Newton Fund, United Kingdom (grant MR/N029399/1); the Ministry of Science and Technology, Vietnam (grant HNQT/SPÐP/04.16); This research was funded in whole by the Wellcome Trust (grant 206194). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.

Here we describe the development and evaluation of a novel an air-dried high-resolution melt (HRM) assay to detect eight major extended spectrum beta-Lactamase (ESBL) (SHV and CTXM groups 1 and 9) and Carbapenemase (NDM, IMP, KPC, VIM and OXA-48) genes that cause antimicrobial resistance. The assay was evaluated using 440 DNA samples extracted from bacterial isolates from Nepal, Malawi and UK and 390 clinical Enterobacteriaceae isolates with known resistance phenotypes from Nepal. The sensitivity and specificity for detecting the ESBL and Carbapenemase genes in comparison to the reference gel-base PCR and sequencing was 94.7% (95%CI: 92.5%-96.5%) and 99.2% (95%CI: 98.8%-99.5%) and 98.5% (95%CI: 97.0%-99.4%) and 98.5% (95%CI: 98.0%-98.9%) when compared to the original wet format. The overall phenotypic agreement was 91.1% (95%CI: 90.0%-92.9%) on predicting resistance to cefotaxime and carbapenems. We observed good inter-machine reproducibility of the air-dried HRM assay using the Rotor-Gene Q, QuantStudio™ 5, CFX96, LightCycler® 480 and MIC. Assay stability upon storage in the fridge (6.2°C ± 0.9), room temperature (20.35°C ± 0.7) and oven (29.7°C ± 1.4) were assessed at six time points for eight months and no loss of sensitivity occurred under all conditions. We present here a ready-to-use air-dried HRM-PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and Carbapenamase genes to improve AMR diagnosis and treatment.

Carriage dynamics of drug-resistant bacteria, especially within households, are poorly understood. This limits the ability to develop effective interventions for controlling the spread of antimicrobial resistance in the community. Two groups consisting of: (i) patients with urinary tract infection requiring antimicrobial treatment; and (ii) patients who were not prescribed antimicrobial treatment were prospectively recruited at three European sites: Antwerp (Belgium), Geneva (Switzerland) and Lodz (Poland). Each index patient and up to three additional household members provided faecal samples at baseline, completion of antimicrobial therapy (or 7-10 days after the first sample for the non-exposed) and 28 days after the second sample. We analysed household-level and individual-level fluoroquinolone resistant Enterobacteriaceae (FQR-E) acquisition and carriage data using Bayesian multi-state Markov models. At the individual level, we estimated a median baseline FQR-E acquisition rate of 0.006 (95%CrI = [0.004, 0.01]) per day, and a median duration of carriage of 24.4 days (95% CrI=[15.23,41.38]). Nitrofurantoin exposure was associated with a reduced rate of FQR-E acquisition (HR=0.28, 95%CrI=[0.14,0.56]), while fluoroquinolone exposure had no clear association with rates of FQR-E acquisition (HR=1.43, 95% CrI=[0.81,2.53]) at individual level. There was evidence that rates of FQR-E acquisition varied by site, and coming from Lodz was associated with a higher acquisition rate (HR=3.56, 95% CrI=[1.92, 6.34]). Prolonged duration of carriage was associated with exposure to fluoroquinolone or nitrofurantoin during the study, use of any antimicrobial agent in the prior 12 months and travel to endemic regions. At household level, we found strong evidence of positive association between FQR-E acquisition and fluoroquinolone exposure (HR=3.43, 95% CrI=[1.51,7.74]). There was weak evidence of negative association between FQR-E acquisition and nitrofurantoin exposure (HR=0.42, 95%CrI=[0.12, 1.24]. Similar to the individual level, carriage duration was also associated with antimicrobial exposure at the household level. Our study has identified within household contacts as an important route for FQR-E transmission and highlights the need for prioritising household focused interventions to control FQR-E spread.

A suspected outbreak of multi-drug resistant (MDR) Klebsiella pneumoniae in a Malawian neonatal unit was investigated using whole-genome sequencing. Strain-types, virulence and resistance genes of K. pneumoniae isolated from patients from the hospital over a four-year period were identified. A MDR ST340 clone was implicated as the likely outbreak cause.

Background The prevalence of bacterial bloodstream infections (BSIs) in sub-Saharan Africa (sSA) is high and antimicrobial resistance is likely to increase mortality from these infections. Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae are of particular concern, given the widespread reliance on ceftriaxone for management of sepsis in Africa. Objectives Reviewing studies from sSA, we aimed to describe the prevalence of 3GC resistance in Escherichia coli, Klebsiella and Salmonella BSIs and the in-hospital mortality from 3GC-R BSIs. Methods We systematically reviewed studies reporting 3GC susceptibility testing of E. coli, Klebsiella and Salmonella BSI. We searched PubMed and Scopus from January 1990 to September 2019 for primary data reporting 3GC susceptibility testing of Enterobacteriaceae associated with BSI in sSA and studies reporting mortality from 3GC-R BSI. 3GC-R was defined as phenotypic resistance to ceftriaxone, cefotaxime or ceftazidime. Outcomes were reported as median prevalence of 3GC resistance for each pathogen. Results We identified 40 articles, including 7 reporting mortality. Median prevalence of 3GC resistance in E. coli was 18.4% (IQR 10.5 to 35.2) from 20 studies and in Klebsiella spp. was 54.4% (IQR 24.3 to 81.2) from 28 studies. Amongst non-typhoidal salmonellae, 3GC resistance was 1.9% (IQR 0 to 6.1) from 12 studies. A pooled mortality estimate was prohibited by heterogeneity. Conclusions Levels of 3GC resistance amongst bloodstream Enterobacteriaceae in sSA are high, yet the mortality burden is unknown. The lack of clinical outcome data from drug-resistant infections in Africa represents a major knowledge gap and future work must link laboratory surveillance to clinical data.

The lung is the site most commonly affected by infectious and non-infectious complications of HIV infection [1], even in individuals on effective antiretroviral therapy (ART). HIV infection is associated with lymphocytic alveolitis [2], a condition characterised by the influx of CD8+ T-cells into the alveolar space. This is thought to occur in response to HIV antigens and forms part of the host response to the presence of HIV in the lung [2, 3]. Excessive influx of inflammatory cells in the lung probably leads to tissue damage, disruption of immune cell homeostasis, impaired gas exchange [4] and predisposition to HIV-associated lung complications., Disruption of lung cytokine networks during chronic HIV infection is incompletely restored in individuals on antiretroviral therapy http://ow.ly/klkF30gTtwf

Background Pneumococcal conjugate vaccines (PCVs) have reduced pneumococcal diseases globally. Pneumococcal genomic surveys elucidate PCV effects on population structure but are rarely conducted in low-income settings despite the high disease burden. Methods We undertook whole-genome sequencing (WGS) of 660 pneumococcal isolates collected through surveys from healthy carriers 2 years from 13-valent PCV (PCV13) introduction and 1 year after rollout in northern Malawi. We investigated changes in population structure, within-lineage serotype dynamics, serotype diversity, and frequency of antibiotic resistance (ABR) and accessory genes. Results In children, After 13-valent pneumococcal conjugate vaccine (PCV13) introduction in Malawi, the frequency of carried vaccine serotypes decreased significantly across lineages; there was an increase of serotypes 7C, 15B/C, 23A, and 28F; antibiotic resistance rates remained stable; and certain accessory genes changed in frequency.

Objectives ESBL-producing Klebsiella pneumoniae (KPN) pose a major threat to human health globally. We carried out a WGS study to understand the genetic background of ESBL-producing KPN in Malawi and place them in the context of other global isolates. Methods We sequenced genomes of 72 invasive and carriage KPN isolates collected from patients admitted to Queen Elizabeth Central Hospital, Blantyre, Malawi. We performed phylogenetic and population structure analyses on these and previously published genomes from Kenya (n = 66) and from outside sub-Saharan Africa (n = 67). We screened for presence of antimicrobial resistance (AMR) genetic determinants and carried out association analyses by genomic sequence cluster, AMR phenotype and time. Results Malawian isolates fit within the global population structure of KPN, clustering into the major lineages of KpI, KpII and KpIII. KpI isolates from Malawi were more related to those from Kenya, with both collections exhibiting more clonality than isolates from the rest of the world. We identified multiple ESBL genes, including blaCTX-M-15, several blaSHV, blaTEM-63 and blaOXA-10, and other AMR genes, across diverse lineages of the KPN isolates from Malawi. No carbapenem resistance genes were detected; however, we detected IncFII and IncFIB plasmids that were similar to the carbapenem resistance-associated plasmid pNDM-mar. Conclusions There are multiple ESBL genes across diverse KPN lineages in Malawi and plasmids in circulation that are capable of carrying carbapenem resistance. Unless appropriate interventions are rapidly put in place, these may lead to a high burden of locally untreatable infection in vulnerable populations.

BackgroundFever is among the most common symptoms of people living in Africa, and clinicians are challenged by the similar clinical features of a wide spectrum of potential aetiologies. AimTo summarise recent studies of fever aetiology in sub-Saharan Africa focusing on causes other than malaria. SourcesA narrative literature review by searching the MEDLINE database, and recent conference abstracts. ContentStudies of multiple potential causes of fever are scarce, and for many participants the infecting organism remains unidentified, or multiple co-infecting microorganisms are identified, and establishing causation is challenging. Among ambulatory patients, self-limiting arboviral infections and viral upper respiratory infections are common, occurring in up to 60% of children attending health centres. Among hospitalised patients there is a high prevalence of potentially fatal infections requiring specific treatment. Bacterial bloodstream infection, and bacterial zoonoses are major causes of fever. In recent years, the prevalence of antimicrobial resistance among bacterial isolates has increased, notably with spread of extended spectrum betalactamase-producing Enterobacteriaceae and fluoroquinolone resistant Salmonella enterica. Among those with HIV infection, Mycobacterium tuberculosis bacteraemia has been confirmed in up to 34.8% of patients with sepsis, and fungal infections such as cryptococcosis and histoplasmosis remain important. ImplicationsUnderstanding the local epidemiology of fever aetiology, and use of diagnostics including malaria and HIV rapid-diagnostic tests, guides healthcare workers in the management of patients with fever. Current challenges for clinicians include assessing which ambulatory patients require antibacterial drugs, and identifying hospitalised patients infected with organisms that are not susceptible to empiric antibacterial regimens.

BACKGROUNDBacterial bloodstream infection is a common cause of morbidity and mortality in sub-Saharan Africa, yet few facilities are able to maintain long-term surveillance. The Malawi-Liverpool-Wellcome Trust Clinical Research Programme has done sentinel surveillance of bacteraemia since 1998. We report long-term trends in bloodstream infection and antimicrobial resistance from this surveillance.METHODS In this surveillance study, we analysed blood cultures that were routinely taken from adult and paediatric patients with fever or suspicion of sepsis admitted to Queen Elizabeth Central Hospital, Blantyre, Malawi from 1998 to 2016. The hospital served an urban population of 920 000 in 2016, with 1000 beds, although occupancy often exceeds capacity. The hospital admits about 10 000 adults and 30 000 children each year. Antimicrobial susceptibility tests were done by the disc diffusion method according to British Society of Antimicrobial Chemotherapy guidelines. We used the Cochran-Armitage test for trend to examine trends in rates of antimicrobial resistance, and negative binomial regression to examine trends in icidence of bloodstream infection over time.FINDINGSBetween Jan 1, 1998, and Dec 31, 2016, we isolated 29 183 pathogens from 194 539 blood cultures. Pathogen detection decreased significantly from 327·1/100 000 in 1998 to 120·2/100 000 in 2016 (pINTERPRETATIONThe rapid expansion of ESBL and fluoroquinolone resistance among common Gram-negative pathogens, and the emergence of MRSA, highlight the growing challenge of bloodstream infections that are effectively impossible to treat in this resource-limited setting.FUNDINGWellcome Trust, H3ABionet, Southern Africa Consortium for Research Excellence (SACORE).

Pathogenic Salmonella strains that cause gastroenteritis are able to colonize and replicate within the intestines of multiple host species. In general, these strains have retained an ability to form the rdar morphotype, a resistant biofilm physiology hypothesized to be important for Salmonella transmission. In contrast, Salmonella strains that are host-adapted or even host-restricted like Salmonella enterica serovar Typhi, tend to cause systemic infections and have lost the ability to form the rdar morphotype. Here, we investigated the rdar morphotype and CsgD-regulated biofilm formation in two non-typhoidal Salmonella (NTS) strains that caused invasive disease in Malawian children, S. Typhimurium D23580 and S. Enteritidis D7795, and compared them to a panel of NTS strains associated with gastroenteritis, as well as S. Typhi strains. Sequence comparisons combined with luciferase reporter technology identified key SNPs in the promoter region of csgD that either shut off biofilm formation completely (D7795) or reduced transcription of this key biofilm regulator (D23580). Phylogenetic analysis showed that these SNPs are conserved throughout the African clades of invasive isolates, dating as far back as 80 years ago. S. Typhi isolates were negative for the rdar morphotype due to truncation of eight amino acids from the C-terminus of CsgD. We present new evidence in support of parallel evolution between lineages of nontyphoidal Salmonella associated with invasive disease in Africa and the archetypal host-restricted invasive serovar; S. Typhi. We hypothesize that the African invasive isolates are becoming human-adapted and ‘niche specialized’ with less reliance on environmental survival, as compared to gastroenteritis-causing isolates., Author summary African clades of nontyphoidal Salmonella cause invasive disease on a daily basis and thousands of deaths each year. Although it is generally accepted that the transmission route for these organisms is fecal-oral, we know very little about their behaviour in the environment between hosts. In this paper, we have identified both a genotype and a phenotype that suggest environmental niche specialization that is distinct from lineages of Salmonella Typhimurium and Salmonella Enteritidis associated with industrialized food supply chains in resource-rich settings. We also compared with strains of Salmonella Typhi, which cause systemic typhoid fever infections exclusively in humans. In each invasive lineage, regulatory or structural gene mutations leading to loss or impairment of biofilm were identified, all associated with curli and cellulose production, the two main structures that comprise the biofilm matrix. This suggests that similar evolutionary pressures are acting on invasive Salmonella isolates. Public health strategies aimed at reducing the burden of invasive Salmonella disease must prevent transmission to vulnerable adults and children via water sanitation and hygiene practices–a process that starts with identification of environmental reservoirs. The results of our study will raise the profile of this neglected aspect of invasive salmonellosis and will challenge researchers and clinicians to search in new places for potential environmental reservoirs of these pathogens.

Highlights • High pneumococcal population diversity in terms of serotypes and sequence types (ST). • Decline in IPD incidence pre-vaccination not associated with specific serotypes. • High prevalence and antibiotic resistance rates in serotype 1 isolates. • High levels of capsule (serotype) switching pre-vaccination. • Surveillance remains crucial to understand pneumococcal epidemiology., Introduction Pneumococcal infections cause a high death toll in Sub Saharan Africa (SSA) but the recently rolled out pneumococcal conjugate vaccines (PCV) will reduce the disease burden. To better understand the population impact of these vaccines, comprehensive analysis of large collections of pneumococcal isolates sampled prior to vaccination is required. Here we present a population genomic study of the invasive pneumococcal isolates sampled before the implementation of PCV13 in Malawi. Materials and methods We retrospectively sampled and whole genome sequenced 585 invasive isolates from 2004 to 2010. We determine the pneumococcal population genetic structure and assessed serotype prevalence, antibiotic resistance rates, and the occurrence of serotype switching. Results Population structure analysis revealed 22 genetically distinct sequence clusters (SCs), which consisted of closely related isolates. Serotype 1 (ST217), a vaccine-associated serotype in clade SC2, showed highest prevalence (19.3%), and was associated with the highest MDR rate (81.9%) followed by serotype 12F, a non-vaccine serotype in clade SC10 with an MDR rate of 57.9%. Prevalence of serotypes was stable prior to vaccination although there was an increase in the PMEN19 clone, serotype 5 ST289, in clade SC1 in 2010 suggesting a potential undetected local outbreak. Coalescent analysis revealed recent emergence of the SCs and there was evidence of natural capsule switching in the absence of vaccine induced selection pressure. Furthermore, majority of the highly prevalent capsule-switched isolates were associated with acquisition of vaccine-targeted capsules. Conclusions This study provides descriptions of capsule-switched serotypes and serotypes with potential to cause serotype replacement post-vaccination such as 12F. Continued surveillance is critical to monitor these serotypes and antibiotic resistance in order to design better infection prevention and control measures such as inclusion of emerging replacement serotypes in future conjugate vaccines.

International audience; The application of genomics technologies to medicine and biomedical research is increasing in popularity, made possible by new high-throughput genotyping and sequencing technologies and improved data analysis capabilities. Some of the greatest genetic diversity among humans, animals, plants, and microbiota occurs in Africa, yet genomic research outputs from the continent are limited. The Human Heredity and Health in Africa (H3Africa) initiative was established to drive the development of genomic research for human health in Africa, and through recognition of the critical role of bioinformatics in this process, spurred the establishment of H3ABioNet, a pan-African bioinformatics network for H3Africa. The limitations in bioinformatics capacity on the continent have been a major contributory factor to the lack of notable outputs in high-throughput biology research. Although pockets of high-quality bioinformatics teams have existed previously, the majority of research institutions lack experienced faculty who can train and supervise bioinformatics students. H3ABioNet aims to address this dire need, specifically in the area of human genetics and genomics, but knock-on effects are ensuring this extends to other areas of bioinformatics. Here, we describe the emergence of genomics research and the development of bioinformatics in Africa through H3ABioNet.

Introductory paragraphInfections caused by carbapenemase-producing enterobacteria (CPE) are a major concern in clinical settings worldwide. Two fundamentally different processes shape the epidemiology of CPE in hospitals: the dissemination of CPE clones from patient to patient (between-patient transfer), and the transfer of carbapenemase-encoding plasmids between enterobacteria in the gut microbiota of individual patients (within-patient transfer). The relative contribution of each process to the overall dissemination of carbapenem resistance in hospitals remains poorly understood. Here, we used mechanistic models combining epidemiological data from more than 9,000 patients with whole genome sequence information from 250 enterobacteria clones to characterise the dissemination routes of the carbapenemase-encoding plasmid pOXA-48 in a hospital setting over a two-year period. Our results revealed frequent between-patient transmission of high-risk pOXA-48-carrying clones, mostly of Klebsiella pneumoniae and sporadically Escherichia coli. The results also identified pOXA-48 dissemination hotspots within the hospital, such as specific wards and individual rooms within wards. Using high-resolution plasmid sequence analysis, we uncovered the pervasive within-patient transfer of pOXA-48, suggesting that horizontal plasmid transfer occurs in the gut of virtually every colonised patient. The complex and multifaceted epidemiological scenario exposed by this study provides new insights for the development of intervention strategies to control the in-hospital spread of CPE.

Highlights • Development and evaluation of a novel air-dried HRM assay to detect eight major ESBL and carbapenemase genes. • Sensitivity and specificity of the air-dried HRM assay versus the reference molecular tests was 94.7–98.3% and 98.5–99.2%. • Assay had overall agreement of 91.1% in predicting phenotypic resistance to cefotaxime and meropenem in Enterobacteriaceae. • Cross-platform validation showed almost perfect reproducibility in five different qPCR platforms. • No loss of sensitivity was observed after 8 months of storage at room, refrigerator and oven temperatures., Objectives This study aimed to develop and evaluate a novel air-dried high-resolution melt (HRM) assay to detect eight major extended-spectrum β-lactamase (ESBL) (blaSHV and blaCTX-M groups 1 and 9) and carbapenemase (blaNDM, blaIMP, blaKPC, blaVIM and blaOXA-48-like) genes that confer resistance to cephalosporins and carbapenems. Methods The assay was evaluated using 439 DNA samples extracted from bacterial isolates from Nepal, Malawi and the UK and 390 clinical isolates from Nepal with known antimicrobial susceptibility. Assay reproducibility was evaluated across five different real-time quantitative PCR (qPCR) instruments [Rotor-Gene® Q, QuantStudioTM 5, CFX96, LightCycler® 480 and Magnetic Induction Cycler (Mic)]. Assay stability was also assessed under different storage temperatures (6.2 ± 0.9°C, 20.4 ± 0.7°C and 29.7 ± 1.4°C) at six time points over 8 months. Results The sensitivity and specificity (with 95% confidence intervals) for detecting ESBL and carbapenemase genes was 94.7% (92.5–96.5%) and 99.2% (98.8–99.5%) compared with the reference gel-based PCR and sequencing and 98.3% (97.0–99.3%) and 98.5% (98.0–98.9%) compared with the original HRM wet PCR mix format. Overall agreement was 91.1% (90.0–92.9%) when predicting phenotypic resistance to cefotaxime and meropenem among Enterobacteriaceae isolates. We observed almost perfect inter-machine reproducibility of the air-dried HRM assay, and no loss of sensitivity occurred under all storage conditions and time points. Conclusion We present a ready-to-use air-dried HRM PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and carbapenemase genes in DNA samples to improve antimicrobial resistance detection.