Whole-genome epidemiology links phage-mediated acquisition of a virulence gene to the clonal expansion of a pandemic Salmonella enterica serovar Typhimurium clone

Epidemic and pandemic clones of bacterial pathogens with distinct characteristics continually emerge, replacing those previously dominant through mechanisms that remain poorly characterized. Here, whole-genome-sequencing-powered epidemiology linked horizontal transfer of a virulence gene, sopE, to the emergence and clonal expansion of a new epidemic Salmonella enterica serovar Typhimurium (S. Typhimurium) clone. The sopE gene is sporadically distributed within the genus Salmonella and rare in S. enterica Typhimurium lineages, but was acquired multiple times during clonal expansion of the currently dominant pandemic monophasic S. Typhimurium sequence type (ST) 34 clone. Ancestral state reconstruction and time-scaled phylogenetic analysis indicated that sopE was not present in the common ancestor of the epidemic clade, but later acquisition resulted in increased clonal expansion of sopE-containing clones that was temporally associated with emergence of the epidemic, consistent with increased fitness. The sopE gene was mainly associated with a temperate bacteriophage mTmV, but recombination with other bacteriophage and apparent horizontal gene transfer of the sopE gene cassette resulted in distribution among at least four mobile genetic elements within the monophasic S. enterica Typhimurium ST34 epidemic clade. The mTmV prophage lysogenic transfer to other S. enterica serovars in vitro was limited, but included the common pig-associated S. enterica Derby (S. Derby). This may explain mTmV in S. Derby co-circulating on farms with monophasic S. Typhimurium ST34, highlighting the potential for further transfer of the sopE virulence gene in nature. We conclude that whole-genome epidemiology pinpoints potential drivers of evolutionary and epidemiological dynamics during pathogen emergence, and identifies targets for subsequent research in epidemiology and bacterial pathogenesis

Single-cell genomics reveals population structures from in vitro evolutionary studies of Salmonella

Single-cell DNA sequencing has the potential to reveal detailed hierarchical structures in evolving populations of cells. Single cell approaches are increasingly used to study clonal evolution in human ageing and cancer but have not yet been deployed to study evolving clonal microbial populations. Here, we present an approach for single bacterial genomic analysis for in vitro evolution experiments using FACS isolation of individual bacteria followed by whole-genome amplification and sequencing. We apply this to the experimental evolution of a hypermutator strain of Salmonella in response to antibiotic stress (ciprofloxacin). By analysing sequence polymorphisms in individual cells from populations we identified the presence and prevalence of sub-populations which have acquired polymorphisms in genes previously demonstrated to be associated with ciprofloxacin susceptibility. We were also able to identify that the population exposed to antibiotic stress was able to develop resistance whilst maintaining diversity. This population structure could not be resolved from bulk sequence data, and our results show how high-throughput single-cell sequencing can enhance experimental studies of bacterial evolution

Faecal carriage of ESBL producing and colistin resistant Escherichia coli in avian species over a 2-year period (2017-2019) in Zimbabwe

DATA AVAILABILITY STATEMENT : The data presented in this study are deposited in the NCBI BioProject Number PRJNA 799483 with accession numbers listed in the Supplementary Datasheet 1.INTRODUCTION : Extended spectrum beta-lactamase (ESBL) producing Escherichia coli have become widespread among food producing animals. These strains serve as a reservoir of antibiotic resistance genes (ARGs) and act as a possible source of infection to humans as transmission can occur by direct or indirect contact. METHODS : This study investigated the faecal carriage of ESBL producing and colistin resistant E. coli in poultry over a 2-year period (2017-2019) from Zimbabwe. A total of 21 ESBL positive isolates from poultry cloacal specimens were selected for whole genome sequencing from animal E. coli isolates biobanked at the National Microbiology Reference laboratory using phenotypic susceptibility testing results from the National Escherichia coli Surveillance Program to provide representation of different geographical regions and year of isolation. Cloacal swabs were collected from 3000 broiler live birds from farm 1 and fromfarm2, 40 backyard chickens and 10 duckswere sampled. Antimicrobial susceptibility and ESBL testing were performed as per Clinical Laboratory Standards Institute guidelines. Whole genome sequencing of ESBL producing isolates was used to determine sequence types (STs), ARGs, and phylogroups. RESULTS : Twenty-one of the included E. coli isolates were confirmed as ESBL producers. Three defined sequence type clonal complexes (CCs) were identified (ST10CC, ST155CC and ST23CC), with ST10CC associated with the most antibiotic resistant profile. The ESBL phenotype was linked to the presence of either cefotaximase-Munich-14 (CTX-M-14) or CTX-M-79. Plasmid mediated quinolone resistant determinants identified were qnrB19 and qnrS1 and one ST10CC isolate from farm 1 broiler chickens harbored a mobile colistin resistance gene (mcr-1). Phylogenetic groups most identified were B1, A and unknown. DISCUSSIONS : The avian ESBL producing E. coli belonged to a diverse group of strains. The detection of several ARGs highlights the importance of implementing enhanced control measures to limit the spread in animals, environment, and humans. This is the first report of mcr-1 in Zimbabwe, which further underscores the importance of the One Health approach to control the spread and development of AMR.The National Health Laboratory Service (NHLS) and the University of Pretoria, South Africa, a strategic partnership between National Microbiology Reference Laboratory and Quadram BioSciences Institute.https://www.frontiersin.org/journals/cellular-and-infection-microbiologyam2023Medical Microbiolog

Phytophthora infestans RXLR effectors act in concert at diverse subcellular locations to enhance host colonization

Oomycetes such as the potato blight pathogen Phytophthora infestans deliver RXLR effectors into plant cells to manipulate host processes and promote disease. Knowledge of where they localize inside host cells is important in understanding their function. Fifty-two P. infestans RXLR effectors (PiRXLRs) up-regulated during early stages of infection were expressed as fluorescent protein (FP) fusions inside cells of the model host Nicotiana benthamiana. FP-PiRXLR fusions were predominantly nucleo-cytoplasmic, nuclear, or plasma membrane-associated. Some also localized to the endoplasmic reticulum, mitochondria, peroxisomes, or microtubules, suggesting diverse sites of subcellular activity. Seven of the 25 PiRXLRs examined during infection accumulated at sites of haustorium penetration, probably due to co-localization with host target processes; Pi16663 (Avr1), for example, localized to Sec5-associated mobile bodies which showed perihaustorial accumulation. Forty-five FP-RXLR fusions enhanced pathogen leaf colonization when expressed in Nicotiana benthamiana, revealing that their presence was beneficial to infection. Co-expression of PiRXLRs that target and suppress different immune pathways resulted in an additive enhancement of colonization, indicating the potential to study effector combinations using transient expression assays. We provide a broad platform of high confidence P. infestans effector candidates from which to investigate the mechanisms, singly and in combination, by which this pathogen causes disease.</p

Genomic epidemiology and the role of international and regional travel in the SARS-CoV-2 epidemic in Zimbabwe: a retrospective study of routinely collected surveillance data.

BACKGROUND: Advances in SARS-CoV-2 sequencing have enabled identification of new variants, tracking of its evolution, and monitoring of its spread. We aimed to use whole genome sequencing to describe the molecular epidemiology of the SARS-CoV-2 outbreak and to inform the implementation of effective public health interventions for control in Zimbabwe. METHODS: We performed a retrospective study of nasopharyngeal samples collected from nine laboratories in Zimbabwe between March 20 and Oct 16, 2020. Samples were taken as a result of quarantine procedures for international arrivals or to test for infection in people who were symptomatic or close contacts of positive cases. Samples that had a cycle threshold of less than 30 in the diagnostic PCR test were processed for sequencing. We began our analysis in July, 2020 (120 days since the first case), with a follow-up in October, 2020 (at 210 days since the first case). The phylogenetic relationship of the genome sequences within Zimbabwe and global samples was established using maximum likelihood and Bayesian methods. FINDINGS: Of 92 299 nasopharyngeal samples collected during the study period, 8099 were PCR-positive and 328 were available for sequencing, with 156 passing sequence quality control. 83 (53%) of 156 were from female participants. At least 26 independent introductions of SARS-CoV-2 into Zimbabwe in the first 210 days were associated with 12 global lineages. 151 (97%) of 156 had the Asp614Gly mutation in the spike protein. Most cases, 93 (60%), were imported from outside Zimbabwe. Community transmission was reported 6 days after the onset of the outbreak. INTERPRETATION: Initial public health interventions delayed onset of SARS-CoV-2 community transmission after the introduction of the virus from international and regional migration in Zimbabwe. Global whole genome sequence data are essential to reveal major routes of spread and guide intervention strategies. FUNDING: WHO, Africa CDC, Biotechnology and Biological Sciences Research Council, Medical Research Council, National Institute for Health Research, and Genome Research Limited.WHO, Africa CDC, Biotechnology and Biological Sciences Research Council, Medical Research Council, National Institute for Health Research, and Genome Research Limite

A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa.

The progression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in Africa has so far been heterogeneous, and the full impact is not yet well understood. In this study, we describe the genomic epidemiology using a dataset of 8746 genomes from 33 African countries and two overseas territories. We show that the epidemics in most countries were initiated by importations predominantly from Europe, which diminished after the early introduction of international travel restrictions. As the pandemic progressed, ongoing transmission in many countries and increasing mobility led to the emergence and spread within the continent of many variants of concern and interest, such as B.1.351, B.1.525, A.23.1, and C.1.1. Although distorted by low sampling numbers and blind spots, the findings highlight that Africa must not be left behind in the global pandemic response, otherwise it could become a source for new variants

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Molecular epidemiology of extended-spectrum beta-lactamase–producing extra-intestinal pathogenic Escherichia coli strains over a 2-year period (2017–2019) from Zimbabwe

This study was designed to characterize extended-spectrum beta-lactamase (ESBL)–producing extra-intestinal pathogenic Escherichia coli (E.coli) (ExPEC) associated with urinary tract infections in nine different geographic regions of Zimbabwe over a 2-year period (2017–2019). A total of 48 ESBL-positive isolates from urine specimen were selected for whole-genome sequencing from 1246 Escherichia coli isolates biobanked at the National Microbiology Reference laboratory using phenotypic susceptibility testing results from the National Escherichia coli Surveillance Programme to provide representation of different geographical regions and year of isolation. The majority of ESBL E. coli isolates produced cefotaximase-Munich (CTX-M)-15, CTX-M-27, and CTX-M-14. In this study, sequence types (ST) 131 and ST410 were the most predominant antimicrobial-resistant clones and responsible for the increase in ESBL–producing E. coli strains since 2017. Novel ST131 complex strains were recorded during the period 2017 to 2018, thus showing the establishment and evolution of this antimicrobial-resistant ESBL clone in Zimbabwe posing an important public health threat. Incompatibility group F plasmids were predominant among ST131 and ST410 isolates with the following replicons recorded most frequently: F1:A2:B20 (9/19, 47%), F2:A1: B (5/19, 26%), and F1:A1:B49 (8/13, 62%). The results indicate the need for continuous tracking of different ESBL ExPEC clones on a global scale, while targeting specific STs (e.g. ST131 and ST410) through control programs will substantially decrease the spread of ESBLs among ExPEC.The National Health Laboratory Service (NHLS), the University of Pretoria, South Africa, and a strategic partnership between National Microbiology Reference Laboratory and Quadrum Institute Biosciences.http://link.springer.com/journal/100962022-11-15hj2022Medical Microbiolog

Salmonella enterica serovar Typhi H58 clone has been endemic in Zimbabwe from 2012 to 2019

Background: Typhoid fever, caused by S. enterica ser. Typhi, continues to be a substantial health burden in developing countries. Little is known of the genotypic diversity of S. enterica ser. Typhi in Zimbabwe, but this is key for understanding the emergence and spread of this pathogen and devising interventions for its control. Objectives: To report the molecular epidemiology of S. enterica ser. Typhi outbreak strains circulating from 2012 to 2019 in Zimbabwe, using comparative genomics. Methods: A review of typhoid cases records from 2012 to 2019 in Zimbabwe was performed. The phylogenetic relationship of outbreak isolates from 2012 to 2019 and emergence of antibiotic resistance was investigated by whole-genome sequence analysis. Results: A total 22 479 suspected typhoid cases, 760 confirmed cases were reported from 2012 to 2019 and 29 isolates were sequenced. The majority of the sequenced isolates were predicted to confer resistance to aminoglycosides, β-lactams, phenicols, sulphonamides, tetracycline and fluoroquinolones (including qnrS detection). The qnrS1 gene was associated with an IncN (subtype PST3) plasmid in 79% of the isolates. Whole-genome SNP analysis, SNP-based haplotyping and resistance determinant analysis showed that 93% of the isolates belonged to a single clade represented by multidrug-resistant H58 lineage I (4.3.1.1), with a maximum pair-wise distance of 22 SNPs. Conclusions: This study has provided detailed genotypic characterization of the outbreak strain, identified as S. Typhi 4.3.1.1 (H58). The strain has reduced susceptibility to ciprofloxacin due to qnrS carried by an IncN (subtype PST3) plasmid resulting from ongoing evolution to full resistance