Biology subjects

Okeke, I. N.

Publications and source records attributed to Okeke, I. N..

At least 19 recordsLinked to original sources

bioRxiv2026

Enteroaggregative Escherichia clade I from Nigeria

Escherichia coli, the Escherichia type species, is present in mammalian and avian intestinal microbiota, and includes both commensals and pathogens. Other Escherichia species are understudied because they are less commonly associated with human disease and because of paucity of tools that can correctly delineate them from E. coli. However, other species of this genus including Escherichia albertii and Escherichia fergusonii are repeatedly reported as diarrhoeagenic. We hypothesized that some bacteria fitting the definition of enteroaggregative E. coli (EAEC) belong to species other than E. coli. We used phylogeny to determine the species of 2,818 Escherichia genomes from diarrhoea epidemiology studies in Nigeria. Phylogeny speciation was confirmed using GTDB-tk and ClermonTyping. Virulence genes were detected using ARIBA/Virulencefinder database and multilocus sequence typing performed using the Achtman scheme. Fourteen non-coli Escherichia genomes were identified-- Escherichia clade I ST485 (11), Escherichia ruysiae ST5792 (2) and Escherichia fergusonii ST5636 (1). All the Escherichia clade I ST485 carry EAEC virulence genes aap, aar, astA and air, as well as hlyF, eatA, tsh, traT, and chuA virulence genes. Interestingly, 62% of enteroaggregative Escherichia clade I ST485 genomes listed on Enterobase are from Africa isolates, despite only 3% of genomes overall coming from the continent. Our results suggest that non-coli Escherichia species are infrequently isolated from human stool, but, when they are, they are misidentified as E. coli so that their significance is largely overlooked. Escherichia clade I ST485 is a globally disseminated enteroaggregative Escherichia clade I lineage that is common in Africa. Author SummaryEscherichia clade I are rarely associated with disease and because of the difficulty in differentiating them from Escherichia coli in routine laboratory, they are often misidentified as Escherichia coli leading to the underestimation of their impact on the burden of disease. Additionally, some clones of Escherichia clade I also carry genetic markers that have been used to define Enteroaggregative Escherichia coli (EAEC), a cause of persistent diarrhoea in developing countries and travellers diarrhoea in developed economies. EAEC has also been associated with malnutrition and poor growth among children in developing economies. We here describe clones of Escherichia clade I (ST485) that carries enteroaggregative genes and in some cases, recovered from diarrhoeal cases. We show from genomes deposited on Enterobase and our study, that this clone is globally disseminated, often associated with human infections and often misidentified as Escherichia coli. We also describe other non-coli Escherichia other than Escherichia clade I isolated from humans. We suggest that the Escherichia clade I clone carrying enteroaggregative genes may be described as Enteroaggregative Escherichia clade I.

microbiology↗

bioRxiv2026

Refining the Serine Protease Autotransporters of Enterobacteriaceae (SPATE) gene detection in Enteroaggregative Escherichia coli genomes uncovers differential SPATE distribution by phylogeny

BackgroundEnteroaggregative Escherichia coli (EAEC) are a heterogenous pathotype, implicated in acute and persistent diarrhoea especially in developing countries. Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) are Type V Secretory System trypsin-like proteases repeatedly reported from EAEC. This study aimed to determine SPATE encoding-gene prevalence among EAEC and their association with diarrhoea. We screened 881 EAEC genomes from four recent epidemiological studies in Nigeria for 23 SPATE-encoding genes, initially using ARIBA and the Virulencefinder database. ResultsInitial screening inflated SPATE gene content, particularly in genomes with multiple SPATEs, due to cross detection of highly similar sequences and other artefacts. We developed and validated refined methodology, which detected 478 of 1,156 original SPATE calls and also identified SPATE miscalls from previous datasets in the literature. The most prevalent SPATE-encoding gene in our EAEC collection was sepA 297(33.71%), closely followed by sat 360 (29.74%). pic, encoding a SPATE with mucinase activity, was found in 65 (7.4%) genomes and associated with diarrhoea (p=0.00004). EAEC strains belonging to E. coli phylogroups A, B1 or C carried, on average, one SPATE gene per genome while >1 was typically detected in phylogroup B2 EAEC. Other EAEC carried few or no SPATE genes. ConclusionsOur study shows that multifunctional genome analysis tools may have to be refined for certain gene families to avoid overestimation. SPATEs are not as prevalent as previously thought but they remain common among EAEC, particularly among phylogroup A, B1, B2 and C, pointing to the possibility that they make lineage-specific contributions to disease.

microbiology↗

bioRxiv2026

Mobile element-mediated carbapenem resistance in Enterobacter hormaechei in a Nigerian intensive care unit

Carbapenem-resistant Gram-negative bacteria pose a critical public health threat. The role of mobile genetic elements in driving their transmission and persistence remains poorly defined. In 2022, we investigated a suspected outbreak of carbapenem-resistant Acinetobacter baumannii (CRAB) in a Nigerian adult intensive care unit (ICU), using short-read whole genome sequencing (WGS) of carbapenem-resistant clinical and environmental isolates during the cluster period. Mobile element dynamics were then inferred from hybrid assemblies of Illumina and Oxford Nanopore reads. The suspected CRAB outbreak was ruled out by WGS but a carbapenem-resistant Enterobacter hormaechei ST114 bloodstream isolate was found to be indistinguishable from two environmental isolates, all recovered during the Acinetobacter surge. Hybrid assemblies revealed a strikingly conserved [~]19 Kb resistance island shared across all ST114 genomes. The island contained a blaNDM-5 cassette alongside many other antimicrobial resistance genes, within class 1 integronns and flanked by insertions sequences, located on a 46,176 bp plasmid. Using the ST114 plasmids hybrid assembly as scaffold, the same plasmid was identified in the genome of a Klebsiella pneumoniae ST15 isolate from the ICU environment during the same period. Additionally, re-interrogation of genomic surveillance data uncovered four clonal 2020 ST109 Enterobacter bloodstream isolates from the same facility that carried the resistance genes in the same context on a large 267,242 bp plasmid. Carbapenem resistance in hospital Enterobacterales is driven by both clonal expansion and horizontal spread of mobile resistance elements. These findings underscore the need to track mobile elements alongside bacterial lineages to inform evidence-based infection control, especially in low-resource settings. Impact StatementCarbapenem resistance among Enterobacterales remains a major public health threat, yet how mobile genetic elements contribute to their persistence and spread in hospital settings is still poorly understood. In this study, we investigated a suspected outbreak of carbapenem-resistant Acinetobacter baumannii in an adult intensive care unit in Nigeria. Although the outbreak was eventually ruled out, genomic analysis has shown the importance of careful interpretation of suspected outbreak cases in hospital settings. Our findings highlight the importance of close monitoring of ICU environments, the implementation of blood culture-based diagnostics, and the value of genomic support in outbreak investigations. These findings demonstrate that carbapenem resistance in hospital Enterobacterales is driven not only by clonal expansion but also by the horizontal dissemination of a highly stable blaNDM-5-associated MDR island capable of integrating into diverse plasmid backbones. This study emphasizes the need for genomic surveillance that tracks both mobile elements and bacterial lineages to strengthen outbreak investigations, especially in low-resource settings. It further underscores the links between clinical and environmental AMR reservoirs and reinforces the value of a One Health approach to controlling carbapenem resistance. Data summaryFASTQ sequences were deposited in the NCBI BioSample database under accession numbers SAMN55915584 - SAMN55915597.

microbiology↗

bioRxiv2026

Identification of Antibiofilm Agents against Salmonella enterica from the Pathogen Box Compound Library

BackgroundBiofilms are central to Salmonella pathogenesis, and targeting their formation is believed to produce less evolutionary pressure of growth inhibition than traditional antibacterials. In this study, we screened the Medicines for Malaria Venture (MMV) Pathogen Box library to identify anti-biofilm agents against S. enterica that possess drug-like properties. Methodology/ Principal FindingsA crystal-violet-based medium-throughput antibiofilm screen of Salmonella enterica serovar Typhimurium ATCC 14028 and a clinical Salmonella enterica serovar Elisabethville isolate was performed on polystyrene surfaces using the 400-compound Pathogen Box library. Compounds that inhibited biofilm formation by >30% and growth by <10% were identified as hits. Salmonella red-dry-rough and motility phenotypes were explored in mechanism of action studies on one hit compound. The Salmonella antibiofilm hit rate was 0.75% for this library. MMV688371 (benzamide) inhibited biofilm formation of S. Typhimurium ATCC 14028 by 33% without inhibiting growth. An ethambutol analogue (MMV687273) and auranofin (MMV688978) met the hit criteria against S. Elisabethville LLD035X. Auranofin showed concentration-dependent, growth-inhibition-independent antibiofilm activity against typhoidal and non-typhoidal Salmonella from Nigeria, and inhibited the motility of S. Elisabethville LLD035X at 5 {micro}M. At 5 {micro}M aurothioglucose, an auranofin gold (I) analogue, and non-gold analogue 1-Thio-beta-D-glucose tetraacetate, inhibited biofilm formation by 61.30% and 11.39%, respectively, pointing to essentiality of the gold (I) moiety for activity. Conclusions/ SignificanceStructurally diverse small molecules can inhibit biofilm formation by Salmonella, and motility inhibition is an important mechanism for this activity. Auranofin inhibits typhoidal and non-typhoidal Salmonella biofilm formation, with its gold content being required for these activities.

microbiology↗

bioRxiv2025

Genomic analysis of extended-spectrum beta-lactamase-producing Enterobacter kobei ST691 strain harboring mcr-10.1 isolated in Yaounde, Cameroon.

ObjectiveEnterobacter species. are opportunistic pathogens commonly responsible for serious, difficult-to-treat hospital-acquired infections. Extended-spectrum {beta}-lactamase (ESBL)-producing and colistin-resistant Enterobacterales are increasingly implicated in human and animal infections worldwide. Here we report a first detection of colistin-resistant ESBL-producing E. kobei strain belonging to ST691 and harbouring mcr-10.1. MethodsThis strain was isolated from the faecal sample of a two-year old child aged, who was diagnosed with gastroenteritis in Yaounde, Cameroon. This ESBL-E. kobei ST691 genome was sequenced using Illumina Miseq (Illumina, San Diego, CA, USA). Mobile genetic elements and antibiotic resistance genes were predicted using AMRFinderPlus, ARIBA and the VFDB and PlasmidFinder databases respectively. ResultsThis strain exhibited phenotypic resistance to numerous antibiotics belonging to penicillin, third generation of cephalosporin and carbapenem families. However, it was susceptible to aminoglycoside and fluoroquinolone. Genome analysis reveals a length of 4 626 300 bp, and N50 of 143 731, GC content of 54.9%. Genes conferring resistance to {beta}-lactams (blaACT-9), polymyxin (mcr-10.1) and phenicol/quinolone (oqxA,B) were detected. Mobile genetic elements including plasmid replicon type [IncFIB(pECLA) and IncFII(pECLA)] and the IS1, IS110, ISEc34, IS1222SC, IS66, IS630, IS3, IS26 insertion sequences were also detected. ConclusionWe report the first colistin-resistant ESBL-producing E. kobei isolated from a two-year old child. It is a high priority potential pathogen showing resistance to last-resort antimicrobials to which there is little access in Cameroon. This underscores the necessity to strengthen genomic surveillance, antimicrobial stewardship and infection prevention and control as well as to heighten awareness of the threat posed by resistant bacteria.

genomics↗

bioRxiv2025

amr.watch - monitoring antimicrobial resistance trends from global genomics data

BackgroundWhole genome sequencing (WGS) is increasingly supporting routine pathogen surveillance at local and national levels, providing comparable data that can inform on the emergence and spread of antimicrobial resistance (AMR) globally. However, the potential for shared WGS data to guide interventions around AMR remains under-exploited, in part due to challenges in collating and transforming the growing volumes of data into timely insights. We present an interactive platform, amr.watch (https://amr.watch), that enables interrogation of AMR trends from public WGS data on an ongoing basis to support research and policy. MethodsThe amr.watch platform incorporates, analyses and visualises high-quality WGS data from WHO-defined priority bacterial pathogens. Analytics are performed using community-standard methods with bespoke species-specific curation of AMR mechanisms. FindingsBy 31 March 2025, amr.watch included data from 620,700 pathogen genomes with geotemporal information, with highly variable representation of different species and geographic regions. By integrating WGS data with sampling information, amr.watch enables users to assess geotemporal trends among genotypic variants (e.g. sequence types) and AMR mechanisms, with implications for interventions including antimicrobial prescribing and drug and vaccine development. Interpretationamr.watch is an information platform for scientists and policy-makers delivering ongoing situational awareness of AMR trends from genomic data. As broad adoption of WGS continues, amr.watch is positioned to monitor both pathogen populations and our global efforts in genomic surveillance, guiding control strategies tailored to each pathogens characteristics. FundingUK National Institute for Health Research & Gates Foundation. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWhole genome sequencing (WGS) approaches enable us to track the spread of bacterial pathogens and antimicrobial resistance (AMR) with high resolution at local, national and global levels. To date, genomic studies assessing AMR dynamics have largely used retrospective data collected for specific research agendas. However, the growing volumes of publicly-shared WGS data, generated increasingly from routine surveillance, provide improved power to detect novel trends and guide interventions. Efforts to collate public bacterial genome data exist, such as AllTheBacteria, although these are aimed at the research community and do not facilitate data usage and interpretation by non-genomics experts, particularly those in public health. To our knowledge, no platforms exist for readily interrogating AMR dynamics from continuously updated public genome data across the array of different WHO priority bacterial pathogens. Of note, however, TyphiNet provides an interactive online dashboard for examining AMR trends exclusively from Salmonella Typhi using a periodically-updated data set from the Pathogenwatch platform. These findings are based on searching PubMed without language restrictions from Jan 1 2000 to December 31 2024, using terms related to "genomic surveillance" and "antimicrobial resistance". Added value of this studyWe have developed amr.watch which, to our knowledge, is the first platform that enables ongoing analysis and visualisation of AMR trends from public genome data across the spectrum of WHO priority bacterial pathogens via an accessible interface. Crucially, the platform incorporates processed genome data via a live always-on stream, enabling insights that are delayed only by the time to data deposition. We reviewed public genomes with available geotemporal information up until 31 March 2025, providing a contemporary global landscape of pathogen genome sequencing. While the number of genomes available annually grew over five-fold globally between 2010 and 2018, we also revealed the extent of differences in geographic representation, with 89.6% of genomes originating from high-income countries and 89 countries contributing no genome data from the priority pathogens. Implications of all the available evidenceRapid generation and sharing of global genome data enables us to more precisely track the spread and define the characteristics of contemporary circulating resistant pathogens. The amr.watch platform provides a solution for retrieving, curating and translating shared genomic data into relevant insights that are accessible and actionable by diverse stakeholders. It thereby forms a basis for monitoring progress in surveillance efforts, alerting on ongoing population changes and guiding enhanced precision for surveillance and interventional development. Additionally, our review of available bacterial genome data highlights the need for additional efforts to increase and sustain the implementation of genomic surveillance of AMR globally, and improve the timely sharing of WGS data and its associated metadata.

genomics↗

bioRxiv2025

Genomic epidemiology of enteropathogenic Escherichia coli (EPEC) in southwestern Nigeria

BackgroundEnteropathogenic Escherichia coli (EPEC) are etiological agents of diarrhea. We studied the genetic diversity and virulence factors of EPEC in southwestern Nigeria, where this pathotype is rarely characterized. Methodology/ Principal findingsEPEC isolates (n=96) recovered from recent southwestern Nigeria diarrhea case-control studies were whole genome-sequenced using Illumina technology. Genomes were assembled using SPAdes and quality was evaluated using QUAST. Virulencefinder, SeroTypefinder, and Resfinder were used to identify virulence genes, serotypes, and resistance genes, while multilocus sequence typing was done by STtyping. Single nucleotide polymorphisms (SNPs) were called out of whole genome alignment using SNP-sites and a phylogenetic tree was constructed using IQtree. Thirty-nine of the 96 (40.6%) EPEC isolates were from cases of diarrhea. Nine isolates from diarrhea patients and four from healthy controls were typical EPEC, harboring bundle-forming pilus (bfp) genes whilst the rest were atypical EPEC. Fifteen isolates were EPEC-EAEC hybrids. Atypical serotypes O71:H19 (16, 16.6%), O108:H21 (6, 6.3%), O157:H39 (5, 5.2%), and O165:H9 (4, 4.2%) were most prevalent; only 8(8.3%) isolates belonged to classical EPEC serovars. The largest clade comprised ST517 isolates, of O71:H19 and O165:H9 serovars harboring multiple siderophore and serine protease autotransporter genes. An O71:H19 subclade comprised isolates <10 SNPs apart, representing a likely outbreak involving 15 children, four presenting with diarrhea. Conclusion/ SignificanceLikely outbreaks, of typical O119:H6(ST28) and atypical O127:H29(ST7798) were additionally identified. EPEC circulating in southwestern Nigeria are diverse and differ substantially from well-characterized lineages seen previously elsewhere. EPEC carriage and outbreaks could be commonplace but are largely undetected, hence, unreported, and require genomic surveillance for identification.

microbiology↗

bioRxiv2025

Transmissible antimicrobial resistance in Escherichia coli isolated from household drinking water in Ibadan, Nigeria

Contaminated household water in peri-urban urban communities is a reservoir for virulent Escherichia coli, but its role in the environmental transmission of antibiotic resistance genes (ARGs) remains poorly understood. This study characterized E. coli from household water and additionally aimed to investigate the transmissibility of ARGs from drug-resistant isolates. Twenty-five E. coli from thirteen household well water sources were tested for resistance to 14 antibiotics by disc diffusion and whole-genome sequenced using the Illumina platform. The ARGs and plasmid replicon types were respectively predicted using ResFinder and PlasmidFinder. Multidrug-resistant strains carrying plasmid replicons found in unrelated strains were conjugated with nalidixic acid-resistant (NALR) E. coli C600 using the solid plate method. Fifteen isolates displayed a multi-drug resistance (MDR) phenotype with 18 possessing ARGs that confer resistance to trimethoprim-sulfamethoxazole, macrolide, sulphonamide, aminoglycoside, chloramphenicol, {beta}-lactams, and tetracycline. Fifteen of the 25 isolates belonged to sequence type detected more than once, and fourteen of these were multidrug resistant. Through solid plate mating, beta-lactam-resistant qnrS1-tet-dfrA14-positive strains bearing IncFI-, IncHI2, and IncHI2A successfully transferred ampicillin resistance to a nalidixic acid-resistant derivative of E. coli-C600. This research highlights the urgent need to safeguard household water sources against fecal contamination to curb the dissemination of ARGs among bacterial populations in this environment.

microbiology↗

bioRxiv2024

Slow-growing Salmonella enterica Typhi mis-identified as Salmonella Gallinarum in Ibadan, Nigeria

Salmonella enterica serovar Typhi is endemic in Nigeria where S. Gallinarum is rarely reported. Probable S. Gallinarum was blood-cultured from three Ibadan patients within 10-days leading to suspicion of an outbreak. All three isolates were re-identified using VITEK-2, whole genome sequenced on Illumina and Oxford Nanopore platforms and confirmed as S. Typhi genotype 3.1.1. Two isolates from the same household had no single nucleotide polymorphisms, suggesting a point-source, but the third was an independent infection event. The isolates formed small colonies, were negative for motility by hanging drop method, and in Sulphide-Indole-Motility medium at 24h, but positive after 48h incubation. Hybrid genome assemblies revealed chromosomal fragment arrangements with imbalances on either side of ori and ter, which has been shown to slow S. Typhi growth. Suspected S. Gallinarum isolates in typhoid endemic areas should be evaluated biochemically and for motility after extended incubation, and verified by serological or molecular methods. Main contributions of the researchO_LIThis study demonstrates that slow-growing Salmonella Typhi can be mis-identified as S. Gallinarum in resource-limited endemic countries. C_LIO_LIExtended motility testing and confirmatory testing can avoid S. Gallinarum miss-calls. C_LIO_LIThe genetic basis for slow-growth may be chromosomal rearrangements at rrn operons, which are known to occur in S. Typhi C_LI

microbiology↗

bioRxiv2024

Genetic basis for antimicrobial resistance in Escherichia coli isolated from household water in municipal Ibadan, Nigeria

Escherichia coli serves as an indicator of recent faecal contamination in water, signaling the potential presence of enteric pathogens. The public health impact of E. coli in water becomes more significant when strains harbor virulence genes, and may themselves be pathogenic, or antimicrobial resistance genes that can be transferred to pathogens. In this study, we used whole genome sequencing (WGS) to characterize E. coli isolated from household water in municipal Ibadan, Nigeria across two seasons. Antimicrobial susceptibility testing was performed on 97 E. coli isolates, and their genomes were assembled using SPAdes. Multi-Locus Sequence Types (MLST), virulence genes and plasmid replicons were determined using ABRicate. Antimicrobial resistance genes (ARGs) were detected using AMRFinderplus. Phylogroups and serotypes were determined using ClermonTyper and ECTyper, respectively. A phylogenetic tree was built using RAxML. Of the 97 isolates, 39(40.2%) were multidrug resistant and 13(15.9%) possessed diarrheagenic E. coli (DEC) virulence genes. Resistance to individual antibiotics was higher and DEC characteristics more frequent among isolates recovered in the dry season compared to the wet season. Thirty-seven resistance genes belonging to nine antibiotic classes were detected. Majority of the isolates belonged to phylogroup A or B1, 35unique Sequence Types (STs) were detected and there were seven expanded clones of four or more isolates. This study determined that multidrug-resistant E. coli, including DEC, were recovered from household water sources in Ibadan. Some isolates were likely derived from point-sources, highlighting the importance of improved water quality management and sanitation in preventing waterborne disease and antimicrobial resistance transmission. IMPORTANCEContamination of household drinking water sources by disease-causing microorganisms is a serious public health concern common in African settings. Escherichia coli, an indicator of faecal contamination, can also be a reservoir for resistance genes. We have previously reported high frequencies of E. coli contamination of household water in municipal Ibadan. In this study we characterized antimicrobial resistance and virulence genes harboured by contaminating isolates. We found potential diarrhoea-causing E. coli in water which often carried antimicrobial resistance genes, irrespective of whether or not they were disease causing. Resistance gene carriage was more common among isolates recovered in the dry, as compared to the wet season. This was attributable to resistant lineages of E. coli bacteria spreading in the dry season. The work shows the importance of monitoring drinking water in urban African cities like Ibadan and that treating ground water sources may be necessary, particularly in the dry season.

microbiology↗

bioRxiv2024

Whole genome Sequencing Reveals Enterobacter hormaechei as a key Bloodstream Pathogen in six tertiary care hospitals in southwestern Nigeria.

Enterobacter spp. are an important cause of healthcare-associated bloodstream infections uncommonly reported in Africa. This study used whole genome sequencing (WGS) to characterise Enterobacter spp. from hospitals in Nigerias antimicrobial resistance (AMR) surveillance system. Blood-culture isolates of Enterobacter from six such tertiary-care hospitals recovered between 2014 and 2020 were re-identified and antimicrobial susceptibility-tested using VITEK2. Illumina technology provided whole genome sequences for genome nomenclature, antimicrobial resistance gene prediction, Single Nucleotide Polymorphism (SNP) phylogeny, and multi-locus sequence typing via publicly available bioinformatics pipelines. Initial biochemical delineation often misclassified Enterobacter, necessitating whole-genome sequencing for accurate classification. Among 98 Enterobacter received, Enterobacter hormaechei subspecies xiangfangensis predominated (43), followed by other E. hormachei subspecies (18), E. cloacae (26), E. roggenkampii (4), E. bugandensis (3), E. kobei (2), E. asburiae (1) and E. cancerogenous (1). Cephalosporins, aminoglycoside, chloramphenicol, macrolide, and carbapenem resistance in E. hormaechei was attributed to known resistance genes. They belonged to clusters III, IV, and VIII based on hsp60 typing and clades A, B, C, and D according to Sutton and Cos nomenclature. These isolates and other Enterobacter species recently reported from Nigeria reveal extensive E. hormaechei diversity, as well as clusters representing potential outbreaks. Enterobacter hormaechei, often misidentified and rarely reported from Nigeria, is this studys most common blood culture isolated Enterobacter spp. Uncovering underappreciated species as important bloodstream pathogens and retrospective detection of likely outbreaks emphasise the value of genomic surveillance in resource-limited settings. DATA SUMMARYAll sequence reads were submitted to the European Nucleotide Archive (ENA) under the project ID PRJEB29739 (https://www.ebi.ac.uk/ena/browser/view/PRJEB29739). Accessions can be found in Table S1. IMPACT STATEMENTAccurate identification of Enterobacter is essential in healthcare settings as misidentification can lead to selecting antimicrobials to the genus is intrinsically resistant resistant before susceptibility testing results are available. Also, misidentification can compromise microbiology support for infection prevention and control. We show that E. hormaechei, which is never reported from clinical laboratories in Nigeria, is frequently misidentified using conventional methods like tube- or strip biochemical testing and VITEK systems. Whole genome sequence data demonstrates that E. hormaechei and E. cloacae are the most common Enterobacter isolated from bloodstream infections in Nigeria. Enhanced identification methods for surveillance play pivotal roles in improving patient care, optimising antibiotic stewardship, and combating the evolving challenges posed by this pathogen. Overall, this study reveals the effectiveness of WGS in correctly identifying this important pathogen.

microbiology↗

bioRxiv2024

Genomic Analysis of Salmonella enterica from cattle, beef and humans in the Greater Tamale Metropolis of Ghana

Salmonella enterica is a bacterial foodborne pathogen notorious for infecting humans and animals. Proper control of Salmonella requires routine surveillance and interventions across the food-production chain. However, due to limited resources the dynamics and transmission of non-typhoidal Salmonella serotypes remain poorly understood in several African settings, including within Ghana. Here, we employed bacterial culture and whole genome sequencing (WGS) to investigate the prevalence, virulence and antimicrobial resistance determinants of Salmonella enterica isolates from beef, cattle blood and human patient stool in Greater Tamale Metropolis, Ghana. Enrichment and culture of the specimens yielded 62 isolates in total from beef (31), bovine blood (28) and human diarrhoeal specimens (3). We identified at least 15 STs and 18 different Salmonella serovars. The most common serovars detected were Poona (n=13), Montevideo (n=10) and Poano (n=7) with S. Montevideo being the most common from cattle blood. Thirty-two isolates belonged to novel sequence types (STs), with ST2609 (n=9) being most common. Four raw beef isolates harboured at least one gene conferring resistance to beta-lactam (blaTEM-1), chloramphenicol (catA), fosfomycin (fosA7), quinolone (qnrD1) or tetracycline (tet(A)). Eight isolates carried at an IncF, IncI and/orCol3M plasmid replicon. This study recovered Salmonella, often belonging to previously undocumented STs, at high frequencies from cattle and beef and demonstrated that isolates from human diarrhoeal patients are closely related to bovine isolates. The data highlight the need for broader and sustained surveillance and the urgent need for food safety interventions in Ghana.

microbiology↗

bioRxiv2024

Typhi Mykrobe: fast and accurate lineage identification and antimicrobial resistance genotyping directly from sequence reads for the typhoid fever agent Salmonella Typhi

BackgroundTyphoid fever results from systemic infection with Salmonella enterica serovar Typhi (Typhi) and causes 10 million illnesses annually. Disease control relies on prevention (water, sanitation, and hygiene interventions or vaccination) and effective antimicrobial treatment. Antimicrobial resistant (AMR) Typhi lineages have emerged and become established in many parts of the world. Knowledge of local pathogen populations informed by genomic surveillance, including of lineages (defined by the GenoTyphi scheme) and AMR determinants, is increasingly used to inform local treatment guidelines and to inform vaccination strategy. Current tools for genotyping Typhi require multiple read alignment or assembly steps and have not been validated for analysis of data generated with Oxford Nanopore Technologies (ONT) long-read sequencing devices. Here, we introduce Typhi Mykrobe, a command line software tool for rapid genotyping of Typhi lineages, AMR determinants, and plasmid replicons direct from sequencing reads. ResultsWe validated Typhi Mykrobe lineage genotyping by comparison with the current standard read mapping-based approach and demonstrated 99.8% concordance across nearly 13,000 genomes sequenced with Illumina platforms. For the few isolates with discordant calls, we show that Typhi Mykrobe results are better supported by the evidence from raw sequence read data than the results generated using the mapping-based approach. We also demonstrate 99.9% concordance for detection of AMR determinants compared with the current standard assembly-based approach, with similar results for plasmid marker detection. Typhi Mykrobe predicts clinical resistance categorisation (S/I/R) for eight drug classes, and we show strong agreement with phenotypic categorisations generated from reference laboratory minimum inhibitory concentration (MIC) data for n=1,572 Illumina-sequenced isolates (>99% agreement within one doubling dilution). We show strong concordance (>96% for genotype and >98% for AMR and plasmid) between calls made from ONT reads and those made from Illumina reads for isolates sequenced on both platforms (n =93 genomes). Typhi Mykrobe takes less than a minute per sample and is available at https://github.com/typhoidgenomics/genotyphi. ConclusionsTyphi Mykrobe provides rapid and sensitive genotyping of Typhi genomes direct from Illumina and ONT reads, although lower accuracy was observed for R9 ONT data. It demonstrated accurate assignment of GenoTyphi lineage, detection of AMR determinants and prediction of corresponding AMR phenotypes, and identification of plasmid replicons.

microbiology↗

bioRxiv2024

Genomic characterization of foodborne Salmonella enterica and Escherichia coli isolates from Saboba district and Bolgatanga Municipality Ghana

Salmonella enterica and Escherichia coli are well-known bacteria commonly associated with foodborne illnesses in humans and animals. Genomic characterization of these pathogens provides valuable insights into their evolution, virulence factors and resistance determinants. This study aimed to characterized previously isolated Salmonella (n = 14) and E. coli (n = 19) from milk, meat and its associated utensils in Ghana using whole-genome sequencing. Most of the Salmonella serovars (Fresno, Plymouth, Infantis, Give and Orleans) identified in this study are yet to be reported in Ghana. Most Salmonella isolates were pan-sensitive, but genes conferring resistance to fosfomycin (fosA7.2) and tetracycline (tet(A)) were detected in one and three isolates, respectively. Seven of the Salmonella isolates carry the IncI1-I(Gamma) plasmid replicon. Although antimicrobial resistance was not common among Salmonella strains, most (11/19) of the E. coli strains had at least one resistance gene, with nearly half (8/19) being multidrug resistant and carrying mobile elements. Three of the 19 E. coli strains belong to serovars commonly associated with enteroaggregative E. coli (EAEC) pathotype. While strains belonging to virulence-associated lineages lacked key plasmid-encoded virulence plasmids, several plasmid replicons were detected in most of the E. coli (14/19) strains. Food contaminated with these pathogens can serve as a vehicle for disease transmission, posing a significant public health risk and necessitating stringent food safety and hygiene practices to prevent outbreaks. Hence, there is need for continuous surveillance and preventive measures to stop the spread of foodborne pathogens and reduce the risk of associated illnesses in Ghana.

microbiology↗

bioRxiv2024

Targeted sequencing of Enterobacterales bacteria using CRISPR-Cas9 enrichment and Oxford Nanopore Technologies

Sequencing DNA directly from patient samples enables faster pathogen characterisation compared to traditional culture-based approaches, but often yields insufficient sequence data for effective downstream analysis. CRISPR-Cas9 enrichment is designed to improve yield of low abundance sequences but has not been thoroughly explored with Oxford Nanopore Technologies (ONT) for use in clinical bacterial epidemiology. We designed CRISPR-Cas9 guide RNAs to enrich for the human pathogen Klebsiella pneumoniae, by targeting multi-locus sequence type (MLST) and transfer RNA (tRNA) genes, as well as common antimicrobial resistance (AMR) genes and the resistance-associated integron gene intI1. We validated enrichment performance in bacterial isolates before comparing enriched and unenriched sequencing of three human faecal samples spiked with K. pneumoniae at varying abundance. Enriched sequencing generated 56x and 11.3x the number of AMR and MLST reads respectively compared to unenriched sequencing and required approximately one third of the computational storage space. Targeting the intI1 gene often led to detection of 10-20 proximal resistance genes due to the long reads produced by ONT sequencing. We demonstrated that CRISPR-Cas9 enrichment combined with ONT sequencing enabled improved genomic characterisation outcomes over unenriched sequencing of patient samples. This method could be used to inform infection control strategies by identifying patients colonised with high-risk strains.

microbiology↗

bioRxiv2024

hAMRonization: Enhancing antimicrobial resistance prediction using the PHA4GE AMR detection specification and tooling

The detection of antimicrobial resistance (AMR) markers directly from genomic or metagenomic data is becoming a standard clinical and public health procedure. This has resulted in the development of a number of different bioinformatic AMR prediction tools. Although many may implement similar principles, these tools differ significantly in their supported inputs, search algorithms, parameterisation, and underlying reference databases. Each of these tools generates a report of detected AMR genes or variants in a distinct, non-standard, format. This presents a huge barrier to the comparison of results and to the modularity of tools for AMR gene prediction within bioinformatic workflows. In collaboration with 17 public health laboratories across 10 countries, the Public Health Alliance for Genomic Epidemiology (PHA4GE) (https://pha4ge.org) data structures working group has developed and piloted a standardized output specification for the bioinformatic detection of AMR from microbial genomes. In this report, we discuss hAMRonization, a python package and command-line utility, which implements PHA4GEs AMR specification to combine the outputs of disparate antimicrobial resistance gene detection tools into a single unified format. hAMRonization can be easily extended and currently supports 18 different tools (both species-agnostic and species-specific) for the detection of genes and/or variants conferring AMR. The harmonized reports are available in tabular form, JSON format or through an interactive HTML file (e.g., https://maguire-lab.github.io/assets/interactive_report_demo.html) that can be opened within the browser for navigable data exploration. As of 2024-03-07 hAMRonization has been downloaded [~]12,500 times, incorporated into >9 public bioinformatic tools and workflows, and been internally adopted by several national and international public health groups. The hAMRonization tool and underlying specification are open-source and freely available through PyPI, conda and GitHub (https://github.com/pha4ge/hAMRonization).

bioinformatics↗

bioRxiv2023

High genetic diversity and blaNDM-1 prevalence among Acinetobacter baumannii in Nigerian hospitals

BackgroundAcinetobacter baumannii cause difficult-to-treat infections mostly among immunocompromised patients. Clinically relevant A. baumannii lineages and their carbapenem resistance mechanisms are sparsely described in Nigeria. ObjectiveThis study aimed to characterise the diversity and genetic mechanisms of carbapenem resistance among A. baumannii strains isolated from hospitals in southwestern Nigeria. MethodsWe sequenced the genomes of all A. baumannii isolates submitted to Nigerias antimicrobial resistance surveillance reference laboratory between 2016 - 2020 on an Illumina platform and performed in silico genomic characterisation. Selected strains were sequenced using the Oxford Nanopore technology to characterise the genetic context of carbapenem resistance genes. ResultsThe 86 A. baumannii isolates were phylogenetically diverse and belonged to 35 distinct sequence types (STs), 16 of which were novel. Thirty-eight (44.2%) isolates belonged to none of the known international clones (ICs). Over 50% of the isolates were phenotypically resistant to 10 of 12 tested antimicrobials. Majority (n=54) of the isolates were carbapenem-resistant, particularly the IC7 (100%) and IC9 (>91.7%) strains. blaOXA-23 (34.9%) and blaNDM-1 (27.9%) were the most common carbapenem resistance genes detected. All blaOXA-23 genes were carried on Tn2006 or Tn2006-like transposons. Our findings suggest that the mobilisation of a 10kb Tn125 composite transposon is the primary means of blaNDM-1 dissemination. ConclusionOur findings highlight an increase in blaNDM-1 prevalence and the widespread transposon-facilitated dissemination of carbapenemase genes in diverse A. baumannii lineages in southwestern Nigeria. We make the case for improving surveillance of these pathogens in Nigeria and other understudied settings. ImportanceAcinetobacter baumannii are increasingly clinically relevant due to their propensity to harbour genes conferring resistance to multiple antimicrobials, as well as their ability to persist and disseminate in hospital environments and cause difficult-to-treat nosocomial infections. Little is known about the molecular epidemiology and antimicrobial resistance profiles of these organisms in Nigeria, largely due to limited capacity for their isolation, identification, and antimicrobial susceptibility testing. Our study characterised the diversity and antimicrobial resistance profiles of clinical A. baumannii in southwestern Nigeria using whole-genome sequencing. We also identified the key genetic elements facilitating the dissemination of carbapenem resistance genes within this species. This study provides key insights into the clinical burden and population dynamics of A. baumannii in hospitals in Nigeria and highlights the importance of routine whole-genome sequencing-based surveillance of this and other previously understudied pathogens in Nigeria and other similar settings.

genomics↗

bioRxiv2021

Antibiofilm agents with therapeutic potential against enteroaggregative Escherichia coli

BackgroundEnteroaggregative Escherichia coli (EAEC) is a predominant but neglected enteric pathogen implicated in infantile diarrhoea and nutrient malabsorption. There are no non-antibiotic approaches to dealing with persistent infection by these exceptional colonizers, which form copious biofilms. We screened the Medicines for Malaria Venture Pathogen Box for chemical entities that inhibit EAEC biofilm formation. MethodologyWe used two EAEC strains, 042 and MND005E, in a medium-throughput crystal violet-based antibiofilm screen. Hits were confirmed in concentration-dependence, growth kinetic and time course assays and activity spectra were determined against a panel of genome-sequenced EAEC. Antibiofilm activity against isogenic EAEC mutants, molecular docking simulations and comparative genomic analysis were used to identify the mechanism of action of one hit. Principal findingsIn all, five compounds (1.25%) reproducibly inhibited biofilm accumulation by at least one strain by 30-85% while inhibiting growth by under 10%. Hits exhibited at least 10-fold greater antibiofilm activity than nitazoxanide, the only known EAEC biofilm inhibitor. Reflective of known EAEC heterogeneity, only one hit was active against both screen isolates, but three hits showed broad antibiofilm activity against a larger panel of strains. Mechanism of action studies point to the EAEC anti-aggregation protein (Aap), dispersin, as the target of compound MMV687800. ConclusionsThis study identified five compounds not previously described as anti-adhesins or Gram-negative antibacterials with significant and specific EAEC antibiofilm activity. One molecule, MMV687800, targets the EAEC Aap. In vitro small-molecule inhibition of EAEC colonization opens a way to new therapeutic approaches to preventing and treating EAEC infection. Author summaryDiarrhoea accounts for over half a million deaths in children under five annually. It additionally contributes to childhood malnutrition as well as growth and development deficiencies, particularly in low-income countries. Enteroaggregative Escherichia coli (EAEC) causes diarrhoea that is often persistent and can also contribute to growth deficiencies in young children. EAEC is a neglected pathogen that is often resistant to antimicrobial drugs. Small molecules that block EAEC colonization may hold the key to interfering with EAEC disease without promoting antimicrobial resistance. We screened the Medicines for Malaria Ventures Pathogen Box for chemicals that can interfere with EAEC biofilm formation, a key colonization indicator. Our screen identified five biofilm-inhibiting molecules that did not interfere with bacterial viability and therefore are unlikely to exert strong pressure for resistance. Molecular biology and computational investigations point to the EAEC anti-aggregative protein, also known as dispersin, as a possible target for one of these hit molecules. Optimizing EAEC antibiofilm hits will create templates that can be employed for resolving EAEC diarrhoea and related infections.

microbiology↗