Search

Abstract Background Vancomycin-resistant enterococci (VRE) are important pathogens categorized as high-priority bacteria in the Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics published by the World Health Organization. The aim of this study was to determine the risk factors, resistance, virulence, mobilomes associated with multidrug-resistant and clonal lineages of Enterococcus faecium and faecalis circulating among hospitalized patients following the health system in South Africa, using whole genome sequencing (WGS). Methods A cross-sectional study was conducted during a two-month periods among hospitalized patients in 2017. Rectal swabs were collected from patients admitted to medical and surgical wards in an urban tertiary hospital, and a rural district hospital in uMgungundlovu district, South Africa. Enterococci were screened for vancomycin resistance on bile esculin azide agar supplemented with 6 mg/L of vancomycin and confirmation of VRE was done using ROSCO kits. Conventional and real-time PCR methods were used to ascertain the presence of VanA, VanB, VanC-2/3 and VanC-1 genes. All six multidrug-resistant Enterococcus faecalis and faecium selected were identified using multiplexed paired-end libraries (2 × 300 bp) with the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and genome sequencing was done using Illumina MiSeq instrument with 100× coverage at the National Institute of Communicable Diseases Sequencing Core Facility, South Africa. Antibiotic resistance genes, virulence factors, plasmids, integrons and CRISPR were characterized using RAST, ResFinder, VirulenceFinder, PlasmidFinder, PHAST and ISFinder respectively. Results Sequencing analysis revealed that these strains harbouring numerous resistance genes to glycopeptides (vanC[100%], vex3[100%], vex2[83,33%] and vanG[16,66%]), macrolides, lincosamides, sterptogramine B (ermB[33,32%], Isa[16,66%], emeA[16,66%]) and tetracyclines (tetM[33,32%]) in both district and tertiary hospitals. Multidrug efflux pumps including MATE, MFS and pmrA conferring resistance to several classes of antibiotics were also identified. The main transposable elements observed were in the Tn3 family, specifically Tn1546. Four single sequence types (STs) were identified among E. faecium in the district hospital, namely ST822, ST636, ST97 along with a novel ST assigned ST1386, while one lineage, ST29 was detected in the tertiary hospital. Conclusion The study reveals the genetic diversity and high pathogenicity of multidrug-resistant Enterococcus faecalis and faecium circulating among hospitalized patients. It underlines the necessity to implement routine screening of admitted patients coupled with infection control procedures, antimicrobial stewardship and awareness should be strengthened to prevent and/or contain the carriage and spread of multidrug resistant E. faecium and E. faecalis in hospitals and communities in South Africa.

IntroductionThis study addresses the rising cardiovascular disease (CVD) rates in the United Arab Emirates (UAE) by investigating the occurrence and impact of genetic variants in CVD-related genes.MethodsWe collected all genes linked to heritable CVD from public and diagnostic databases and mapped them to their corresponding biological processes and molecular pathways. We then evaluated the types and burden of genetic variants within these genes in 343 individuals from the Emirati Mendelian Study Cohort and 3,007 national electronic health records.ResultsWe identified a total of 735 genes associated with heritable CVD, covering a range of cardiovascular conditions. Enrichment analysis revealed key biological processes and pathways, including Apelin, FoxO, and Ras signaling, that are implicated across all forms of heritable CVD. Analysis of a UAE cohort of 3,350 individuals showed a predominance of rare and unique CVD variants specific to the population. The study found a significant burden of pathogenic variants in families with CVD within the Emirati Mendelian cohort and re-assessed the pathogenicity of 693 variants from national health records, leading to the discovery of new CVD-causing variants.DiscussionThis study underscores the importance of continuously updating our understanding of genes and pathways related to CVD. It also highlights the significant underrepresentation of the UAE population in public databases and clinical literature on CVD genetics, offering valuable insights that can inform future research and intervention strategies.

We whole-genome sequence a drug-resistant Pseudomonas aeruginosa strain (PANU108) isolated in 2020 from an elderly male patient admitted to an intensive care unit with bloodstream infection in Khartoum, Sudan. The analysis of the sequenced data revealed that the strain is sequence type 319 (ST319) and carrying 11 acquired resistance genes belonging to 5 drug classes. Interestingly, we found that PANU108 strain harbouring blaTMB-1 resistance gene. To the best of our knowledge, this the first report of a P. aeruginosa producing Tripoli Metallo-β-lactamase 1 (TMB-1) resistance determinant.

Abstract Background Clopidogrel is a widely prescribed prodrug that requires activation via specific pharmacogenes to exert its anti-platelet function. Genetic variations in the genes encoding its transporter, metabolizing enzymes, and target receptor lead to variability in its activation and platelet inhibition and, consequently, its efficacy. This variability increases the risk of secondary cardiovascular events, and therefore, some variations have been utilized as genetic biomarkers when prescribing clopidogrel. Methods Our study examined clopidogrel-related genes (CYP2C19, ABCB1, PON1, and P2Y12R) in a cohort of 298 healthy Emiratis individuals. The study used whole exome sequencing (WES) data to comprehensively analyze pertinent variations of these genes, including their minor allele frequencies, haplotype distribution, and their resulting phenotypes. Results Our data shows that approximately 37% (n = 119) of the cohort are likely to benefit from the use of alternative anti-platelet drugs due to their classification as intermediate or poor CYP2C19 metabolizers. Additionally, more than 50% of the studied cohort exhibited variants in ABCB1, PON1, and P2YR12 genes, potentially influencing clopidogrel’s transport, enzymatic clearance, and receptor performance. Conclusions Recognizing these alleles and genotype frequencies may explain the clinical differences in medication response across different ethnicities and predict adverse events. Our findings underscore the need to consider genetic variations in prescribing clopidogrel, with potential implications for implementing personalized anti-platelet therapy among Emiratis based on their genetic profiles.

Anopheles arabiensis is a highly adaptable member of the An. gambiae complex. Its flexible resting behaviour and diverse feeding habits make conventional vector control methods less effective in controlling this species. Another emerging challenge is its adaptation to breeding in polluted water, which impacts various life history traits relevant to epidemiology. The gut microbiota of mosquitoes play a crucial role in their life history, and the larval environment significantly influences the composition of this bacterial community. Consequently, adaptation to polluted breeding sites may alter the gut microbiota of adult mosquitoes. This study aimed to examine how larval exposure to metal pollution affects the gut microbial dynamics of An. arabiensis adults. Larvae of An. arabiensis were exposed to either cadmium chloride or copper nitrate, with larvae reared in untreated water serving as a control. Two laboratory strains (SENN: insecticide unselected, SENN-DDT: insecticide selected) and F1 larvae sourced from KwaZulu-Natal, South Africa, were exposed. The gut microbiota of the adults were sequenced using the Illumina Next Generation Sequencing platform and compared. Larval metal exposure affected alpha diversity, with a more marked difference in beta diversity. There was evidence of core microbiota shared between the untreated and metal-treated groups. Bacterial genera associated with metal tolerance were more prevalent in the metal-treated groups. Although larval metal exposure led to an increase in pesticide-degrading bacterial genera in the laboratory strains, this effect was not observed in the F1 population. In the F1 population, Plasmodium-protective bacterial genera were more abundant in the untreated group compared to the metal-treated group. This study therefore highlights the importance of considering the larval environment when searching for local bacterial symbionts for paratransgenesis interventions.

Background Infants suffering from lower respiratory tract infections (LRTIs) have distinct nasopharyngeal (NP) microbiome profiles that correlate with severity of disease. Whether these profiles precede the infection or are a consequence of it, is unknown. In order to answer this question, longitudinal studies are needed. Methods We conducted a retrospective analysis of NP samples collected in a longitudinal birth cohort study of Zambian mother-infant pairs. Samples were collected every two weeks from 1-week through 14-weeks of age. Ten of the infants in the cohort who developed LRTI were matched 1:3 with healthy comparators. We completed 16S rRNA gene sequencing on the samples each of these infants contributed and compared the NP microbiome of the healthy infants to infants who developed LRTI. Results The infant NP microbiome maturation was characterized by transitioning from Staphylococcus dominant to respiratory-genera dominant profiles during the first three months of life, similar to what is described in the literature. Interestingly, infants who developed LRTI had distinct NP microbiome characteristics before infection, in most cases as early as the first week of life. Their NP microbiome was characterized by the presence of Novosphingobium, Delftia, high relative abundance of Anaerobacillus, Bacillus, and low relative abundance of Dolosigranulum, compared to the healthy controls. Mothers of infants with LRTI also had low relative abundance of Dolosigranulum in their baseline samples compared to mothers of infants that did not develop an LRTI. Conclusions Our results suggest that specific characteristics of the NP microbiome precede LRTI in young infants and may be present in their mothers as well. Early dysbiosis may play a role in the causal pathway leading to LRTI or could be a marker of underlying immunological, environmental, or genetic characteristics that predispose to LRTI.

We used whole genome sequencing (WGS) as an epidemiologic surveillance tool to elucidate the transmission dynamics of Shiga toxin-producing Escherichia coli (STEC) strains along the beef production chain in South Africa. Isolates were obtained from a cattle farm, abattoirs and retail outlets. Isolates were analysed using WGS on a MiSeq platform (Illumina, San Diego, CA, USA) and phylogenetic analysis was carried out. Of the 85 isolates, 39% (33) carried the stx gene and 61% (52) had lost the stx gene. The prevalence of stx subtypes was as follows; stx1a 55% (18/33), stx1b 52% (17/33), stx2a 55% (18/33), stx2b 27% (9/33), stx2dB 30% (10/33) and stx2d1A 15% (5/33). Thirty-five different serogenotypes were detected, of which 65% (56) were flagellar H-antigens and 34% (29) were both O-antigens and flagellar H-antigens. We identified 50 different sequence types (STs), and only nine of the isolates were assigned to three different clonal complexes. Core genome phylogenetic analysis revealed genetic relatedness, and isolates clustered mainly according to their STs and serogenotypes regardless of stx subtypes. This study provides evidence of horizontal transmission and recirculation of STEC strains in Gauteng province and demonstrates that every stage of the beef production chain plays a significant role in STEC entry into the food chain.

Background: Hearing loss (HL) is an impairment of auditory function with identified genetic forms that can be syndromic (30%) or non-syndromic (70%). HL is genetically heterogeneous, with more than 1,000 variants across 150 causative genes identified to date. The genetic diagnostic rate varies significantly depending on the population being tested. Countries with a considerably high rate of consanguinity provide a unique resource for studying rare forms of recessive HL. In this study, we identified genetic variants associated with bilateral sensorineural HL (SNHL) using whole-exome sequencing (WES) in 11 families residing in the United Arab Emirates (UAE).Results: We established the molecular diagnosis in six probands, with six different pathogenic or likely pathogenic variants in the genes MYO15A, SLC26A4, and GJB2. One novel nonsense variant, MYO15A:p.Tyr1962Ter*, was identified in a homozygous state in one family, which has not been reported in any public database. SLC26A4 and GJB2 were found to be the most frequently associated genes in this study. In addition, six variants of uncertain significance (VUS) were detected in five probands in the genes CDH23, COL11A1, ADGRV1, NLRP3, and GDF6. In total, 12 variants were observed in eight genes. Among these variants, eight missense variants (66.7%), three nonsense variants (25.0%), and one frameshift (8.3%) were identified. The overall diagnostic rate of this study was 54.5%. Approximately 45.5% of the patients in this study came from consanguineous families.Conclusion: Understanding the genetic basis of HL provides insight for the clinical diagnosis of hearing impairment cases through the utilization of next-generation sequencing (NGS). Our findings contribute to the knowledge of the heterogeneous genetic profile of HL, especially in a population with a high rate of consanguineous marriage in the Arab population.

Salmonella enterica serovar Rissen is an emerging and important Salmonella serovar prevalent in live animals and foods from retail markets worldwide. Here, we describe the whole-genome sequence of Salmonella enterica Serovar Rissen Sequence Type 8877 isolated from a cracked table egg in Sudan. The whole-genome sequencing was obtained using Illumina Miseq platform. The quality of the sequenced read, the De novo assembly, and the sequencing typing was conducted by JEKESA pipeline (https://github.com/stanikae/jekesa). The assembled genome was also uploaded to the Center for Genomic Epidemiology web server to determine acquired antibiotic resistance genes, predict the serovar, and the antigenic profile. The genome of Salmonella enterica serovar Rissen 1-M1 was found to harbor 4,689 protein-coding genes, 96 RNA genes, and 115 pseudogenes, as predicted by NCBI Prokaryotic Genome Annotation Pipeline. This whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under accession JAPSFB000000000.

Abstract A pathogenic strain of Macrococcus caseolyticus (M. caseolyticus) was isolated from wounds infection during an investigation on donkeys in Khartoum State. (122) samples were collected from external wounds (head, abdomen, back and leg) during different seasons. One isolate (124B) was identified using whole-genome sequence analysis. RAST software identified 31 virulent genes of disease and defense, including methicillin-resistant genes, TatR family and ANT(4′)-Ib. Plasmid rep22 was identified by PlasmidFindet-2.0 Server and a CRISPR. MILST-2.0 predicted many novel alleles. NCBI notated the genome as a novel M. caseolyticus strain (DaniaSudan). The MLST−tree−V1 revealed that DaniaSudan and KM0211a strains were interrelated. Strain DaniaSudan was resistant to ciprofloxacin, ceftazidime, erythromycin, oxacillin, clindamycin and kanamycin. Mice modeling showed bacteremia and many clinical signs (swelling, allergy, wounds, and hair loss). Enlargement, hyperemia, adhesions and abscesses were observed in many organs. Constructive conclusion The prevalence of the strain was 4.73%, with significant differences between collection seasons and locations of wounds. A highly significant association between doses (105 CFU/ml, 102 CFU/ml, Intra-peritoneum and sub-cutaneous) and swelling, developing of allergy and loss of hair (p = 0.001, p = 0.000 and p = 0.005) respectively were seen. This result represents the first report of pathogenic strains of M. caseolyticus worldwide.

Abstract The gut microbiota of mosquitoes plays a critical role in the life history of the animal. There is a growing body of research characterising the gut microbiota of a range of mosquito species, but there is still a paucity of information on some members of the Anopheles gambiae complex. In this study, the gut microbiota of four laboratory strains were characterised. SENN (Anopheles arabiensis—insecticide susceptible major vector), SENN DDT (Anopheles arabiensis—insecticide resistant major vector), MAFUS (Anopheles merus—minor vector) and SANGWE (Anopheles quadriannulatus—non-vector) were used in this study. The microbiota of fourth instar larvae, 3-day old, 15-day old non-blood fed and 15-day old blood fed females were characterised by MALDI-TOF mass spectroscopy and 16 s rRNA gene sequencing by next generation sequencing. The four strains differed in species richness but not diversity. The major vectors differ in β-diversity from that of the minor and non-vectors. There was no difference in α- or β-diversity in 15 non-blood fed females and 15-day old females that had 3 blood meals before day 15. These differences may be related to a mixture of the effect of insecticide resistance phenotype as well as a potential relationship to vector competence to a limited extent. Bacterial diversity is affected by species and age. There is also a potential relationship between the differences in gut microbiota and capacity to transmit parasites. This genetic background of the mosquitoes, however, play a major role, and must be considered in this relationship.

Enterococci are among the most common opportunistic hospital pathogens. This study used whole-genome sequencing (WGS) and bioinformatics to determine the antibiotic resistome, mobile genetic elements, clone and phylogenetic relationship of Enterococcus faecalis isolated from hospital environments in South Africa. This study was carried out from September to November 2017. Isolates were recovered from 11 frequently touched sites by patients and healthcare workers in different wards at 4 levels of healthcare (A, B, C, and D) in Durban, South Africa. Out of the 245 identified E. faecalis isolates, 38 isolates underwent whole-genome sequencing (WGS) on the Illumina MiSeq platform, following microbial identification and antibiotic susceptibility tests. The tet(M) (31/38, 82%) and erm(C) (16/38, 42%) genes were the most common antibiotic-resistant genes found in isolates originating from different hospital environments which corroborated with their antibiotic resistance phenotypes. The isolates harboured mobile genetic elements consisting of plasmids (n = 11) and prophages (n = 14) that were mostly clone-specific. Of note, a large number of insertion sequence (IS) families were found on the IS3 (55%), IS5 (42%), IS1595 (40%), and Tn3 transposons the most predominant. Microbial typing using WGS data revealed 15 clones with 6 major sequence types (ST) belonging to ST16 (n = 7), ST40 (n = 6), ST21 (n = 5), ST126 (n = 3), ST23 (n = 3), and ST386 (n = 3). Phylogenomic analysis showed that the major clones were mostly conserved within specific hospital environments. However, further metadata insights revealed the complex intraclonal spread of these E. faecalis major clones between the sampling sites within each specific hospital setting. The results of these genomic analyses will offer insights into antibiotic-resistantE. faecalis in hospital environments relevant to the design of optimal infection prevention strategies in hospital settings.

In South Africa, Candida auris was the third most common cause of candidemia in 2016–2017. We performed single nucleotide polymorphism (SNP) genome-wide analysis of 115 C. auris isolates collected between 2009 and 2018 from national laboratory-based surveillance, an environmental survey at four hospitals and a colonization study during a neonatal unit outbreak. The first known South African C. auris strain from 2009 clustered in clade IV. Overall, 98 strains clustered within clade III (85%), 14 within clade I (12%) and three within clade IV (3%). All environmental and colonizing strains clustered in clade III. We also identified known clade-specific resistance mutations in the ERG11 and FKS1 genes. Identification of clade I strains between 2016 and 2018 suggests introductions from South Asia followed by local transmission. SNP analysis characterized most C. auris strains into clade III, the clade first reported from South Africa, but the presence of clades I and IV strains also suggest early introductions from other regions.

Abstract Background We aimed to describe an outbreak of cutaneous abscesses caused by Panton-Valentine leukocidin (PVL)-producing methicillin-susceptible Staphylococcus aureus (MSSA) among gold mine workers. Methods In February 2018, we retrospectively reviewed a random sample of 50 medical records from 243 cases and conducted face-to-face interviews using a structured questionnaire. Pus aspirates were sent to the National Institute for Communicable Diseases from prospectively-identified cases (November 2017–March 2018). Nasopharyngeal swabs were collected during a colonisation survey in February 2018. Staphylococcus aureus isolates were screened with a conventional PCR for lukS/F-PV. Pulsed-field gel electrophoresis (PFGE) was performed to determine the genetic relatedness among the isolates. A sample of isolates were selected for whole genome sequencing (WGS). We conducted an assessment on biological risks associated with mining activities. Results From January 2017 to February 2018, 10% (350/3582) of mine workers sought care for cutaneous abscesses. Forty-seven medical files were available for review, 96% were male (n = 45) with a mean age of 43 years (SD = 7). About 52% (24/46) were involved in stoping and 28% (13/47) worked on a particular level. We cultured S. aureus from 79% (30/38) of cases with a submitted specimen and 14% (12/83) from colonisation swabs. All isolates were susceptible to cloxacillin. Seventy-one percent of S. aureus isolates (30/42) were PVL-PCR-positive. Six PFGE clusters were identified, 57% (21/37) were closely related. WGS analysis found nine different sequence types. PFGE and WGS analysis showed more than one cluster of S. aureus infections involving closely related isolates. Test reports for feed and product water of the mine showed that total plate counts were above the limits of 1000 cfu/ml, coliform counts > 10 cfu/100 ml and presence of faecal coliforms. Best practices were poorly implemented as some mine workers washed protective clothing with untreated water and hung them for drying at the underground surface. Conclusions PVL-producing MSSA caused an outbreak of cutaneous abscesses among underground workers at a gold mining company. To our knowledge, no other outbreaks of PVL-producing S. aureus involving skin and soft tissue infections have been reported in mining facilities in South Africa. We recommend that worker awareness of infection prevention and control practices be strengthened.

Background. Antimicrobial resistance is limiting treatment options for Neisseria gonorrhoeae infections. To aid or replace culture and the syndromic management approach, molecular assays are required for antimicrobial susceptibility testing to guide appropriate and rapid treatment. Objective. We aimed to detect single-nucleotide polymorphisms and plasmids associated with antimicrobial resistance from N. gonorrhoeae isolates from a clinic population in South Africa, using real-time PCR as a rapid test for AMR detection. Methods. N. gonorrhoeae isolates, from female and male patients presenting for care at a sexually transmitted infections clinic in Durban, South Africa, were analysed using phenotypic and genotypic methods for identification and antibiotic susceptibility testing (AST). Real-time PCR and high-resolution melting analysis were used to detect porA pseudogene (species-specific marker) and resistance-associated targets. Whole-genome sequencing was used as the gold standard for the presence of point mutations. Results. The real-time porA pseudogene assay identified all N. gonorrhoeae-positive isolates and specimens. Concordance between molecular detection (real-time PCR and HRM) and resistance phenotype was ≥92% for blaTEM (HLR penicillin), rpsJ_V57M (tetracycline), tetM (tetracycline), and gyrA_S91F (ciprofloxacin). Resistance determinants 16SrRNA_C1192U (spectinomycin), mtrR_G45D (azithromycin), and penA_D545S, penA_mosaic (cefixime/ceftriaxone) correlated with the WHO control isolates. Conclusions. Eight resistance-associated targets correlated with phenotypic culture results. The porA pseudogene reliably detected N. gonorrhoeae. Larger cohorts are required to validate the utility of these targets as a convenient culture-free diagnostic tool, to guide STI management in a South African population.

Introduction. Treatment of gonorrhoea infection is limited by the increasing prevalence of multidrug-resistant strains. Cost-effective molecular diagnostic tests can guide effective antimicrobial stewardship. The aim of this study was to correlate mRNA expression levels in Neisseria gonorrhoeae antibiotic target genes and efflux pump genes to antibiotic resistance in our population. Methods. This study investigated the expression profile of antibiotic resistance-associated genes (penA, ponA, pilQ, mtrR, mtrA, mtrF, gyrA, parC, parE, rpsJ, 16S rRNA, and 23S rRNA) and efflux pump genes (macAB, norM, and mtrCDE), by quantitative real-time PCR, in clinical isolates from KwaZulu-Natal, South Africa. Whole-genome sequencing was used to determine the presence or absence of mutations. Results. N. gonorrhoeae isolates, from female and male patients presenting for care at clinics in KwaZulu-Natal, South Africa, were analysed. As determined by binomial regression and ROC analysis, the most significant (p≤0.05) markers for resistance prediction in this population, and their cutoff values, were determined to be mtrC (p=0.024; cutoff 7.754). Conclusion. Antimicrobial stewardship includes exploring options to conserve currently available drugs for gonorrhoea treatment. There is the potential to predict an isolate as either susceptible or nonsusceptible based on the mRNA expression level of specific candidate markers, to inform patient management. This real-time qPCR approach, with few targets, can be further investigated for use as a potentially cost-effective diagnostic tool to detect resistance.

The genogroup II genotype 4 (GII.4) noroviruses are a major cause of viral gastroenteritis. Since the emergence of the Sydney_2012 variant, no novel norovirus GII.4 variants have been reported. The high diversity of noroviruses and periodic emergence of novel strains necessitates continuous global surveillance. The aim of this study was to assess the diversity of noroviruses in selected wastewater samples from Pretoria, South Africa (SA) using amplicon-based next-generation sequencing (NGS). Between June 2018 and August 2020, 200 raw sewage and final effluent samples were collected fortnightly from two wastewater treatment plants in Pretoria. Viruses were recovered using skimmed milk flocculation and glass wool adsorption-elution virus recovery methods and screened for noroviruses using a one-step real-time reverse-transcription PCR (RT-PCR). The norovirus BC genotyping region (570–579 bp) was amplified from detected norovirus strains and subjected to Illumina MiSeq NGS. Noroviruses were detected in 81% (162/200) of samples. The majority (89%, 89/100) of raw sewage samples were positive for at least one norovirus, compared with 73% (73/100) of final effluent samples. Overall, a total of 89 different GI and GII RdRp-capsid combinations were identified, including 51 putative novel recombinants, 34 previously reported RdRp-capsid combinations, one emerging novel recombinant and three Sanger-sequencing confirmed novel recombinants.

This study investigated the genetic differences between Aedes aegypti subspecies (Aedes aegypti aegypti (Aaa) and Aedes aegypti formosus (Aaf)) from Sudan using the NADH dehydrogenase subunit 4 (ND4) mitochondrial gene marker. Nineteen distinct haplotypes of the ND4 were identified in female Aedes aegypti mosquitoes from the study sites. The phylogenetic relationship of the 19 ND4 haplotypes was demonstrated in a median-joining haplotype network tree with Aaa and Aaf populations found to share three haplotypes. The genetic variance (Pairwise FST values) was estimated and found to range from 0.000 to 0.811. Isolation by distance test revealed that geographical distance was correlated to genetic variation (coefficient value (r) = 0.43). The Polar maximum likelihood tree showed the phylogenetic relationship of 91 female Aaa and Aaf from the study sites, with most of the Aaf haplotypes clustered in one group while most of the Aaa haplotypes gathered in another group, but there was an admixture of the subspecies in both clusters, especially the Aaa cluster. The Spatial Analysis of Molecular Variance (SAMOVA) test revealed that the eight populations clustered into two phylogeographic groups/clusters of the two subspecies populations. The 2 Aedes aegypti subspecies seemed not to be totally separated geographically with gene flow among the populations.

Anopheles merus can breed in a range of saltwater concentrations. The consequences of this ability on the life history of adult An. merus are poorly understood. This study examined the effects of exposure to 0, 2.1875, 4.375, 8.75, and 17.5 g/L of sodium chloride on An. merus. The effects on larval development, adult longevity, fertility, and fecundity, as well as deltamethrin tolerance were examined. The effect of larval salt exposure on the expression of defensin-1 in adults was examined by quantitative Real-Time PCR. Finally, the effect of the larval salt concentration on microbial dynamics was assessed by 16S Next Generation Sequencing. High concentrations of saltwater increased larval development time and number of eggs laid, as well as deltamethrin tolerance. Larval exposure to salt also reduced the expression of defensin-1. The exposure also had a significant effect on microbial diversity in larvae and adults. The diversity of larvae decreased once adults emerged. Salt-tolerant bacterial genera predominated in larvae but were absent in adults. High salt concentrations resulted in greater abundance of Plasmodium-protective genera in adults. Although this study was conducted on a laboratory strain of An. merus, these data suggest that osmoregulation has a significant effect on the life history of the species with potential epidemiological consequences.

Objectives: The underlying resistance mechanisms, defence systems, mobilome, virulome, clonality and global phylogenetic relationship of a novel sequence type (ST) 658 Aeromonas hydrophilia (A34a) isolated from a pig abattoir in South Africa was determined using whole-genome sequence (WGS) technology. Methods: Following isolation on chromogenic agar (CHROMID® CARBA SMART), microbial identification and antibiotic susceptibility testing were performed using a VITEK®2 platform. Genotyping involved WGS performed with an Illumina MiSeq platform. Results: The antibiotic resistome agreed with the resistance phenotype of the isolate and included antibiotic resistance determinants for β-lactams (blaCPHA3 and blaOXA-724). BLASTn analysis of resistome-encoding contigs affirmed chromosomally-mediated resistance. BURST algorithmic analysis identified the novel ST658 as a satellite variant. Virulome analysis predicted virulence genes of Aeromonas whose expression are critical for establishing infection in the host. Global phylogenomic analyses showed strain A34a is closely related to two international isolates from Sri Lanka (Ae25) and the USA (RU34A), although there is little to suggest that it was imported from abroad. Conclusion: This is the first report on the genomic analysis of a novel ST658 A. hydrophilia, offering useful insights into its pathogenicity and global phylogenetics.

The increasing occurrence of multidrug-resistant (MDR) extended-spectrum β-lactamase- (ESBL) and/or AmpC β-lactamase- (AmpC) producing Enterobacterales in irrigation water and associated irrigated fresh produce represents risks related to the environment, food safety, and public health. In South Africa, information about the presence of ESBL/AmpC-producing Enterobacterales from non-clinical sources is limited, particularly in the water–plant-food interface. This study aimed to characterize 19 selected MDR ESBL/AmpC-producing Escherichia coli (n=3), Klebsiella pneumoniae (n=5), Serratia fonticola (n=10), and Salmonella enterica (n=1) isolates from spinach and associated irrigation water samples from two commercial spinach production systems within South Africa, using whole genome sequencing (WGS). Antibiotic resistance genes potentially encoding resistance to eight different classes were present, with blaCTX-M-15 being the dominant ESBL encoding gene and blaACT-types being the dominant AmpC encoding gene detected. A greater number of resistance genes across more antibiotic classes were seen in all the K. pneumoniae strains, compared to the other genera tested. From one farm, blaCTX-M-15-positive K. pneumoniae strains of the same sequence type 985 (ST 985) were present in spinach at harvest and retail samples after processing, suggesting successful persistence of these MDR strains. In addition, ESBL-producing K. pneumoniae ST15, an emerging high-risk clone causing nosocomical outbreaks worldwide, was isolated from irrigation water. Known resistance plasmid replicon types of Enterobacterales including IncFIB, IncFIA, IncFII, IncB/O, and IncHI1B were observed in all strains following analysis with PlasmidFinder. However, blaCTX-M-15 was the only β-lactamase resistance gene associated with plasmids (IncFII and IncFIB) in K. pneumoniae (n=4) strains. In one E. coli and five K. pneumoniae strains, integron In191 was observed. Relevant similarities to human pathogens were predicted with PathogenFinder for all 19 strains, with a confidence of 0.635–0.721 in S. fonticola, 0.852–0.931 in E. coli, 0.796–0.899 in K. pneumoniae, and 0.939 in the S. enterica strain. The presence of MDR ESBL/AmpC-producing E. coli, K. pneumoniae, S. fonticola, and S. enterica with similarities to human pathogens in the agricultural production systems reflects environmental and food contamination mediated by anthropogenic activities, contributing to the spread of antibiotic resistance genes.

Botulism, a rare life-threatening toxemia, is probably underdiagnosed in all of its forms in Africa. This study reports the first laboratory-supported case of infant botulism on the African continent. A 10-week-old, previously well infant presented with progressive global weakness, feeding difficulty, and aspiration pneumonia. During a lengthy hospitalization, a rare bivalent Clostridium botulinum strain, producing subtype B3 and F8 toxins and with a new multilocus sequence type, was isolated from stool. The infant was successfully treated with a heptavalent botulinum antitoxin infusion and pyridostigmine. Despite the relative rarity of infant botulism, this case illustrates the importance of maintaining a high level of clinical suspicion when assessing hypotonic infants. The value of modern diagnostic modalities in identifying and characterizing this under-recognized condition is also demonstrated.

We investigated the antibiotic resistome, mobilome, virulome, and phylogenomic lineages of Enterococcus spp. obtained from a wastewater treatment plant and its associated waters using whole-genome sequencing (WGS) and bioinformatics tools. The whole genomes of Enterococcus isolates including Enterococcus faecalis (n = 4), Enterococcus faecium (n = 5), Enterococcus hirae (n = 2), and Enterococcus durans (n = 1) with similar resistance patterns from different sampling sites and time points were sequenced on an Illumina MiSeq machine. Multilocus sequence typing (MLST) analysis revealed two E. faecalis isolates that had a common sequence type ST179; the rest had unique sequence types ST841, and ST300. The E. faecium genomes belonged to 3 sequence types, ST94 (n = 2), ST361 (n = 2), and ST1096 (n = 1). Detected resistance genes included those encoding tetracycline [tet(S), tet(M), and tet(L)], and macrolides [msr(C), msr(D), erm(B), and mef(A)] resistance. Antibiotic resistance genes were associated with insertion sequences (IS6, ISL3, and IS982), and transposons (Tn3 and Tn6000). The tet(M) resistance gene was consistently found associated with a conjugative transposon protein (TcpC). A total of 20 different virulence genes were identified in E. faecalis and E. faecium including those encoding for sex pheromones (cCF10, cOB1, cad, and came), adhesion (ace, SrtA, ebpA, ebpC, and efaAfs), and cell invasion (hylA and hylB). Several virulence genes were associated with the insertion sequence IS256. No virulence genes were detected in E. hirae and E. durans. Phylogenetic analysis revealed that all Enterococcus spp. isolates were more closely related to animal and environmental isolates than clinical isolates. Enterococcus spp. with a diverse range of resistance and virulence genes as well as associated mobile genetic elements (MGEs) exist in the wastewater environment in South Africa.

Objectives: This study delineated the clonal lineages, antibiotic resistome and plasmid replicon types in multidrug-resistant K. pneumoniae isolates from a teaching hospital in Ghana. Methods: Identification and antibiotic susceptibility testing were done using the MALDI-TOF MS and Vitek-2 automated system. Genomic DNA extraction was carried out using the NucliSens easyMAG® (BioMérieux) kits and the DNA was subjected to whole genome sequencing (WGS) using the Illumina MiSeq platform. Results: Of the 200 isolates obtained, 37 were identified as K. pneumoniae of which 9 were resistant to all second and third-generation cephalosporins. These 9 isolates selected for further genomic analysis were characterized by the presence of 8 diverse sequence types (STs), capsular polysaccharide serotypes (K types and wzi allelic types) and multiple genes encoding resistance to β-lactams (blaCTX-M-15, blaSHV-11, blaTEM-1B, blaOXA-1), aminoglycosides (aac(3)-IIa, strB, strA, aadA16), fluoroquinolones/quinolones (qnrB66, oqxA, oqxB) and other antibiotic classes. Resistance genes were associated with plasmids, predominantly IncFIB(K) and ColRNAI. Multiple and diverse mutations in quinolone resistance-determining regions of gyrA (S83Y, D87A) and parC (S80I, N304S) in isolates resistant to ciprofloxacin (MIC ≥ 4 mg/mL) were found. Global phylogenomic analysis affirmed the diverse clonal clustering and origin of these isolates. Conclusions: The varied clonal clusters and resistome identified in the multidrug-resistant K. pneumoniae isolates is a major threat to the management of infections in Ghana. The molecular characterization of antibiotic resistance is thus imperative to inform strategies for containment. Keywords: Whole genome sequencing, Multidrug-resistant, ESBL, Klebsiella pneumoniae, Resistome, Ghana

Bagaza virus (BAGV), a member of the Ntaya serogroup in the Flavivirus genus of the Flaviviridae, was isolated from the brain tissue of a Himalayan monal pheasant that died following neurological signs in Pretoria, South Africa in 2016. Next-generation sequencing was carried out on this isolate resulting in a genome sequence of 10980nt. The full genome sequence of this isolate, designated ZRU96-16, shared 98% nucleotide identity with a BAGV isolate found in Culex univitattus mosquitoes from Namibia and 97% nucleotide identity with a Spanish BAGV sequence isolated from an infected partridge. In total, seven amino acid variations were unique to ZRU96-16 after alignment with other BAGV and Israel turkey meningoencephalomyelitis (ITV) genomes. The 3′UTR sequence of ZRU96-16 was resolved with sufficient detail to be able to annotate the variable and conserved sequence elements within this region. Multiple sequence alignment of the 3′UTR suggested that it could be useful in lineage designation as more similar viruses carried similar mutations across this region, while also retaining certain unique sites. Maximum likelihood phylogenetic analysis revealed two clusters containing both BAGV and ITVs from Europe, the Middle East and Africa. Broadly, temporal clustering separated isolates into two groups, with one cluster representing viruses from the 1960–2000’s and the other from 2010 onwards. This suggests that there is consistent exchange of BAGV and ITV between Europe and Africa. This investigation provides more information on the phylogenetics of an under-represented member of the Flaviviridae and provides an avenue for more extensive research on its pathogenesis and geographic expansion.

The resistome, virulome and mobilome of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-Ec) isolated from pigs in Cameroon and South Africa were assessed using whole genome sequencing (WGS). Eleven clonally related phenotypic ESBL-Ec isolates were subjected to WGS. The prediction of antibiotic resistance genes, virulence factors (VFs) and plasmids was performed using ResFinder, VirulenceFinder and PlasmidFinder, respectively. Diverse sequence types (STs) were detected with ST2144 and ST88 being predominant and blaCTX-M-15 (55%) being the principal ESBL gene. All except two isolates harboured various aminoglycoside resistance genes, including aph(3″)-Ib (6/11, 55%) and aph(6)-1d (6/11, 55%), while the qnrS1 gene was identified in four of the isolates. The ESBL-Ec isolates showed a 93.6% score of being human pathogens. The fim, ehaB, ibeB/C were the leading virulence factors detected. All isolates harboured at least three extraintestinal pathogenic E. coli (ExPEC) VFs, with one isolate harbouring up to 18 ExPEC VFs. Five isolates (45.45%) harboured the plasmid incompatibility group IncF (FII, FIB, FIC, FIA). The study revealed that there is an urgent need to implement effective strategies to contain the dissemination of resistant and virulent ESBL-Ec through the food chain in Cameroon and South Africa.

There is limited information on the comparative genomic diversity of antibiotic-resistant Escherichia coli from wastewater. We sought to characterize environmental E. coli isolates belonging to various pathotypes obtained from a wastewater treatment plant (WWTP) and its receiving waters using whole-genome sequencing (WGS) and an array of bioinformatics tools to elucidate the resistomes, virulomes, mobilomes, clonality, and phylogenies. Twelve multidrug-resistant (MDR) diarrheagenic E. coli isolates were obtained from the final effluent of a WWTP, and the receiving river upstream and downstream of the WWTP were sequenced on an Illumina MiSeq machine. The multilocus sequence typing (MLST) analysis revealed that multiple sequence types (STs), the most common of which was ST69 (n = 4) and ST10 (n = 2), followed by singletons belonging to ST372, ST101, ST569, ST218, and ST200. One isolate was assigned to a novel ST ST11351. A total of 66.7% isolates were positive for β-lactamase genes with 58.3% harboring the blaTEM1B gene and a single isolate the blaCTX−M−14 and blaCTX−M−55 extended-spectrum β-lactamase (ESBL) genes. One isolate was positive for the mcr-9 mobilized colistin resistance gene. Most antibiotic resistance genes (ARGs) were associated with mobile genetic support: class 1 integrons (In22, In54, In191, and In369), insertion sequences (ISs), and/or transposons (Tn402 or Tn21). A total of 31 virulence genes were identified across the study isolates, including those responsible for adhesion (lpfA, iha, and aggR), immunity (air, gad, and iss), and toxins (senB, vat, astA, and sat). The virulence genes were mostly associated with IS (IS1, IS3, IS91, IS66, IS630, and IS481) or prophages. Co-resistance to heavy metal/biocide, antibiotics were evident in several isolates. The phylogenomic analysis with South African E. coli isolates from different sources (animals, birds, and humans) revealed that isolates from this study mostly clustered with clinical isolates. Phylogenetics linked with metadata revealed that isolates did not cluster according to source but according to ST. The occurrence of pathogenic and MDR isolates in the WWTP effluent and the associated river is a public health concern.

No abstract available.

Background: Acinetobacter baumannii’s (A. baumannii) growing resistance to all available antibiotics is of concern. The study describes a colistin-resistant A. baumannii isolated at a clinical facility from a tracheal aspirate sample. Furthermore, it determines the isolates’ niche establishment ability within the tertiary health facility. Methods: An antimicrobial susceptibility test, conventional PCR, quantitative real-time PCR, phenotypic evaluation of the efflux pump, and whole-genome sequencing and analysis were performed on the isolate. Results: The antimicrobial susceptibility pattern revealed a resistance to piperacillin/tazobactam, ceftazidime, cefepime, cefotaxime/ceftriaxone, imipenem, meropenem, gentamycin, ciprofloxacin, trimethoprim/sulfamethoxazole, tigecycline, and colistin. A broth microdilution test confirmed the colistin resistance. Conventional PCR and quantitative real-time PCR investigations revealed the presence of adeB, adeR, and adeS, while mcr-1 was not detected. A MIC of 0.38 µg/mL and 0.25 µg/mL was recorded before and after exposure to an AdeABC efflux pump inhibitor. The whole-genome sequence analysis of antimicrobial resistance-associated genes detected beta-lactam: blaOXA-66; blaOXA-23; blaADC-25; blaADC-73; blaA1; blaA2, and blaMBL; aminoglycoside: aph(6)-Id; aph(3″)-Ib; ant(3″)-IIa and armA) and a colistin resistance-associated gene lpsB. The whole-genome sequence virulence analysis revealed a biofilm formation system and cell–cell adhesion-associated genes: bap, bfmR, bfmS, csuA, csuA/B, csuB, csuC, csuD, csuE, pgaA, pgaB, pgaC, and pgaD; and quorum sensing-associated genes: abaI and abaR and iron acquisition system associated genes: barA, barB, basA, basB, basC, basD, basF, basG, basH, basI, basJ, bauA, bauB, bauC, bauD, bauE, bauF, and entE. A sequence type classification based on the Pasteur scheme revealed that the isolate belongs to sequence type ST2. Conclusions: The mosaic of the virulence factors coupled with the resistance-associated genes and the phenotypic resistance profile highlights the risk that this strain is at this South African tertiary health facility.

In 2015, a cluster of respiratory diphtheria cases was reported from KwaZulu-Natal Province in South Africa. By using whole-genome analysis, we characterized 21 Corynebacterium diphtheriae isolates collected from 20 patients and contacts during the outbreak (1 patient was infected with 2 variants of C. diphtheriae). In addition, we included 1 cutaneous isolate, 2 endocarditis isolates, and 2 archived clinical isolates (ca. 1980) for comparison. Two novel lineages were identified, namely, toxigenic sequence type (ST) ST-378 (n = 17) and nontoxigenic ST-395 (n = 3). One archived isolate and the cutaneous isolate were ST-395, suggesting ongoing circulation of this lineage for >30 years. The absence of preexisting molecular sequence data limits drawing conclusions pertaining to the origin of these strains; however, these findings provide baseline genotypic data for future cases and outbreaks. Neither ST has been reported in any other country; this ST appears to be endemic only in South Africa.

Objectives: Here we describe the draft genome sequence of a clinical Acinetobacter haemolyticus isolate (A109R1B4) from a rectal swab of a hospitalised patient in South Africa. Methods: Genomic DNA from the isolate was sequenced using an Illumina MiSeq platform. Generated reads were de novo assembled using Qiagen CLC Genomics Workbench. The assembled contigs were annotated and antimicrobial resistance genes and sequence type were identified. Results: The genome comprised 281 contigs with a total assembly length of 3 371 389 bp, a G + C content of 39.9% and an N50 value of 58 196 bp, and reference coverage was 93.08 while the coverage was 88.08. A total of 3387 genes, 3292 coding sequences (CDS), 3175 coding genes and 95 RNA genes were detected. The antimicrobial resistance genes blaOXA-264 and aac(6ʹ)-Ig were detected. Conclusion: The genome sequence reported will serve as a reference point for molecular epidemiological studies of antibiotic-resistant A. haemolyticus in Africa.

Aedes aegypti is the most important arboviral disease vector worldwide. In Africa, it exists as two morphologically distinct forms, often referred to as subspecies, Aaa and Aaf. There is a dearth of information on the distribution and genetic diversity of these two forms in Sudan and other African Sahelian region countries. This study aimed to explore the distribution and genetic diversity of Aedes aegypti subspecies using morphology and Cytochrome oxidase-1 mitochondrial marker in a large Sahelian zone in Sudan. An extensive cross-sectional survey of Aedes aegypti in Sudan was performed. Samples collected from eight locations were morphologically identified, subjected to DNA extraction, amplification, sequencing, and analyses. We classified four populations as Aaa and the other four as Aaf. Out of 140 sequence samples, forty-six distinct haplotypes were characterized. The haplotype and nucleotide diversity of the collected samples were 0.377–0.947 and 0.002–0.01, respectively. Isolation by distance was significantly evident (r = 0.586, p = 0.005). The SAMOVA test indicated that all Aaf populations are structured in one group, while the Aaa clustered into two groups. AMOVA showed 53.53% genetic differences within populations and 39.22% among groups. Phylogenetic relationships indicated two clusters in which the two subspecies were structured. Thus, the haplotype network consisted of three clusters.

The pathogenomics of carbapenem-resistant Aeromonas veronii (A. veronii) isolates recovered from pigs in KwaZulu-Natal, South Africa, was explored by whole genome sequencing on the Illumina MiSeq platform. Genomic functional annotation revealed a vast array of similar central networks (metabolic, cellular, and biochemical). The pan-genome analysis showed that the isolates formed a total of 4349 orthologous gene clusters, 4296 of which were shared; no unique clusters were observed. All the isolates had similar resistance phenotypes, which corroborated their chromosomally mediated resistome (blaCPHA3 and blaOXA-12) and belonged to a novel sequence type, ST657 (a satellite clone). Isolates in the same sub-clades clustered according to their clonal lineages and host. Mobilome analysis revealed the presence of chromosome-borne insertion sequence families. The estimated pathogenicity score (Pscore ≈ 0.60) indicated their potential pathogenicity in humans. Furthermore, these isolates carried several virulence factors (adherence factors, toxins, and immune evasion), in different permutations and combinations, indicating a differential ability to establish infection. Phylogenomic and metadata analyses revealed a predilection for water environments and aquatic animals, with more recent reports in humans and food animals across geographies, making A. veronii a potential One Health indicator bacterium.

Bacillus oleronius strain DSM 9356 isolated from the termite Reticulitermes santonensis was sequenced to gain insights in relation to its closest phylogenetic neighbor Bacillus sporothermodurans. The draft genome of strain DSM 9356 contains 5,083,966 bp with an estimated G + C content of 35%, 4899 protein-coding genes, 116 tRNAs and 18 rRNAs. The RAST annotation assigned these genes into 462 subsystems, with the maximum number of genes associated with amino acids and derivatives metabolism (14.84%), followed by carbohydrates (13.89%) and protein metabolism subsystems (9.10%). The draft genome sequence and annotation has been deposited at NCBI under the accession number MTLA00000000.

Bacillus sporothermodurans currently possesses one of the most highly heat-resistant spores (HRS), which can withstand ultra-high temperature (UHT) processing. Determination of multiple whole genome sequences of B. sporothermodurans provided an opportunity to perform the first comparative genome analysis between strains and with B. oleronius, B. cereus, and B. subtilis groups. In this study, five whole genome sequences of B. sporothermodurans strains, including those belonging to the HRS clone (SAD and BR12) normally isolated from UHT milk, were compared with the aforementioned Bacillus species for gene clusters responsible for heat resistance. In the phylogenomic analysis, B. sporothermodurans, with its closest phylogenetic neighbor, B. oleronius, clustered with B. thermoamylovorans and B. thermotolerans. Heat shock proteins GrpE, GroES, GroEL, and DnaK presented identical sequences for all B. sporothermodurans strains, indicating that differences in functional efficiency are not involved in the thermal resistance variations. However, comparing all species evaluated, B. sporothermodurans exhibited a different gene configuration in the chromosomal region of the heat shock protein GrpE. Furthermore, only B. sporothermodurans strains presented the stage II sporulation protein P gene located in this region. Multisequence alignment and phylogenetic analysis of the ClpB protein showed differences for HRS and non-HRS strains. The study identified ClpC, ClpE, and ClpX as the three ATPases putatively involved in protein disaggregation in B. sporothermodurans. Bacillussporothermodurans exhibits high homology with other Bacillus species in the DnaK, DnaJ, GroEL, and GroES cluster of genes involved in heat resistance. The data presented here pave the way to select and evaluate the phenotypic effects of genes putatively involved in heat resistance.

Whole-genome sequence (WGS) analyses were employed to investigate the genomic epidemiology of extensively drug-resistant Klebsiella pneumoniae strains, focusing on the carbapenem resistance-encoding determinants, mobile genetic support, clonal and epidemiological relationships. A total of ten isolates were obtained from patients admitted to the intensive care unit (ICU) in a public hospital in South Africa. Five isolates were from rectal swabs of colonized patients and five from blood cultures of patients with invasive carbapenem-resistant infections. Following microbial identification and antibiotic susceptibility tests, the isolates were subjected to WGS on the Illumina MiSeq platform. All the isolates showed genotypic resistance to tested β-lactams (NDM-1, OXA-1, CTX-M-15, TEM-1B, SHV-1) and other antibiotics. All but one isolate belonged to the ST152 with a novel sequence type, ST3136, differing by a single-locus variant. The isolates had the same plasmid multilocus sequence type (IncF[K12:A-:B36]) and capsular serotype (KL149), supporting the epidemiological linkage between the clones. Resistance to carbapenems in the 10 isolates was conferred by the blaNDM-1 mediated by the acquisition of multi-replicon [ColRNAI, IncFIB(pB171), Col440I, IncFII, IncFIB(K) and IncFII(Yp)] p18-43_01 plasmid. These findings suggest that the acquisition of blaNDM-1-bearing plasmid structure (p18-43_01), horizontal transfer and clonal dissemination facilitate the spread of carbapenemases in South Africa. This emphasizes the importance of targeted infection control measures to prevent dissemination.

Pathogenomic analysis was performed on a novel carbapenem-resistant Citrobacter freundii isolate (H2730R) from a rectal swab of an adult male patient admitted to a tertiary hospital, Durban, South Africa. H2730R was identified using selective media and API 20e kit. Confirmatory identification and antibiotic susceptibility testing were performed using the VITEK II. H2730R was whole-genome sequenced on the Illumina MiSeq platform. H2730R was resistant to all tested antibiotics except tigecycline and was defined as ST498 by the C. freundii multilocus sequence typing (MLST) database. The estimated pathogenic potential predicted a higher probability (Pscore ≈ 0.875), supporting H2730R as a human pathogen. H2730R harbored 25 putative acquired resistance genes, 4 plasmid replicons, 4 intact prophages, a class 1 integron (IntI1), 2 predominant insertion sequences (IS3 and IS5), numerous efflux genes, and virulome. BLASTn analysis of the blaNDM-1 encoding contig (00022) and its flanking sequences revealed the blaNDM-1 was located on a plasmid similar to the multireplicon p18-43_01 plasmid reported for the spread of carbapenem resistance in South Africa. Phylogenomic analysis showed clustering of H2730R with CF003/CF004 strains in the same clade, suggesting a possible association between C. freundii strains/clones. Acquiring the p18-43_01 plasmid containing blaNDM-1, the diversity, and complex resistome, virulome, and mobilome of this pathogen makes its incidence very worrying regarding mobilized resistance. This study presents the background genomic information for future surveillance and tracking of the spread of carbapenem-resistant Enterobacteriaceae in South Africa.

Background and objectives: Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae is a serious public health issue globally. In this study, the antibiotic resistance genes, virulence factors, mobile genetic elements, and genetic lineages of circulating ESBL-producing K. pneumoniae strains isolated from pigs and humans in Cameroonian abattoirs were investigated using whole genome sequencing (WGS), in order to ascertain zoonotic transmission (viz. from animals to humans and/or vice-versa) in the food chain.Methods: During March–October 2016, 288 nasal and rectal pooled samples from 432 pigs as well as nasal and hand swabs from 82 humans were collected from Cameroon and South Africa. Seven ESBL-producing K. pneumoniae circulating in Cameroonian pig abattoirs were selected and their genomic DNA sequenced using an Illumina MiSeq platform. Generated reads were de novo assembled using the Qiagen CLC Genomics Workbench and SPAdes. The assembled contigs were annotated using RAST and antibiotic resistance genes, virulence factors, plasmids, and bacteriophages were identified with ResFinder, Virulence Finder, PlasmidFinder, and PHAST, respectively.Results: ESBL-producing K. pneumoniae were detected in pigs (34/158; 21.52%) and exposed workers (8/71; 11.26%) in Cameroon only. The circulating K. pneumoniae strains were dominated principally by the sequence type (ST) 14 and 39. In addition, the “high-risk” ST307 clone and two novel STs assigned ST2958 and ST2959 were detected. Genomic analysis identified various antibiotic resistance genes associated with resistance to β-lactams, aminoglycosides, fluoroquinolones, macrolide, lincosamide and streptogramins, rifampicin, sulfonamides, trimethoprim, phenicols and tetracycline. None of the ESBL-producing K. pneumoniae harbored virulence genes. Intermingled K. pneumoniae populations were observed between pig- and human-source within and across abattoirs in the country.Conclusion: Our study shows that ESBL-producing K. pneumoniae is actively disseminating in pigs and occupationally exposed workers in Cameroonian pig abattoirs and is probably underestimated in the absence of molecular epidemiological studies. It suggests pigs, abattoir workers and food products as potential reservoirs and sources of zoonotic transmission in Cameroon. Our findings underline the existence of a potential unheeded food safety and public health threat associated with these resistant strains and reinforce the crucial importance of implementing appropriate food safety measures and promoting rational antibiotic use.

This study undertook genome mining and comparative genomics to gain genetic insights into the dominance of the methicillin-resistant Staphylococcus aureus (MRSA) endemic clone ST612-CC8-t1257-SCCmec_IVd(2B), obtained from the poultry food chain in South Africa. Functional annotation of the genome revealed a vast array of similar central metabolic, cellular and biochemical networks within the endemic clone crucial for its survival in the microbial community. In-silico analysis of the clone revealed the possession of uniform defense systems, restriction-modification system (type I and IV), accessory gene regulator (type I), arginine catabolic mobile element (type II), and type 1 clustered, regularly interspaced, short palindromic repeat (CRISPR)Cas array (N = 7 ± 1), which offer protection against exogenous attacks. The estimated pathogenic potential predicted a higher probability (average Pscore ≈ 0.927) of the clone being pathogenic to its host. The clone carried a battery of putative virulence determinants whose expression are critical for establishing infection. However, there was a slight difference in their possession of adherence factors (biofilm operon system) and toxins (hemolysins and enterotoxins). Further analysis revealed a conserved environmental tolerance and persistence mechanisms related to stress (oxidative and osmotic), heat shock, sporulation, bacteriocins, and detoxification, which enable it to withstand lethal threats and contribute to its success in diverse ecological niches. Phylogenomic analysis with close sister lineages revealed that the clone was closely related to the MRSA isolate SHV713 from Australia. The results of this bioinformatic analysis provide valuable insights into the biology of this endemic clone.

We generated genome sequences from 218 cases of Ebola virus disease (EVD) in Sierra Leone (SLE) during 2014–2015 to complement available datasets, particularly by including cases from a period of low sequence coverage during peak transmission of Ebola virus (EBOV) in the highly-affected Western Area division of SLE. The combined dataset was utilized to produce phylogenetic and phylodynamic inferences, to study sink–source dynamics and virus dispersal from highly-populated transmission hotspots. We identified four districts in SLE where EBOV was introduced and transmission occurred without onward exportation to other districts. We also identified six districts that substantially contributed to the dispersal of the virus and prolonged the EVD outbreak: five of these served as major hubs, with lots of movement in and out, and one acted primarily as a source, exporting the virus to other areas of the country. Positive correlations between case numbers, inter-district transition events, and district population sizes reaffirm that population size was a driver of EBOV transmission dynamics in SLE. The data presented here confirm the role of urban hubs in virus dispersal and of a delayed laboratory response in the expansion and perpetuation of the EVD outbreak in SLE.