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The efficacy of 23 bacterial isolates obtained from surface-sterilized stems and leaves of three medicinal plants (Aloe barbadensis Miller, Artemisia afra, and Moringa oleifera) was investigated in an endeavour to prevent the growth of Mycobacterium bovis using the cross-streak method. Endophytes were isolated by incubating sterile plant materials on nutrient agar at 30 °C for 5 days. Two isolates showing activity were subsequently utilized to produce the extracts. Whole-genome sequencing (WGC) was used to identify the isolates. Secondary metabolites produced after 7 days of growth in nutrient broth were harvested through extraction with ethyl acetate. The extracts were chemically profiled using gas chromatography–high resolution time-of-flight mass spectrometry (GC–HRTOF-MS). NCBI BLAST search results revealed that the isolated endophytes belonged to the Pseudomonas and Enterobacter genera, based on WGC. Two endophytes, Aloe I4 and Aloe I3–I5 from Aloe barbadensis, exhibited potency based on the cross-streak method. The metabolite profiling of the selected endophytes identified 34 metabolites from Aloe I4, including ergotamine, octadecane, L-proline and 143 other metabolites including quinoline and valeramide, which inhibit microbial quorum sensing. These findings suggest that bacterial endophytes from medicinal plants, particularly Aloe barbadensis, hold promise as sources of antimycobacterial agents for human health applications. [ABSTRACT FROM AUTHOR]

Background: We describe the genotypic characteristics and antimicrobial resistance (AMR) determinants of Salmonella enterica serovar Isangi (Salmonella Isangi) clinical isolates in South Africa from 2020 through 2021. Methods: During the years 2020 to 2021, the Centre for Enteric Diseases of the National Institute for Communicable Diseases, a national reference centre in South Africa for human infections resulting from enteric bacterial pathogens, investigated a total of 3549 clinical isolates of Salmonella species. Whole genome sequencing (WGS) was performed using Illumina NextSeq Technology. WGS data was analyzed using Centre for Genomic Epidemiology-based tools and EnteroBase web-based platform. Genotypic relatedness and cluster analysis was investigated based on core-genome multilocus sequence typing. Results: Forty-nine isolates were confirmed to be Salmonella Isangi, with most submitted from Gauteng Province (24/49, 49%). The most prevalent sequence type was ST335 (48/49, 98%), and the remaining 1 isolate was ST216. All ST335 isolates were genotypically multidrug-resistant (MDR), with resistance to fluoroquinolones, chloramphenicol, trimethoprim-sulfamethoxazole and tetracycline; the ST216 isolate was resistant only to aminoglycosides. All ST335 isolates carried ESBL genes, the most common being blaCTX-M-15. Five clusters (consisting of isolates related within five allele differences) were detected, all being ST335. Conclusions: Most Salmonella Isangi isolates in South Africa are MDR and ESBL-positive. Ongoing monitoring of the epidemiology and AMR profile of this serovar is important for public health and treatment guidelines. [ABSTRACT FROM AUTHOR]

The emergence of antimicrobial-resistant, livestock-associated Enterococcus faecalis represents a public health concern. Here, we report the isolation, molecular detection of virulence and antimicrobial resistance determinants, in addition to the phylogenetic analyses of 20 Enterococcus species using whole genome sequencing analysis of 15 Enterococcus faecalis strains including six strains of three novel sequence types, three Enterococcus faecium and two Enterococcus durans. All strains were isolated from food chain animals in South Africa. Enterococcus strains were isolated on bile aesculin azide agar, followed by identification using MALDI-TOF MS analysis. Antibiotic susceptibility testing was performed using the Kirby–Bauer disk diffusion method. The genomic DNA of the isolates was extracted and sequencing was performed using the Illumina MiSeq platform. Sequence reads were trimmed and de novo assembled. The assembled contigs were analyzed for antimicrobial resistance genes and chromosomal mutations, extra-chromosomal plasmids, and multi-locus sequence type (MLST). Multidrug antimicrobial resistance genes conferring resistance to aminoglycosides (ant(6)-Ia, aph(3′)-IIIa, sat4, and spw), lincosamides (lnu(B), lsa(A), and lsa(E)), macrolides (erm(B)), trimethoprim (dfrG) and tetracyclines (tet(L) and tet(M)) were identified. Plasmid replicons were detected in seven E. faecalis and three E. faecium isolates. The sequence type (ST) of each isolate was determined using the Enterococcus PubMLST database. Ten STs were identified in the collection, three of which (ST1240, ST1241, and ST1242) have not been previously reported and are described in the present study for the first time. To compare the sequenced strains to other previously sequenced E. faecalis strains, assembled sequences of E. faecalis from livestock were downloaded from the PubMLST database. Core genome-based phylogenetic analysis was performed using ParSNP. The detection of multiple drug-resistance in Enterococcus including E. faecalis and E. faecium highlights the significance of genomic surveillance to monitor the spread of antimicrobial resistance in food chain animals. In addition, the genome sequences of Enterococcus strains reported in the present study will serve as a reference point for future molecular epidemiological studies of livestock-associated and antibiotic-resistant E. faecalis in Africa. In addition, this study enables the in-depth analysis of E. faecalis genomic structure, as well as provides valuable information on the phenotypic and genotypic antimicrobial resistance, and the pathogenesis of livestock-associated E. faecalis and E. faecium. [ABSTRACT FROM AUTHOR]

We report the genetic characterization of two potentially novel rabies-related lyssaviruses identified from bats in Limpopo province, South Africa. Matlo bat lyssavirus (MBLV) was identified in two Miniopterus natalensis (Natal long-fingered) bats in 2015 and 2016, and Phala bat lyssavirus (PBLV) was identified in a Nycticeinops schlieffeni (Schlieffen's) bat in 2021. The distribution of both of these bat species is largely confined to parts of Africa, with limited reports from the Arabian Peninsula. MBLV and PBLV were demonstrated to group with the unassigned and phylogroup I lyssaviruses, respectively. MBLV was most closely related to Lyssavirus caucasicus (WCBV), whereas PBLV was most closely related to Lyssavirus formosa (TWBLV-1) and Taiwan bat lyssavirus 2 (TWBLV-2), based on analysis of the N and G genes, the concatenated N + P + M + G + L coding sequence, and the complete genome sequence. Based on our analysis, MBLV and WCBV appeared to constitute a phylogroup separate from Lyssavirus lleida (LLEBV) and Lyssavirus ikoma (IKOV). Analysis of the antigenic sites suggests that PBLV will likely be serologically distinguishable from established lyssaviruses in virus-neutralization tests, whereas MBLV appeared to be antigenically highly similar to WCBV. Taken together, the findings suggested that, while PBLV is likely a new lyssavirus species, MBLV is likely related to WCBV. [ABSTRACT FROM AUTHOR]

Simple Summary: Climate change plays a major role in livestock production. Hence the utilization of crossbreeding strategies allows for the improvement of animal production during harsh environmental conditions. The aim of this study was to identify the genetic differences in the F1 Nguni × Bonsmara and its reciprocal cross (Bonsmara × Nguni). This was achieved by studying the changes in structural variation, such as copy number variants in these two crosses. The major findings from this study have revealed several genes relating to adaption in these crossbred cattle. Crossbreeding forms part of Climate-Smart beef production and is one of the strategies to mitigate the effects of climate change. Two Nguni-sired and three Bonsmara-sired crossbred animals underwent whole genome sequencing. Following quality control and file preparation, the sequence data were investigated for genome-wide copy number variation (CNV) using the panelcn.MOPS tool. A total of 355 CNVs were identified in the crossbreds, of which 274 were unique in Bonsmara-sired crossbreds and 81 unique in the Nguni-sired crossbreds. Genes that differed in copy number in both crossbreds included genes related to growth (SCRN2, LOC109572916) and fertility-related factors (RPS28, LOC1098562432, LOC109570037). Genes that were present only in the Bonsmara-sired crossbreds included genes relating to lipid metabolism (MAF1), olfaction (LOC109569114), body size (HES7), immunity (LOC10957335, LOC109877039) and disease (DMBT1). Genes that were present only in the Nguni-sired crossbreds included genes relating to ketosis (HMBOX1) and amino acid transport (LOC109572916). Results of this study indicate that Nguni and Bonsmara cattle can be utilized in crossbreeding programs as they may enhance the presence of economically important traits associated with both breeds. This will produce crossbred animals that are good meat producers, grow faster, have high fertility, strong immunity and a better chance of producing in South Africa's harsh climate conditions. Ultimately, this study provides new genetic insights into the adaptability of Nguni and Bonsmara crossbred cattle. [ABSTRACT FROM AUTHOR]

Intra-host diversity studies are used to characterise the mutational heterogeneity of SARS-CoV-2 infections in order to understand the impact of virus-host adaptations. This study investigated the frequency and diversity of the spike (S) protein mutations within SARS-CoV-2 infected South African individuals. The study included SARS-CoV-2 respiratory samples, from individuals of all ages, received at the National Health Laboratory Service at Charlotte Maxeke Johannesburg Academic hospital, Gauteng, South Africa, from June 2020 to May 2022. Single nucleotide polymorphism (SNP) assays and whole genome sequencing were performed on a random selection of SARS-CoV-2 positive samples. The allele frequency (AF) was determined using TaqMan Genotyper software for SNP PCR analysis and galaxy.eu for analysis of FASTQ reads from sequencing. The SNP assays identified 5.3% (50/948) of Delta cases with heterogeneity at delY144 (4%; 2/50), E484Q (6%; 3/50), N501Y (2%; 1/50) and P681H (88%; 44/50), however only heterogeneity for E484Q and delY144 were confirmed by sequencing. From sequencing we identified 9% (210/2381) of cases with Beta, Delta, Omicron BA.1, BA.2.15, and BA.4 lineages that had heterogeneity in the S protein. Heterogeneity was primarily identified at positions 19 (1.4%) with T19IR (AF 0.2–0.7), 371 (92.3%) with S371FP (AF 0.1–1.0), and 484 (1.9%) with E484AK (0.2–0.7), E484AQ (AF 0.4–0.5) and E484KQ (AF 0.1–0.4). Mutations at heterozygous amino acid positions 19, 371 and 484 are known antibody escape mutations, however the impact of the combination of multiple substitutions identified at the same position is unknown. Therefore, we hypothesise that intra-host SARS-CoV-2 quasispecies with heterogeneity in the S protein facilitate competitive advantage of variants that can completely/partially evade host's natural and vaccine-induced immune responses. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 lineages and variants of concern (VOC) have gained more efficient transmission and immune evasion properties with time. We describe the circulation of VOCs in South Africa and the potential role of low-frequency lineages on the emergence of future lineages. Whole genome sequencing was performed on SARS-CoV-2 samples from South Africa. Sequences were analysed with Nextstrain pangolin tools and Stanford University Coronavirus Antiviral & Resistance Database. In 2020, 24 lineages were detected, with B.1 (3%; 8/278), B.1.1 (16%; 45/278), B.1.1.348 (3%; 8/278), B.1.1.52 (5%; 13/278), C.1 (13%; 37/278) and C.2 (2%; 6/278) circulating during the first wave. Beta emerged late in 2020, dominating the second wave of infection. B.1 and B.1.1 continued to circulate at low frequencies in 2021 and B.1.1 re-emerged in 2022. Beta was outcompeted by Delta in 2021, which was thereafter outcompeted by Omicron sub-lineages during the 4th and 5th waves in 2022. Several significant mutations identified in VOCs were also detected in low-frequency lineages, including S68F (E protein); I82T (M protein); P13L, R203K and G204R/K (N protein); R126S (ORF3a); P323L (RdRp); and N501Y, E484K, D614G, H655Y and N679K (S protein). Low-frequency variants, together with VOCs circulating, may lead to convergence and the emergence of future lineages that may increase transmissibility, infectivity and escape vaccine-induced or natural host immunity. [ABSTRACT FROM AUTHOR]

The iconic Cape buffalo has experienced several documented population declines in recent history. These declines have been largely attributed to the late 19th century rinderpest pandemic. However, the effect of the rinderpest pandemic on their genetic diversity remains contentious, and other factors that have potentially affected this diversity include environmental changes during the Pleistocene, range expansions and recent human activity. Motivated by this, we present analyses of whole genome sequencing data from 59 individuals from across the Cape buffalo range to assess present‐day levels of genome‐wide genetic diversity and what factors have influenced these levels. We found that the Cape buffalo has high average heterozygosity overall (0.40%), with the two southernmost populations having significantly lower heterozygosity levels (0.33% and 0.29%) on par with that of the domesticated water buffalo (0.29%). Interestingly, we found that these lower levels are probably due to recent inbreeding (average fraction of runs of homozygosity 23.7% and 19.9%) rather than factors further back in time during the Pleistocene. Moreover, detailed investigations of recent demographic history show that events across the past three centuries were the main drivers of the exceptional loss of genetic diversity in the southernmost populations, coincident with the onset of colonialism in the southern extreme of the Cape buffalo range. Hence, our results add to the growing body of studies suggesting that multiple recent human‐mediated impacts during the colonial period caused massive losses of large mammal abundance in southern Africa. [ABSTRACT FROM AUTHOR]

Background: Rifampicin resistance missed by commercial rapid molecular assays but detected by phenotypic assays may lead to discordant susceptibility results and affect patient management. Objective: This study was conducted to evaluate the causes of rifampicin resistance missed by the GenoType MTBDR plus and its impact on the programmatic management of tuberculosis in KwaZulu-Natal, South Africa. Methods: We analysed routine tuberculosis programme data from January 2014 to December 2014 on isolates showing rifampicin susceptibility on the GenoType MTBDR plus assay but resistance on the phenotypic agar proportion method. Whole-genome sequencing was performed on a subset of these isolates. Results: Out of 505 patients with isoniazid mono-resistant tuberculosis on the MTBDR plus , 145 (28.7%) isolates showed both isoniazid and rifampicin resistance on the phenotypic assay. The mean time from MTBDR plus results to initiation of drug-resistant tuberculosis therapy was 93.7 days. 65.7% of the patients had received previous tuberculosis treatment. The most common mutations detected in the 36 sequenced isolates were I491F (16; 44.4%) and L452P (12; 33.3%). Among the 36 isolates, resistance to other anti-tuberculosis drugs was 69.4% for pyrazinamide, 83.3% for ethambutol, 69.4% for streptomycin, and 50% for ethionamide. Conclusion: Missed rifampicin resistance was mostly due to the I491F mutation located outside the MTBDR plus detection area and the L452P mutation, which was not included in the initial version 2 of the MTBDR plus. This led to substantial delays in the initiation of appropriate therapy. The previous tuberculosis treatment history and the high level of resistance to other anti-tuberculosis drugs suggest an accumulation of resistance. [ABSTRACT FROM AUTHOR]

Samples showing blotchy mottle symptoms were collected from soybeans in North-West province, South Africa. The assembly of high-throughput sequencing data from three samples yielded contigs of 13,426 to 13,435nt, which represent the first complete genome sequences of soybean blotchy mosaic virus (SbBMV). SbBMV shows a typical cytorhabdovirus gene organization (3′-N-P-P3-M-G-L-5'), with each putative gene product being most similar, but with only 49.1-71.1% sequence identity, to those of cucurbit cytorhabdovirus 1. Given the species demarcation thresholds for rhabdoviruses, SbBMV is thus a distinct member of the genus Cytorhabdovirus. [ABSTRACT FROM AUTHOR]

Background: Rifampicin-resistant tuberculosis (RR-TB) remains an important global health problem. Ideally, the complete drug-resistance profile guides individualized treatment for all RR-TB patients, but this is only practised in high-income countries. Implementation of whole genome sequencing (WGS) technologies into routine care in low and middle-income countries has not become a reality due to the expected implementation challenges, including translating WGS results into individualized treatment regimen composition.Methods: This trial is a pragmatic, single-blinded, randomized controlled medical device trial of a WGS-guided automated treatment recommendation strategy for individualized treatment of RR-TB. Subjects are 18 years or older and diagnosed with pulmonary RR-TB in four of the five health districts of the Free State province in South Africa. Participants are randomized in a 1:1 ratio to either the intervention (a WGS-guided automated treatment recommendation strategy for individualized treatment of RR-TB) or control (RR-TB treatment according to the national South African guidelines). The primary effectiveness outcome is the bacteriological response to treatment measured as the rate of change in time to liquid culture positivity during the first 6 months of treatment. Secondary effectiveness outcomes include cure rate, relapse rate (recurrence of RR-TB disease) and TB free survival rate in the first 12 months following RR-TB treatment completion. Additional secondary outcomes of interest include safety, the feasibility of province-wide implementation of the strategy into routine care, and health economic assessment from a patient and health systems perspective.Discussion: This trial will provide important real-life evidence regarding the feasibility, safety, cost, and effectiveness of a WGS-guided automated treatment recommendation strategy for individualized treatment of RR-TB. Given the pragmatic nature, the trial will assist policymakers in the decision-making regarding the integration of next-generation sequencing technologies into routine RR-TB care in high TB burden settings.Trial Registration: ClinicalTrials.gov NCT05017324. Registered on August 23, 2021. [ABSTRACT FROM AUTHOR]

The poxvirus lumpy skin disease virus (LSDV) is the causative agent of the vexatious lumpy skin disease, which predominantly affects cattle and water buffalo. It has been endemic to South Africa since the 1950s, and in 1960, a live attenuated vaccine was commercially released for use in the country to mitigate the spread of this transboundary disease. This vaccine (Neethling/vaccine/LW-1959) was generated from serial passages of the prototype lumpy skin disease virus strain Neethling-WC/RSA/1957, which was isolated in 1957 from an outbreak in the Western Cape province of South Africa and was subsequently used to prove the infectious nature of the virus and the resulting disease in cattle. In this study, we determined the complete genome sequence of the LSDV prototype strain Neethling-WC/RSA/1957, as well as three other LSDV isolates from the 1950s, one wild-type isolate from the 1970s, and a commercial vaccine produced in 1988 (LW-1959). Phylogenomic analysis showed that all six sequences were in cluster 1.1, along with previous sequences of the vaccine strain, the oldest known isolate (LSDV/Haden/RSA/1954), and virulent viruses isolated in the 1990s from South Africa. Seven single-nucleotide polymorphisms were identified between the Neethling-WC/RSA/1957 strain and the vaccine strain (LW-1959), providing new insights into virus attenuation and possible markers for DIVA assays. [ABSTRACT FROM AUTHOR]

African wild dogs (Lycaon pictus) have undergone severe population reductions and are listed as endangered on the International Union for Conservation of Nature Red List. Small, isolated populations have the potential to suffer from threats to their genetic diversity that may impact species viability and future survival. This study provides the first set of population-wide genomic data to address conservation concerns for this endangered species. Whole genome sequencing data were generated for 71 free-ranging African wild dogs from the Kruger National Park (KNP), South Africa, and used to estimate important population genomic parameters. Genomic diversity metrics revealed that variation levels were low; however, this African wild dog population showed low levels of inbreeding. Very few first- and second-order relationships were observed in this cohort, with most relationships falling into the third-order or distant category. Patterns of homozygosity could have resulted from historical inbreeding or a loss in genome variation due to a population bottleneck. Although the results suggest that this stronghold African wild dog population maintains low levels of inbreeding, likely due to their cooperative breeding system, it may lead to a continuous population decline when a reduced number of suitable mates are available. Consequently, the low genomic variation may influence species viability over time. This study highlights the importance of assessing population genomic parameters to set conservation priorities. Future studies should include the investigation of the potential of this endangered species to adapt to environmental changes considering the low genomic diversity in this population. [ABSTRACT FROM AUTHOR]

Although Old World alphaviruses, Middelburg- (MIDV) and Sindbis virus (SINV), have previously been detected in horses and wildlife with neurologic disease in South Africa, the pathogenesis and clinical presentation of MIDV and SINV infections in animals are not well documented. Clinical samples from horses across South Africa with acute or fatal neurologic and febrile infections submitted between 2014–2018 were investigated. In total, 69/1084 (6.36%) and 11/1084 (1.01%) horses tested positive for MIDV and SINV, respectively, by real-time reverse transcription (RT) PCR. Main signs/outcomes for MIDV (n = 69): 73.91% neurological, 75.36% fever, 28.99% icterus and anorexia, respectively, 8.70% fatalities; SINV (n = 11): 54.54% neurological, 72.73% fever, 36.36% anorexia and 18.18% fatalities. MIDV cases peaked in the late summer/autumn across most South African provinces while SINV cases did not show a clear seasonality and were detected in fewer South African provinces. MIDV could still be detected in blood samples via RT-PCR for up to 71,417 and 21 days after onset of signs in 4 horses respectively, suggesting prolonged replication relative to SINV which could only be detected in the initial sample. Phylogenetic analyses based on partial sequences of the nsP4 (MIDV n = 59 and SINV n = 7) and E1 (MIDV n = 45) genes, as well as full genome sequences (MIDV n = 6), clustered the MIDV and SINV strains from the present study with previously detected strains. MIDV infection appears to be more prevalent in horses than SINV infection based on RT-PCR results, however, prevalence estimates might be different when also considering serological surveillance data. [ABSTRACT FROM AUTHOR]

Research into the phylogenetic relationships of lumpy skin disease virus (LSDV) strains was long overlooked, partially due to its original restricted distribution to sub‐Saharan Africa. However, recent incursions into northern latitudes, and a rapid spread causing major economic losses worldwide, have intensified additional research on the disease and the causative virus. This study delineates the phylogeny of LSDV in the context of full genome sequences of strains recovered in the field, as well as strains highly passaged in cell culture. We sequenced the oldest known field strain to date (isolate LSDV/Haden/RSA/1954 [South Africa] recovered from an outbreak in 1954), a recent field isolate (LSDV/280‐KZN/RSA/2018 [South Africa] sequenced directly from blood during an outbreak in 2018) and strain LSDV/Russia/Dagestan‐75 (a high‐passaged cell culture strain derived from the field strain, LSDV/Russia/Dagestan/2015 [Russia]). Sequence analysis placed the field strain LSDV/Haden/RSA/1954 in the same cluster (cluster 1.1) with attenuated Neethling‐type commercial vaccine viruses, with eight SNP differences, discrediting the previously held hypothesis that cluster 1.1 vaccine strains were derived from cluster 1.2 field viruses via the process of attenuation between them. In contrast, the recent LSDV/280‐KZN/RSA/2018 isolate grouped with other recent field isolates in cluster 1.2, providing evidence that cluster 1.1 strains were displaced by cluster 1.2 strains in South Africa. Based on the field isolates between 1954 and 2018, the substitution rate of 7.4 × 10–6 substitutions/site/year was established, with mutations occurring in either synonymous sites or intergenic regions. This is the first evolutionary metric recorded for LSDV. Comparing the genome sequences of high‐passage strains of LSDV showed that propagation in vitro without animal host selective pressure generates mainly non‐synonymous SNPs in virus‐replication genes. These results improve our understanding of LSDV evolution and demonstrate that the population dynamics of circulating isolates is not constant, with LSDV associated with different genetic clusters dominating the landscape during specific periods in time. [ABSTRACT FROM AUTHOR]

Bagaza virus (BAGV) is a mosquito-borne orthoflavivirus known to occur in regions of southern Europe, Africa, India and the Middle East. The virus has been associated with neurological disease and fatalities in various wild bird species. Association with human disease is not confirmed although limited serological evidence has suggested human infection. Surveillance programs for screening mosquitoes for evidence of arbovirus infection play an important role in providing information regarding the circulation and spread of viruses in specific regions. BAGV was detected in a mosquito pool during surveillance of mosquitoes collected in central South Africa between November 2019 and March 2023. Homogenized mosquito pools were screened for flaviviral RNA using conventional RT-PCR and virus isolation was attempted on positive samples. BAGV was detected and subsequently isolated using cell culture. A multiplex tiling PCR method for targeted enrichment using a PCR based or amplicon sequencing approach of the complete genome of BAGV was developed and optimized. Primers were designed using alignment of complete genome sequence data retrieved from GenBank to identify suitable primer sites that would generate overlapping fragments spanning the complete genome. Six forward primers and eight reverse primers were identified that target the complete genome and amplified nine overlapping fragments, that ranged in length from 1954 to 2039 with an overlap ranging from 71 to 711 base pairs. The design strategy included multiple forward and reverse primer pairs for the 5' and 3' ends. Phylogenetic analysis with other isolates was performed and BAGV isolate VBD 74/23/3 was shown to share high similarity with previous BAGV isolates from all regions, with genetic distance ranging from 0.026 to 0.083. VBD 74/23/3 was most closely related to previous isolates from southern Africa, ZRU96/16/2 isolated from a post-mortem sample from a pheasant in 2016 and MP-314-NA-2018 isolated from mosquitoes in northwestern Namibia with genetic distance 0.0085 and 0.016 respectively. Currently there is limited complete genome sequence data available for many of the arboviruses circulating in Africa. The multiplex tiling method provided a simple and cost-effective method for obtaining complete genome sequence. This method can be readily applied to other viruses using sequence data from publicly available databases and would have important application facilitating genomic surveillance of arboviruses in low resource countries. • Detection and isolation of Bagaza virus from a mosquito pool in central South Africa. • Evidence for circulation of Bagaza virus in central South Africa in Aedes mosquitoes. • Complete genome sequencing of Bagaza virus isolated in cell culture using multiplex PCR tiling method for target enrichment. [ABSTRACT FROM AUTHOR]

• NGS detection of a new mutations in early emerging SARS-CoV-2 Omicron variant. • Weakly and transient Y170W spike mutation during emergence of Omicron variant. • Absence of Y170W spike mutation impact in binding to the SARS-CoV-2 ACE2 receptor. • Absence of Y170W mutation impact onto neutralizing antibodies sites interactions. To assess the genetic characteristics of the early emerging SARS-CoV-2 Omicron variant strains, we retrospectively analyzed a collection of 150 nasopharyngeal samples taken from a series of outpatient cases tested positive by a referenced qRT-PCR assay during the reported period of Omicron variant emergence in December 2021, in northeastern region of France. Next Generation Sequencing (NGS) analysis of SARS-CoV-2 spike sequences revealed that only 3 (2 %) of these detected strains were Omicron variants, while 147 (98 %) were identified as previously described delta variants. Our phylogenetic analyzes of SARS-CoV-2 RNA genomes showed that these French early emerging Omicron variants may have originated from South Africa or India. In addition, whole viral genome sequences NGS comparison analyzes allowed us to identify an original and uncharacterized Y170W spike mutation that was weakly and transiently detected during the period of SARS-CoV-2 Omicron variant emergence in human populations. Molecular modeling and docking experiments indicated that this original mutated residue Y170W was neither directly involved in binding to the SARS-CoV-2 receptor ACE2 nor in interacting with known neutralizing antibody sites. However, this new mutation may be responsible for preventing the transition from the closed to the open Spike conformation, thus promoting the early emergence of the Omicron variant. Overall, these results underscore the epidemiological utility of a routine whole-genome viral NGS strategy that enables genotypic characterization of emerging or mutant SARS-CoV-2 variants, which could have significant implications for public health policy. [ABSTRACT FROM AUTHOR]

The rapid emergence and spread of numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants across the globe underscores the crucial need for continuous SARS-CoV-2 surveillance to ensure that potentially more pathogenic variants are detected early and contained. Whole genome sequencing (WGS) is currently the gold standard for COVID-19 surveillance; however, it remains cost-prohibitive and requires specialized technical skills. To increase surveillance capacity, especially in resource-scarce settings, supplementary methods that are cost- and time-effective are needed. Real-time multiplex PCR genotyping assays offer an economical and fast solution for screening circulating and emerging variants while simultaneously complementing existing WGS approaches. In this study we evaluated the AllplexTM SARS-CoV-2 Variants II multiplex real-time PCR genotyping assay, Seegene (South Korea), and implemented it in retrospectively characterizing circulating SARS-CoV-2 variants in a rural South African setting between April and October 2021, prior to the emergence of the Omicron variant in South Africa. The AllplexTM SARS-CoV-2 Variants II real-time PCR assay demonstrated perfect concordance with whole-genome sequencing in detecting Beta and Delta variants and exhibited high specificity, sensitivity and reproducibility. Implementation of the assay in characterization of SARS-CoV-2 variants between April and October 2021 in a rural South African setting revealed a rapid shift from the Beta to the Delta variant between April and June. All specimens successfully genotyped in April were Beta variants and the Delta variant was not detected until May. By June, 78% of samples genotyped were Delta variants and in July >95% of all genotyped samples were Delta variants. The Delta variant continued to predominate through to the end of our analysis in October 2021. Taken together, a commercial SARS-CoV-2 variant genotyping assay detected the rapid rate at which the Delta variant displaced the Beta variant in Limpopo, an under-monitored province in South Africa. Such assays provide a quick and cost-effective method of monitoring circulating variants and should be used to complement genomic sequencing for COVID-19 surveillance especially in resource-scarce settings. [ABSTRACT FROM AUTHOR]

Background: The genus Planococcus is comprised of halophilic bacteria generally reported for the production of carotenoid pigments and biosurfactants. In previous work, we showed that the culturing of the orange-pigmented Planococcus sp. CP5-4 isolate increased the evaporation rate of industrial wastewater brine effluent, which we attributed to the orange pigment. This demonstrated the potential application of this bacterium for industrial brine effluent management in evaporation ponds for inland desalination plants. Here we identified a C30-carotenoid biosynthetic gene cluster responsible for pigment biosynthesis in Planococcus sp. CP5-4 through isolation of mutants and genome sequencing. We further compare the core genes of the carotenoid biosynthetic gene clusters identified from different Planococcus species' genomes which grouped into gene cluster families containing BGCs linked to different carotenoid product chemotypes. Lastly, LC–MS analysis of saponified and unsaponified pigment extracts obtained from cultures of Planococcus sp. CP5-4, revealed the structure of the main (predominant) glucosylated C30-carotenoid fatty acid ester produced by Planococcus sp. CP5-4. Results: Genome sequence comparisons of isolated mutant strains of Planococcus sp. CP5-4 showed deletions of 146 Kb and 3 Kb for the non-pigmented and "yellow" mutants respectively. Eight candidate genes, likely responsible for C30-carotenoid biosynthesis, were identified on the wild-type genome region corresponding to the deleted segment in the non-pigmented mutant. Six of the eight candidate genes formed a biosynthetic gene cluster. A truncation of crtP was responsible for the "yellow" mutant phenotype. Genome annotation revealed that the genes encoded 4,4′-diapolycopene oxygenase (CrtNb), 4,4′- diapolycopen-4-al dehydrogenase (CrtNc), 4,4′-diapophytoene desaturase (CrtN), 4,4′- diaponeurosporene oxygenase (CrtP), glycerol acyltransferase (Agpat), family 2 glucosyl transferase 2 (Gtf2), phytoene/squalene synthase (CrtM), and cytochrome P450 hydroxylase enzymes. Carotenoid analysis showed that a glucosylated C30-carotenoid fatty acid ester, methyl 5-(6-C17:3)-glucosyl-5, 6′-dihydro-apo-4, 4′-lycopenoate was the main carotenoid compound produced by Planococcus sp. CP5-4. Conclusion: We identified and characterized the carotenoid biosynthetic gene cluster and the C30-carotenoid compound produced by Planococcus sp. CP5-4. Mass-spectrometry guided analysis of the saponified and unsaponified pigment extracts showed that methyl 5-glucosyl-5, 6-dihydro-apo-4, 4′-lycopenoate esterified to heptadecatrienoic acid (C17:3). Furthermore, through phylogenetic analysis of the core carotenoid BGCs of Planococcus species we show that various C30-carotenoid product chemotypes, apart from methyl 5-glucosyl-5, 6-dihydro-apo-4, 4′-lycopenoate and 5-glucosyl-4, 4-diaponeurosporen-4′-ol-4-oic acid, may be produced that could offer opportunities for a variety of applications. [ABSTRACT FROM AUTHOR]

Background Rift Valley fever virus (RVFV) is an arbovirus that causes epizootics and epidemics among livestock population and humans. Our surveillance system has revealed multiple emergences and re-emergences of RVFV in West Africa over the last decade. Methods The Sentinel Syndromic Surveillance Network in Senegal (4S) has been implemented since 2011. Samples from human suspected arbovirus infection in 4S sentinel sites were sent to Institut Pasteur de Dakar (IPD), where arbovirus diagnosis by enzyme-linked immunosorbent assay (ELISA), real-time reverse transcription polymerase chain reaction (RT-PCR), and virus isolation was performed. Overall, IPD has received a total of 1149 samples from arboviral suspected patients through the 4S network from January to December 2020. These samples were screened for 7 arboviruses including RVFV. Whole-genome sequencing of positive RVFV samples by RT-PCR was performed using the Illumina Miseq platform followed by genome assembly. Phylogenetic analyses were performed using MEGA X. Results Out of the 1149 arbovirus suspected cases, 4 RVFV-positive samples were detected with RT-PCR while 5 RVFV-positive samples were detected by ELISA. Complete genome sequences were obtained for 3 strains among the 4 positive samples by RT-PCR. Phylogenetic analyses indicated an emergence of a virus first described in South Africa during a major outbreak. Conclusions This strong surveillance system allowed the detection of an RVFV outbreak in Senegal in 2020. The obtained genomes clustered with strains from South Africa belonging to lineage H. This calls for implementation of a strong surveillance system for wild animals, humans, and livestock simultaneously in all African countries. [ABSTRACT FROM AUTHOR]

Background: The Old world Alphavirus, Middelburg virus (MIDV), is not well known and although a few cases associated with animal illness have previously been described from Southern Africa, there has been no investigation into the association of the virus with human illness. The current study aimed to investigate possible association of MIDV infection with febrile or neurological manifestations in hospitalized or symptomatic patients fromGauteng, South Africa. Methods: This study is a descriptive retrospective and prospective laboratory based study. Archived cerebrospinal fluid (CSF) samples submitted to the National Health Laboratory Service (NHLS), Tshwane Academic division for viral investigation from public sector hospitals in Gauteng as well as EDTA (ethylenediaminetetraacetic acid) whole blood samples from ad hoc cases of veterinary students, presenting with neurological and febrile illness, were selected and screened for the presence of alphaviruses using real-time reverse transcription(rtRT) PCR.Virus isolations from rtRT-PCR positive samples were conducted in Vero cell culture and used to obtain full genome sequences. Basic descriptive statistical analysis was conducted using EpiInfo. Results: MIDV was detected by rtRT-PCR in 3/187 retrospective CSF specimens obtained from the NHLS from hospitalised patients in the Tshwane region of Gauteng and 1/2 EDTA samples submitted in the same year (2017) from ad hoc query arbovirus cases from veterinary students from the Faculty of Veterinary Science University of Pretoria.Full genome sequences were obtained for virus isolates from two cases; one from an EDTA whole blood sample (ad hoc case) and another from a CSF sample (NHLS sample).Two of the four Middelburg virus positive cases,for which clinical information was available, had other comorbidities or infections at the time of infection. Conclusion: Detection of MIDV in CSF of patients with neurological manifestations suggests that the virus should be investigated as a human pathogen with the potential of causing or contributing to neurological signs in children and adults. Author summary: Old World alphaviruses are mainly associated with polyarthritis in humans. Global emergence of chikungunya virus raises concerns regarding disease potential of neglected African arboviruses.New World alphaviruses are associated with severe neurological disease in humans and horses in the Americas with a high mortality rate. Middelburg virus (MIDV) is an Old World alphavirus previously associated with neurological disease and fatalities in horses and wildlife in southern Africa but not yet linked to disease in humans. This study investigated MIDV in humans presenting with acute neurological symptoms in South Africa. MIDV infection was confirmed in 4/189 (2%) such cases using a generic Alphavirus real-time RT-PCR and phylogenetic analysis. MIDV full genomes were obtained from a cerebrospinal fluid sample of a 2-year-old child and a whole blood sample of a veterinary student, both presenting with neurological signs. This study suggests MIDV may be associated with unsolved neurological disease in humans in Africa. [ABSTRACT FROM AUTHOR]

Klebsiella grimontii is a recently identified species that has been implicated in clinical infections. Few or no reports on environmental K. grimontii exist from Africa. This study was part of a broader longitudinal research that aimed to assess the distribution, antibiotic resistance patterns, and genomics of Enterobacterales obtained from raw and treated wastewater and the associated river water of a wastewater treatment plant (WWTP) in KwaZulu-Natal, South Africa. We reported the genomics of an antibiotic resistant Klebsiella grimontii isolate obtained from the raw influent of the WWTP. Following phenotypic characterisation and antibiotic susceptibility testing, the isolate was sequenced on an Illumina MiSeq machine. Raw reads were assembled de novo by using SPAdes (v3.6.2) prior to bioinformatics analysis. The assembled K. grimontii INF139 genome was 6,369,878 bp, with 113 contiguous sequences (>200 bp) and 55.4 % GC content. The isolate was assigned a novel sequence type ST350. Genomic analysis revealed the presence of chromosomally encoded ß-lactamase (blaOXY-6-1) and fluoroquinolone (oqxB) resistance genes. Virulence factors encoding antiphagocytic, iron acquisition, and Intracellular survival properties were identified. Mobilome analysis revealed the presence of plasmid replicons (ColRNAI, FIA (pBK30683), IncFII, IncFII(Yp)) and insertion sequences (ISIX2, ISSen9, ISIS, IS1A). To our knowledge, this is the first description of antibiotic resistant K. grimontii from the water environment from Africa. The presence of this antibiotic resistant and potentially pathogenic isolate in the water environment is worrying as it may be disseminated into river systems used by informal settlements. [ABSTRACT FROM AUTHOR]

At Wuhan, in December 2019, the SRAS-CoV-2 outbreak was detected and it has been the pandemic worldwide. This study aims to investigate the mutations in sequence of the SARS-CoV-2 genome and characterize the mutation patterns in Egyptian COVID-19 patients during different waves of infection. The samples were collected from 250 COVID-19 patients and the whole genome sequencing was conducted using Next Generation Sequencing. The viral sequence analysis showed 1115 different genome from all Egyptian samples in the second wave mutations including 613 missense mutations, 431 synonymous mutations, 25 upstream gene mutations, 24 downstream gene mutations, 10 frame-shift deletions, and 6 stop gained mutation. The Egyptian genomic strains sequenced in second wave of infection are different to that of the first wave. We observe a shift of lineage prevalence from the strain B.1 to B.1.1.1. Only one case was of the new English B.1.1.7. Few samples have one or two mutations of interest from the Brazil and South Africa isolates. New clade 20B appear by March 2020 and 20D appear by May 2020 till January 2021. [ABSTRACT FROM AUTHOR]

Objectives: Colistin is a last-resort antibiotic for the treatment of carbapenem-resistant Gram-negative infections. Colistin resistance thus poses a threat to human health. Colistin resistance is most commonly encoded by mutations in chromosomal pmrA, pmrB, phoP, phoQ, ccrB, and mgrB genes, and the presence of plasmid-mediated mcr genes. This study describes colistin resistance mechanisms in clinical Enterobacterales isolates from the Western Cape, South Africa. Results:Escherichia coli (n = 22) and Klebsiella spp. (n = 7) isolates, from nine health care facilities, were confirmed to be colistin resistant during 2016 and 2017. mcr-1 was present in 55% (12/22) of E. coli and 71% (5/7) of Klebsiella spp. isolates. Colistin resistance mutations in pmrB were identified in 8/10 mcr-negative E. coli isolates using whole-genome sequencing, with pmrB Pro-94→Gln being the most frequent with presence in 4 isolates. One mcr-negative Klebsiella spp. isolate had a complete deletion of the mgrB and one contained an insertion sequence (IS1) in mgrB. Conclusion: A reduction in the proportion of colistin-resistant isolates harboring mcr-1 from 2016 to 2017 was observed. Colistin-resistant E. coli attributed by chromosomal mutations in pmrB in 2017 were mostly clonal related, which contrasts with the 2016 unrelated mcr-1-positive isolates. The diverse strains, hospitals, and resistance mechanisms may suggest that selective pressure is the main driver of colistin resistance. [ABSTRACT FROM AUTHOR]

Enterococcus faecalis is a ubiquitous bacterium found in various environments, including processed beef meat, and is known for its importance in both food safety and public health. This pivotal significance stems not solely from its virulence but also from its adeptness in eliciting multidrug-resistant infections in humans. The aim of this study was to investigate the population structure, resistome, mobilome, and virulome of E. faecalis obtained from processed beef meat sources in South Africa. A total of eight genomes sequenced in this study were examined, alongside 78 publicly available, high-quality genomes of E. faecalis , with a comprehensive analysis conducted to identify antimicrobial resistance (AMR) determinants, virulence factors, and mobile genetic elements (MGE). Six distinct sequence types (STs) (ST79, ST860, ST40, ST238, ST21, and ST700) and 41 core virulence factors were found across all the genomes. The virulence factors included genes encoding adherence (ace, asa1, Ef0485, ebpA, ebpB, ebpC, srtC); exoenzyme (Ef3023, Ef0818, gelE, sprE); immunomodulation (cpsA, cpsB, cpsC, cpsD, cpsE, cpsF, cpsG, cpsH, cpsI, cpsK), and biofilm formation (bopD, fsrA, fsrB, fsrC). In addition, AMR genes were identified across all genomes, which include aminoglycoside resistance (ant(6)-Ia), trimethoprim resistance (dfrA), drug and biocide resistance (efrA and efrB), multidrug efflux pump (emeA), clindamycin quinupristin-dalfopristin, dalfopristin resistance (lsaA), and tetracycline resistance (tetM). The genomes of E. faecalis sequenced here contained a variety of MGEs, including Insertion Sequences (ISs), transposons, prophages, and plasmids, which may have facilitated genetic exchange within and between these species. The results highlight that beef meat products act as a reservoir for virulent E. faecalis strains possessing antibiotic-resistance traits. This study provides insight into the genomic characteristics, antimicrobial resistance genes, virulence factors, and genetic mobile elements associated with eight E. faecalis isolates from processed beef meat in the Gauteng province of South Africa. • Enterococcus faecalis is a ubiquitous bacterium found in various environments, including processed beef meat, and is known for its importance in both food safety and public health. This pivotal significance stems not solely from its virulence but also from its adeptness in eliciting multidrug-resistant infections in humans. The aim of this study was to investigate the population structure, resistome, mobilome, and virulome of E. faecalis obtained from processed beef meat sources in South Africa. A total of eight genomes sequenced in this study were examined, alongside 78 publicly available, high-quality genomes of E. faecalis , with a comprehensive analysis conducted to identify antimicrobial resistance (AMR) determinants, virulence factors, and mobile genetic elements (MGE).Six distinct sequence types (STs) (ST79, ST860, ST40, ST238, ST21, and ST700) and 41 core virulence factors were found across all the genomes. The virulence factors included genes encoding adherence (ace, asa1, Ef0485, ebpA, ebpB, ebpC, srtC); exoenzyme (Ef3023, Ef0818, gelE, sprE); immunomodulation (cpsA, cpsB, cpsC, cpsD, cpsE, cpsF, cpsG, cpsH, cpsI, cpsK), and biofilm formation (bopD, fsrA, fsrB, fsrC). In addition, AMR genes were identified across all genomes, which include aminoglycoside resistance (ant(6)-Ia), trimethoprim resistance (dfrA), drug and biocide resistance (efrA and efrB), multidrug efflux pump (emeA), clindamycin quinupristin-dalfopristin, dalfopristin resistance (lsaA), and tetracycline resistance (tetM). The genomes of E. faecalis sequenced here contained a variety of MGEs, including Insertion Sequences (ISs), transposons, prophages, and plasmids replicons, which may have facilitated genetic exchange within and between these species. The results highlights that beef meat products act as a reservoir for virulent E. faecalis strains possessing antibiotic-resistance traits. This study provides insight into the genomic characteristics, antimicrobial resistance genes, virulence factors, and genetic mobile elements associated with eight E. faecalis isolates from processed beef meat in the Gauteng province of South Africa. [ABSTRACT FROM AUTHOR]

Patterns of transmission of drug-resistant tuberculosis (TB) remain poorly understood, despite over half a million incident cases worldwide in 2017. Modeling TB transmission networks can provide insight into drivers of transmission, but incomplete sampling of TB cases can pose challenges for inference from individual epidemiologic and molecular data. We assessed the effect of missing cases on a transmission network inferred from Mycobacterium tuberculosis sequencing data on extensively drug-resistant TB cases in KwaZulu-Natal, South Africa, diagnosed in 2011–2014. We tested scenarios in which cases were missing at random, missing differentially by clinical characteristics, or missing differentially by transmission (i.e. cases with many links were under- or oversampled). Under the assumption that cases were missing randomly, the mean number of transmissions per case in the complete network needed to be larger than 20, far higher than expected, to reproduce the observed network. Instead, the most likely scenario involved undersampling of high-transmitting cases, and models provided evidence for super-spreading. To our knowledge, this is the first analysis to have assessed support for different mechanisms of missingness in a TB transmission study, but our results are subject to the distributional assumptions of the network models we used. Transmission studies should consider the potential biases introduced by incomplete sampling and identify host, pathogen, or environmental factors driving super-spreading. [ABSTRACT FROM AUTHOR]

Background Tuberculosis (TB) is the leading infectious cause of death globally, and drug-resistant TB strains pose a serious threat to controlling the global TB epidemic. The clinical features, locations, and social factors driving transmission in settings with high incidences of drug-resistant TB are poorly understood. Methods We measured a network of genomic links using Mycobacterium tuberculosis whole-genome sequences. Results Patients with 2–3 months of cough or who spent time in urban locations were more likely to be linked in the network, while patients with sputum smear–positive disease were less likely to be linked than those with smear-negative disease. Associations persisted using different thresholds to define genomic links and irrespective of assumptions about the direction of transmission. Conclusions Identifying factors that lead to many transmissions, including contact with urban areas, can suggest settings instrumental in transmission and indicate optimal locations and groups to target with interventions. [ABSTRACT FROM AUTHOR]

Introduction: Klebsiella pneumoniae with OXA-48-like enzymes were introduced into Tshwane Tertiary Hospital (TTH) (Pretoria, South Africa) during September 2015, causing nosocomial outbreaks.Methods: PCR methodologies and WGS were used to characterize K. pneumoniae with carbapenemases (n = 124) from TTH (July 2015-December 2016).Results: PCR was used to track K. pneumoniae ST307 with OXA-181 among 60% of carbapenemase-positive isolates in different wards/units over time and showed the transmission of IncX3 plasmids to other K. pneumoniae clones. WGS identified different ST307 clades: 307_OXA181 (consisting of two lineages, A and B) with OXA-181 on IncX3 plasmids (named p72_X3_OXA181) and clade 307_VIM with VIM-1 on IncFII plasmids. Clade 307_OXA181 lineage B was responsible for the rapid increase and transmission of OXA-181 K. pneumoniae in various wards/units throughout TTH, while the numbers of clade 307_OXA181 lineage A and clade 307_VIM remained low. Separate outbreaks were due to K. pneumoniae ST17 and ST29 with p72_X3_OXA181 plasmids.Conclusions: The high-risk clone K. pneumoniae ST307 with OXA-181 rapidly spread to different wards/units despite infection and prevention measures. ST307 clades and lineages seemingly acted differently in outbreak situations. This study also highlighted the threat of promiscuous plasmids such as p72_X3_OXA181. [ABSTRACT FROM AUTHOR]

Background: Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations.Methods: We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants.Results: Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role.Conclusion: The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile. [ABSTRACT FROM AUTHOR]

Food animals are considered reservoirs of methicillin‐resistant Staphylococcus aureus (MRSA) and are implicated in their zoonotic transmission in the farm‐to‐plate continuum. LA‐MRSA has been reported as a zoonotic agent that has the potential to spread to humans and may cause infections in at‐risk groups. In this study, whole genome sequencing was used to describe the genetic environment (resistance mechanisms, virulence factors and mobile genetic elements) and investigate the genetic lineages of MRSA isolates from pigs in Cameroonian and South African abattoirs. 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. Genomic DNA was 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, and antibiotic resistance genes, virulence factors, plasmids, SCCmec and phage elements were identified with ResFinder, Virulence Finder, PlasmidFinder, SCCmec Finder and PHAST, respectively. Core genome single nucleotide analysis was undertaken to assess clonal relatedness among isolates. A lower MRSA prevalence was observed in pigs in Cameroon (n = 1/13; 0.07%) compared with South Africa (n = 4/22; 18.18%), and none of the workers were colonized by MRSA. Genome analysis identified various antibiotic resistance genes along with six virulence factors in all isolates. All MRSA isolates belonged to the clonal lineage ST398 (spa‐type t011) and harboured the type Vc SCCmec and several plasmids. Our study shows that the livestock‐associated MRSA clonal lineage ST398 is already present in both Cameroon and South Africa and is probably underestimated in the absence of molecular epidemiological studies. It reveals the serious food safety and public health threat associated with this animal strain and underscores the need for interventions to contain this resistant clone. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is sporadically detected on a range of ready to eat fresh produce lines, such as spinach and rocket, and is a threat to public health. However, little is known about the diversity of L. monocytogenes present on fresh produce and their potential pathogenicity. In this work, fifteen Listeria monocytogenes isolates from the UK fresh produce supply chain were characterised using whole genome sequencing (WGS). Additionally, isolates were characterised based on their ability to form biofilm. Whole genome sequencing data was used to determine the sequence type of isolates based on multi-locus sequence typing (MLST), construct a core single nucleotide polymorphism (SNP) phylogeny and determine the presence of virulence and resistance associated genes. MLST revealed 9 distinct sequence types (STs) spanning 2 lineages (I & II) with one isolate belonging to the ST6 subtype, strains from which have been recently implicated in two large, food-associated L. monocytogenes outbreaks in South Africa and across Europe. Although most of the 15 isolates were different, comparison of core genome SNPs showed 4 pairs of 'indistinguishable' strains (<5 SNPs difference). Virulence profiling revealed that some isolates completely lacked the Listeria pathogenicity island-3 (LIPI-3) amongst other virulence factors. Investigation of the inlA gene showed that no strains in this study contained a premature stop codon (PMSC), an indicator of attenuated virulence. Assessment of biofilm production showed that isolates found in the fresh produce supply chain differ in their ability to form biofilm. This trait is considered important for L. monocytogenes to persist in environments associated with food production and processing. Overall the work indicates that a genetically diverse range of L. monocytogenes strains is present in the UK fresh produce supply chain and the virulence profiles found suggests that at least some of the strains are capable of causing human illness. Interestingly, the presence of some genetically indistinguishable isolates within the 15 isolates examined suggests that cross-contamination in the fresh produce environment does occur. These findings have useful implications in terms of food safety and for informing microbial surveillance programmes in the UK fresh produce supply chain. • The Lm population in the UK leafy produce supply chain is genetically diverse • Isolates are related to epidemic strains and possess the necessary genes for disease • Biofilm production differs amongst closely related isolates • WGS provides valuable information on isolates that is of interest to stakeholders [ABSTRACT FROM AUTHOR]

Rift Valley fever (RVF) is a re-emerging zoonotic disease responsible for major losses in livestock production, with negative impact on the livelihoods of both commercial and resource-poor farmers in sub-Sahara African countries. The disease remains a threat in countries where its mosquito vector thrives. Outbreaks of RVF usually follow weather conditions which favour increase in mosquito populations. Such outbreaks are usually cyclical, occurring every 10–15 years. Recent outbreaks of the disease in South Africa have occurred unpredictably and with increased frequency. In 2008, outbreaks were reported in Mpumalanga, Limpopo and Gauteng provinces, followed by 2009 outbreaks in KwaZulu-Natal, Mpumalanga and Northern Cape provinces and in 2010 in the Eastern Cape, Northern Cape, Western Cape, North West, Free State and Mpumalanga provinces. By August 2010, 232 confirmed infections had been reported in humans, with 26 confirmed deaths.To investigate the evolutionary dynamics of RVF viruses (RVFVs) circulating in South Africa, we undertook complete genome sequence analysis of isolates from animals at discrete foci of the 2008–2010 outbreaks. The genome sequences of these viruses were compared with those of the viruses from earlier outbreaks in South Africa and in other countries. The data indicate that one 2009 and all the 2008 isolates from South Africa and Madagascar (M49/08) cluster in Lineage C or Kenya-1. The remaining of the 2009 and 2010 isolates cluster within Lineage H, except isolate M259_RSA_09, which is a probable segment M reassortant. This information will be useful to agencies involved in the control and management of Rift Valley fever in South Africa and the neighbouring countries. Author summary: A single RVF virus serotype exists, yet differences in virulence and pathogenicity of the virus have been observed. This necessitates the need for detailed genetic characterization of various isolates of the virus. Some of the RVF virus isolates that caused the 2008–2010 disease outbreaks in South Africa were most probably reassortants resulting from exchange of portions of the genome, particularly those of segment M. Although clear association between RVFV genotype and phenotype has not been established, various amino acid substitutions have been implicated in the phenotype. Viruses with amino acid substitutions from glycine to glutamic acid at position 277 of segment M have been shown to be more virulent in mice in comparison to viruses with glycine at the same position. Phylogenetic analysis carried out in this study indicated that the viruses responsible for the 2008–2010 RVF outbreaks in South Africa were not introduced from outside the country, but mutated over time and caused the outbreaks when environmental conditions became favourable. [ABSTRACT FROM AUTHOR]

Horizontal acquisition of mobile genetic elements is a powerful evolutionary driving force that can profoundly affect pathogens epidemiology and their interactions with the environment and host. In the last decade, the role of the epidemic megaplasmid, pESI was demonstrated in the global emergence of multi-drug resistant (MDR) Salmonella enterica serovar Infantis strains, but it was unknown if this was a one-time phenomenon, or that pESI can drive the emergence of other pathogens. Epidemiological, molecular, whole genome sequencing, de-novo assembly, bioinformatics and genetic approaches were used to analyze the emergence of a pESI-positive Salmonella enterica serovar Muenchen strain in Israel. Since 2018, we report the emergence and high prevalence of S. Muenchen in Israel, which consisted at 2020, 40% (1055/2671) of all clinical Salmonella isolates. We show that the emergence of S. Muenchen is dominated by a clonal MDR strain, report its complete assembled genome sequence, and demonstrate that in contrast to preemergent strains, it harbors the epidemic megaplasmid, pESI, which can be self-mobilized into E. coli and other Salmonella serovars. Additionally, we identified bioinformatically highly similar genomes of clinical isolates that were recently collected in South Africa, UK and USA. This is a second documented case of a pathogen emergence associated with pESI acquisition. Considering the genetic cargo of pESI that enhances resistance, stress tolerance and virulence, and its ability to conjugate into prevalent Salmonella serovars, we provide further support that pESI facilities the emergence and spreading of new Salmonella strains. [ABSTRACT FROM AUTHOR]

Background: The capsular polysaccharide is the principal virulence factor of Streptococcus pneumoniae and a target for current pneumococcal vaccines. However, some pathogenic pneumococci are serologically nontypeable [nontypeable pneumococci (NTPn)]. Due to their relative rarity, NTPn are poorly characterized, and, as such, limited data exist which describe these organisms. We aimed to describe disease and genotypically characterize NTPn causing invasive pneumococcal disease in South Africa. Results: Isolates were detected through national, laboratory-based surveillance for invasive pneumococcal disease in South Africa and characterized by whole genome analysis. We predicted ancestral serotypes (serotypes from which NTPn may have originated) for Group I NTPn using multilocus sequence typing and capsular region sequence analyses. Antimicrobial resistance patterns and mutations potentially causing nontypeability were identified. From 2003-2013, 39 (0.1 %, 39/32,824) NTPn were reported. Twenty-two (56 %) had partial capsular genes (Group I) and 17 (44 %) had complete capsular deletion of which 15 had replacement by other genes (Group II). Seventy-nine percent (31/39) of our NTPn isolates were derived from encapsulated S. pneumoniae. Ancestral serotypes 1 (27 %, 6/22) and 8 (14 %, 3/22) were most prevalent, and 59 % (13/22) of ancestral serotypes were serotypes included in the 13-valent pneumococcal conjugate vaccine. We identified a variety of mutations within the capsular region of Group I NTPn, some of which may be responsible for the nontypeable phenotype. Nonsusceptibility to tetracycline and erythromycin was higher in NTPn than encapsulated S. pneumoniae. Conclusions: NTPn are currently a rare cause of invasive pneumococcal disease in South Africa and represent a genetically diverse collection of isolates. [ABSTRACT FROM AUTHOR]

• SARS-CoV-2 Gene Target Failure for Delta variants. • Detection of SARS CoV-2 Delta variant without genomic sequencing. • Emergence of SARS-CoV-2 variant in South Africa. • SARS-CoV-2 variant dynamics in South Africa. Routine SARS-CoV-2 surveillance in the Western Cape region of South Africa (January-August 2021) found a reduced RT-PCR amplification efficiency of the RdRp-gene target of the Seegene, Allplex 2019-nCoV diagnostic assay from June 2021 when detecting the Delta variant. We investigated whether the reduced amplification efficiency denoted by an increased RT-PCR cycle threshold value (RΔE) can be used as an indirect measure of SARS-CoV-2 Delta variant prevalence. We found a significant increase in the median RΔE for patient samples tested from June 2021, which coincided with the emergence of the SARS-CoV-2 Delta variant within our sample set. Whole genome sequencing on a subset of patient samples identified a highly conserved G15451A, non-synonymous mutation exclusively within the RdRp gene of Delta variants, which may cause reduced RT-PCR amplification efficiency. While whole genome sequencing plays an important in identifying novel SARS-CoV-2 variants, monitoring RΔE value can serve as a useful surrogate for rapid tracking of Delta variant prevalence. [ABSTRACT FROM AUTHOR]

Atypical enteropathogenic E. coli (aEPEC) strains are emerging pathogens responsible for fatal diarrhoea in humans worldwide. The purpose of this study was to investigate genetic diversity, virulence and antimicrobial resistance profiles of aEPEC O177 strains isolated from faeces of cattle reared in intensive and extensive production systems in South Africa. A total of 96 multidrug resistant (MDR) aEPEC O177 isolates were typed using enterobacterial repetitive intergenic consensus (ERIC) and random amplified polymorphism DNA (RAPD) typing. The resistome, virulome and mobilome of two aEPEC O177 isolates were investigated using WGS analysis. The ERIC typing was efficient and reproducible with a discriminatory index of 0.95. RAPD typing had poor reproducibility with satisfactory discriminatory power of 0.859. The dendrograms constructed based on ERIC and RAPD banding patterns produced 9 and 8 clusters, respectively, which indicate genetic variation among E. coli O177 isolates. WGS analysis revealed that CF-154-A and CF-335-B) isolates belonged to the O177 serotype with H7 and H21, respectively. Both isolates harboured several virulome genes such as intimin (eaeA), haemolysin (hlyA and hlyE), translocated iron receptor (tir), Type III secretion system (eprH , gspL and prgH), bssR and bssS. However, genes encoding shiga toxins were not found in either isolate. Antibiotic resistance genes such as ampC , tet , ermB , sul2 , strB AcrD , aph (6)- Ic , aph (6)- Ib , aph (3″) - I , ant (3″) - 1a AcrA and acrE were found in the E. coli O177 strains. Furthermore, genome annotation results indicated that both isolates carried plasmids, insertion sequences, prophages and cluster of regularly interspaced short palindromic repeats (CRISPR) type I. Based on in silico multi locus typing (MLST) analysis, the two isolates were assigned to different sequence types (CF-154-A, ST-1308 and CF-335-B, ST-58). Whole genome multi locus typing tree showed that our isolates clustered with E. coli O177:H21 (reference), suggesting the close genomic relatedness among the strains. Overall, these findings showed that cattle carry genetically diverse E. coli O177 strains, which harbour a repertoire of virulome, resistome and mobilome genes. This highlights a need for multidrug resistant E. coli O177 strain surveillance in cattle. • Cattle harbour genetically diverse atypical enteropathogenic E. coli O177 strains • Complete genome sequence analysis of two E. coli O177 strains revealed a high number of virulence and antimicrobial resistance profiles. • The two E. coli O177 strains were ascribed to different MLST sequence types (ST-1308 and ST-58) • WgMLST analysis confirmed genomic relatedness of the two E. coli O177 strains and the reference strain (E. coli O177:H21) [ABSTRACT FROM AUTHOR]

The study investigated carbapenemase-producing Klebsiella pneumoniae (CPKP) isolates of patients in an intensive care unit (ICU) in a public hospital in the KwaZulu-Natal province, South Africa using whole-genome sequencing (WGS). Ninety-seven rectal swabs, collected from all consenting adult patients (n = 31) on days 1, 3, and 7 and then weekly, were screened for carbapenemase-production using Chrome-ID selective media. Antibiotic susceptibility was determined for the fourteen positive CPKP isolates obtained using the VITEK 2 automated system. All isolates (100%) were resistant to ertapenem and meropenem, and 71.4% (n = 10) were resistant to imipenem. All CPKP isolates were subjected to ERIC/PCR, and a sub-sample of isolates was selected for WGS based on their antibiograms and clonality. All sequenced isolates harbored the blaOXA-181 carbapenemase (100%) and co-carried other β-lactamase genes such as blaOXA-1, blaCTX-M-15, blaTEM-1B, and blaSHV-1. IncF, IncX3, and Col plasmid replicons groups and class I integrons (ln191 and ln27) were detected. All isolates belonged to the same sequence type ST307 and capsular serotypes (K102, O2v2). All the isolates carried the same virulence repertoire, reflecting the epidemiological relationship between isolates. blaOXA-181 was located on a multi-replicon plasmid similar to that of E. coli p010_B-OXA181, and isolates were aligned with several South African and international clades, demonstrating horizontal and vertical transboundary distribution. The findings suggest that blaOXA-181 producing K. pneumoniae is endemic in this ICU, colonizing the patients. CRE screening and enhanced infection prevention and control measures are urgently required. [ABSTRACT FROM AUTHOR]

The article focuses on the powerful toolkit of exponential random graph models (ERGMs) to the analysis of transmission networks derived from whole-genome sequence (WGS) data collected during an outbreak of extensively drug-resistant tuberculosis (XDR-TB) in KwaZulu-Natal, South Africa; and mentions that ERGMs provide estimates of the likelihood that connections will form between individuals as a function of both their individual and contextual attributes.