Genome analysis of multidrug resistant Enterococcus faecium and Enterococcus faecalis circulating among hospitalized patients in uMgungundlovu District, KwaZulu-Natal, South Africa.

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.

Metagenomics insights into microbiome and antibiotic resistance genes from free living amoeba in chlorinated wastewater effluents.

Free living amoeba (FLA) are among the organisms commonly found in wastewater and are well-established hosts for diverse microbial communities. Despite its clinical significance, there is little knowledge on the FLA microbiome and resistome, with previous studies relying mostly on conventional approaches. In this study we comprehensively analyzed the microbiome, antibiotic resistome and virulence factors (VFs) within FLA isolated from final treated effluents of two wastewater treatment plants (WWTPs) using shotgun metagenomics. Acanthamoeba has been identified as the most common FLA, followed by Entamoeba. The bacterial diversity showed no significant difference (p > 0.05) in FLA microbiomes obtained from the two WWTPs. At phylum level, the most dominant taxa were Proteobacteria, followed by Firmicutes and Actinobacteria. The most abundant genera identified were Enterobacter followed by Citrobacter, Paenibacillus, and Cupriavidus. The latter three genera are reported here for the first time in Acanthamoeba. In total, we identified 43 types of ARG conferring resistance to cephalosporins, phenicol, streptomycin, trimethoprim, quinolones, cephalosporins, tigecycline, rifamycin, and kanamycin. Similarly, a variety of VFs in FLA metagenomes were detected which included flagellar proteins, Type IV pili twitching motility proteins (pilH and rpoN), alginate biosynthesis genes AlgI, AlgG, AlgD and AlgW and Type VI secretion system proteins and general secretion pathway proteins (tssM, tssA, tssL, tssK, tssJ, fha, tssG, tssF, tssC and tssB, gspC, gspE, gspD, gspF, gspG, gspH, gspI, gspJ, gspK, and gspM). To the best of our knowledge, this is the first study of its kind to examine both the microbiomes and resistome in FLA, as well as their potential pathogenicity in treated effluents. Additionally, this study showed that FLA can host a variety of potentially pathogenic bacteria including Paenibacillus, and Cupriavidus that had not previously been reported, indicating that their relationship may play a role in the spread and persistence of antibiotic resistant bacteria (ARBs) and antibiotic resistance genes (ARGs) as well as the evolution of novel pathogens.

Spectrum of genetic variants in bilateral sensorineural hearing loss.

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.

The diversity and clinical implications of genetic variants influencing clopidogrel bioactivation and response in the Emirati population.

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.

Extreme resistance to the novel siderophore-cephalosporin cefiderocol in an extensively drug-resistant Klebsiella pneumoniae strain causing fatal pneumonia with sepsis: genomic analysis and synergistic combinations for resistance reversal.

Cefiderocol (CFDC) is the first-in-class siderophore-cephalosporin. Klebsiella pneumoniae strain that is extremely resistant to CFDC (MIC: 256 µg/ml) was isolated for the first time in the United Arab Emirates from a patient with pneumonia and sepsis. It belonged to sequence-type 14 (ST14), with a novel core genome ST. Resistance was driven by the co-expression of ß-lactamases (blaNDM-1, blaOXA-232 and blaCTX-M-15) and a mutation in catecholate-siderophore receptor, utilized by CFDC to enter the bacterial cell. Synergistic combinations (ß-lactamase inhibitors, aztreonam plus CFDC) re-sensitized the bacteria to CFDC. Although CFDC resistance is multifactorial, the combination with ß-lactamase inhibitors represents a promising approach in resistance reversal for fighting superbugs.

A Genomic Snapshot of Antibiotic-ResistantEnterococcus faecalis within Public Hospital Environments in South Africa.

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.

Whole-genome sequence data of a Salmonella enterica serovar Rissen sequence type 8877 isolated from cracked table egg in Sudan.

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.

Multiple Novel Human Norovirus Recombinants Identified in Wastewater in Pretoria, South Africa by Next-Generation Sequencing.

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.

Genetic Polymorphism and Phylogenetics of Aedes aegypti from Sudan Based on ND4 Mitochondrial Gene Variations.

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.

Marked Effects of Larval Salt Exposure on the Life History and Gut Microbiota of the Malaria Vector Anopheles merus (Diptera: Culicidae).

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.

Assessment of Antibiotic Resistance and Efflux Pump Gene Expression in Neisseria Gonorrhoeae Isolates from South Africa by Quantitative Real-Time PCR and Regression Analysis.

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 <0.089), gyrA (p = 0.027; cutoff <0.0518), parE (p = 0.036; cutoff <0.0033), rpsJ (p = 0.047; cutoff <0.0012), and 23S rRNA (p = 0.042; 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.

Phylogenetic Characterisation of the Full Genome of a Bagaza Virus Isolate from Bird Fatalities in South Africa.

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.

Prevalence, Genomic Characterization, and Risk Assessment of Human Pathogenic Vibrio Species in Seafood.

ABSTRACT: Pathogenic Vibrio species are largely responsible for human diseases associated with consumption of contaminated seafood. The aim of this study was to determine the prevalence, population densities, species diversity, and molecular characteristics of pathogenic Vibrio in various seafood commodities and the health risks associated with consumption of these contaminated commodities. Samples of finfish and shellfish (oysters and sea urchins) were collected from various regions and analyzed for Vibrio with the most-probable-number (MPN) technique. Genomic DNA of putative Vibrio isolates was analyzed by whole genome sequencing for taxonomic identification and identification of virulence and antimicrobial resistance genes. The risk of Vibrio-related illnesses due to the consumption of contaminated seafood was assessed with Risk Ranger. Populations of presumptive Vibrio were 2.6 to 4.4 log MPN/g and correlated with season; Vibrio levels were significantly higher (P < 0.05) in the summer. Fifteen Vibrio isolates were identified as Vibrio alginolyticus (five isolates), Vibrio parahaemolyticus (six isolates), Vibrio harveyi (two isolates), and Vibrio diabolicus (two isolates). Two of the six V. parahaemolyticus isolates (ST 2504 and ST 2505) from oysters harbored either the tdh gene for thermostable direct hemolysin or the trh gene for thermostable direct hemolysin-related hemolysin. In addition to virulence genes, the shellfish isolates also harbored genes encoding resistance to multiple antibiotics, including tetracycline, penicillin, quinolone, and ß-lactams, thus arousing concern. The risk assessment predicted that an estimated 21 cases of V. parahaemolyticus-associated gastroenteritis could occur in the general population annually due to consumption of contaminated oysters. This study highlights both the wide prevalence and diversity of Vibrio in seafood and the potential for certain strains to threaten public health.

Genome Analysis of ESBL-Producing Escherichia coli Isolated from Pigs.

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.

Whole-Genome Sequencing of a Colistin-Resistant Acinetobacter baumannii Strain Isolated at a Tertiary Health Facility in Pretoria, South Africa.

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.

A prevalence and molecular characterization of novel pathogenic strains of Macrococcus caseolyticus isolated from external wounds of donkeys in Khartoum State -Sudan.

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 conclusionThe 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.

Characteristics in the whole-genome sequence of Klebsiella pneumoniae ST147 from Turkey.

The study aimed to analyze antibiotic resistance determinants in a carbapenem-resistant Klebsiella pneumoniae by whole-genome sequencing (WGS). K. pneumoniae was isolated from a urine sample and it was characterized by 16S rDNA sequencing in Turkey. This strain was named as Kpn Rize-53-TR. Antimicrobial susceptibility testing was performed for seventeen antibiotics by VITEK-2 and the result was confirmed by MIC. The whole genome of isolate was sequenced by Illumina and was analysed by bioinformatic tools for MLST, replicon types, and antimicrobial resistance genes. The whole genome data was submitted to NCBI. The isolate was found to be resistant to all tested ß-lactam antibiotics and the highest MIC values were found for piperacillin, piperacillin/tazobactam (≥128). No resistance to colistin and moderate susceptibility to amikacin and tetracycline was observed. The isolate carried 12 resistance genes belonging to 10 resistance classes; ere(A), fosA, oqxB, cmlA1, aac(a)-IIa, bla KPC-2, bla TEM-1A, bla SHV-67, bla CTX-M-15, bla OXA-1-2-9. Mutations were detected in gyrA (83Y) and parC (80I) genes. Clonal subtype of the isolate was ST147, and it had wzi420 and wzc38 alleles. Its serotype was O3/O3a. The bla KPC-2 was firstly found in both ST147 clonal group in Turkey and in serotype O3/O3a in the world. By plasmid replicon typing, five plasmids IncFII(K), Col(BS512), IncR, IncFIA(HI1) and IncFIB(pQil) were determined in Kpn Rize-53-TR and bla KPC-2 was located on IncFII(K) plasmid. The presence of bla KPC-2 on the plasmid with other resistance genes accelerates its own spread together with other resistance genes.

The dynamic gut microbiota of zoophilic members of the Anopheles gambiae complex (Diptera: Culicidae).

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 ß-diversity from that of the minor and non-vectors. There was no difference in α- or ß-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.

High-Resolution Melting Analysis to Detect Antimicrobial Resistance Determinants in South African Neisseria gonorrhoeae Clinical Isolates and Specimens.

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 bla TEM (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.

Mobile genetic elements-mediated Enterobacterales-associated carbapenemase antibiotic resistance genes propagation between the environment and humans: A One Health South African study.

We, (1) studied carbapenem-resistant Enterobacterales (CRE) in the environment, humans, and animals, within the same geographical area and, (2) delineated the isolates' resistome, mobilome, virulome, and phylogeny. Following ethical approval, 587 samples (humans = 230, pigs = 345, and water = 12) were collected and cultured on CRE selective media. Confirmatory identification and antibiotic susceptibility testing were performed using the VITEK 2 automated platform. The resistomes, virulomes, mobilomes, and phylogenies were ascertained by whole genome sequencing. Nineteen (3.2%), i.e., 15/19 humans and 4/19 environmental, but no pig, CRE were obtained. CREs included Klebsiella pneumoniae 9/19 (47%), Enterobacter hormaechei 6/19 (32%), Klebsiella quasipneumoniae 2/19 (11%), a novel ST498 Citrobacter freundii 1/19 (5%) and Serratia marcescens 1/19 (5%). Eleven isolates were extensively drug-resistant; eight were multidrug-resistant. Sixteen CRE harbored the blaOXA-181, blaOXA-48, blaOXA-484, blaNDM-1, and blaGES-5 genes. Multiple species/clones carried blaOXA-48 and blaNDM-1 carbapenemase-encoding genes with respective mobile genetic elements (MGEs). The IncFIB(K) plasmid replicon was found in most human K. pneumoniae strains (7/9) and all environmental K. quasipneumoniae isolates; most K. pneumoniae produced OXA-181 (5/9). The (Col440I) plasmid replicon, identified in 11 (26.82%) isolates, mainly E. hormaechei (n = 6), predominated both sectors. Most ß-lactamase-encoding genes were associated with class 1 integrons IntI1, insertion sequences (IS) (IS91, IS5075, IS30, IS3000, IS3, IS19, ISKpn19, IS5075) and transposons (Tn3). The IncL/M(pMU407) and IncL/M(pOXA48) plasmid replicons were found exclusively in K. pneumoniae; all but one of these strains produced OXA-181. Also, the Klebsiella spp. harbored 80 virulence genes. Phylogenomic clustered identified isolates with other carbapenemase-producing K. pneumoniae, E. hormaechei, S. marcescens, and C. freundii from different South African sources (animals, environment, and humans). We delineated the resistome, mobilome, virulome, and phylogeny of carbapenemase-producing Enterobacterales in humans and environment, highlighting antibiotic resistance genes propagation via MGEs across sectors, emphasizing a One Health approach to AMR.