Genomic characterization of multi drug resistant ESBL-producing Escherichia coli isolates from patients and patient environments in a teaching hospital in Ghana.

BACKGROUND: ESBL-producing Escherichia coli pose a growing health risk in community and healthcare settings. We investigated the resistome, virulome, mobilome, and genetic relatedness of multidrug-resistant (MDR) E. coli isolates from patients and their environment in a Ghanaian teaching hospital. MATERIALS AND METHODS: Twenty-three MDR ESBL-producing or carbapenem-resistant E. coli isolates from a collection of MDR Gram-negative bacteria (GNB) from patients and environments were selected for genomic analyses. Whole genome sequencing and bioinformatics tools were used to analyze genomic characteristics and phylogeny. RESULTS: The prevalence and incidence of rectal carriage of ESBL E. coli among patients were 13.65% and 11.32% respectively. The ß-lactamase genes, blaTEM-1B (10 isolates) and blaCTX-M-15 (12 isolates) were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Insertion sequences, transposons, and class I integrons were found with blaCTX-M-15. Carriage and environmental isolates carried multiple virulence genes, with terC being the most prevalent in 21 isolates. Seventeen sequence types (STs) were identified, including a novel ST (ST13846). Phylogenetic analysis grouped the isolates into four main clusters, with one outlier. High genetic relatedness was observed between two carriage isolates of ST940 and between a carriage isolate and an environmental isolate of ST648. Isolates with different STs, collected at different times and locations, also showed genetic similarities. CONCLUSION: We identified ESBL-producing E. coli with diverse genomic characteristics circulating in different hospital directorates. Clonal relatedness was observed among isolates from patients and the environment, as well as between different patients, suggesting transmission within and between sources.

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.

Carbapenem Resistance Determinants Acquired through Novel Chromosomal Integrations in Extensively Drug-Resistant Pseudomonas aeruginosa.

Two novel blaDIM-1- or blaIMP-1-containing genomic islands (GIs) were discovered by whole-genome sequence analyses in four extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates from inpatients at a tertiary hospital in Ghana. The strains were of sequence type 234 (ST234) and formed a phylogenetic clade together with ST111, which is recognized as a global high-risk clone. Their carbapenem resistance was encoded by two Tn402-type integrons, In1592 (blaDIM-1) and In1595 (blaIMP-1), both carrying complete tni mobilization modules. In1595 was bound by conserved 25-bp inverted repeats (IRs) flanked by 5-bp direct repeats (DRs) associated with target site duplication. The integrons were embedded in two GIs that contained cognate integrases and were distinguished by a lower GC content than the chromosomal average. PAGI-97A (52.659 bp; In1592), which encoded a P4-type site-specific integrase of the tyrosine recombinase family in its 3' border, was integrated into tRNA-Pro(ggg) and bracketed by a 49-bp perfect DR created by 3'-end target duplication. GIs with the same structural features, but diverse genetic content, were identified in 41/226 completed P. aeruginosa genomes. PAGI-97B (22,636 bp; In1595), which encoded an XerC/D superfamily integrase in its 5' border, was inserted into the small RNA (sRNA) PrrF1/PrrF2 locus. Specific insertions into this highly conserved locus involved in iron-dependent regulation, all leaving PrrF1 intact, were identified in an additional six phylogenetically unrelated P. aeruginosa genomes. Our molecular analyses unveiled a hospital-associated clonal dissemination of carbapenem-resistant ST234 P. aeruginosa in which the XDR phenotype resulted from novel insertions of two GIs into specific chromosomal sites.

Spread of Plasmid-Encoded NDM-1 and GES-5 Carbapenemases among Extensively Drug-Resistant and Pandrug-Resistant Clinical Enterobacteriaceae in Durban, South Africa.

Whole-genome sequence analyses revealed the presence of blaNDM-1 (n = 31), blaGES-5 (n = 8), blaOXA-232 (n = 1), or blaNDM-5 (n = 1) in extensively drug-resistant and pandrug-resistant Enterobacteriaceae organisms isolated from in-patients in 10 private hospitals (2012 to 2013) in Durban, South Africa. Two novel NDM-1-encoding plasmids from Klebsiella pneumoniae were circularized by PacBio sequencing. In p19-10_01 [IncFIB(K); 223.434 bp], blaNDM-1 was part of a Tn1548-like structure (16.276 bp) delineated by IS26 The multireplicon plasmid p18-43_01 [IncR_1/IncFIB(pB171)/IncFII(Yp); 212.326 bp] shared an 80-kb region with p19-10_01, not including the blaNDM-1-containing region. The two plasmids were used as references for tracing NDM-1-encoding plasmids in the other genome assemblies. The p19-10_01 sequence was detected in K. pneumoniae (n = 7) only, whereas p18-43_01 was tracked to K. pneumoniae (n = 4), Klebsiella michiganensis (n = 1), Serratia marcescens (n = 11), Enterobacter spp. (n = 7), and Citrobacter freundii (n = 1), revealing horizontal spread of this blaNDM-1-bearing plasmid structure. Global phylogeny showed clustering of the K. pneumoniae (18/20) isolates together with closely related carbapenemase-negative ST101 isolates from other geographical origins. The South African isolates were divided into three phylogenetic subbranches, where each group had distinct resistance and replicon profiles, carrying either p19-10_01, p18-10_01, or pCHE-A1 (8,201 bp). The latter plasmid carried blaGES-5 and aacA4 within an integron mobilization unit. Our findings imply independent plasmid acquisition followed by local dissemination. Additionally, we detected blaOXA-232 carried by pPKPN4 in K. pneumoniae (ST14) and blaNDM-5 contained by a pNDM-MGR194-like genetic structure in Escherichia coli (ST167), adding even more complexity to the multilayer molecular mechanisms behind nosocomial spread of carbapenem-resistant Enterobacteriaceae in Durban, South Africa.

Building an antimicrobial stewardship model for a public-sector hospital: a pre-implementation study.

Introduction. Antimicrobial resistance (AMR) is recognized as an important global health risk, associated with increased mortality, morbidity and healthcare costs. Antimicrobial stewardship (AMS) involves a coherent set of processes that promote the rational use of antimicrobials.Gap statement. An AMS programme should be adapted and developed according to the available resources of a facility. This requires an analysis of the core AMS elements that are already in place and the resources available.Aim. This study aimed to assess the readiness of a tertiary healthcare facility and staff towards implementing an antimicrobial stewardship programme (ASP).Methodology. This study focused on two aspects during an AMS pre-implementation phase. A situational or strengths, weaknesses, opportunities, and threats analysis was conducted based on (1) a questionnaire on attitudes and perceptions of pharmacists, clinicians and nurses towards AMR and AMS and (2) a situational analysis on the readiness of the facility.Results. The questionnaire, which was available for completion between September 2021 and December 2021, was sent to a total of 3100 healthcare professionals (HCPs). Thirty-two (1.0 %) HCPs comprising 2 pharmacists, 16 clinicians and 14 nurses completed the questionnaire. Of the total participants, 31 (96.9 %) viewed AMR as a problem in South African hospitals and 29 (90.6 %) perceived AMR as a problem at their facility. The majority (n = 29, 90.6 %) of the participants were familiar with the term AMS, and 26 (81.3 %) participants agreed to willingly participate in any initiatives involving antimicrobial use at the facility. The situational analysis depicted existing strengths in terms of AMS structures such as the formation of an AMS committee and information and technology systems at the HCP's disposal. Weaknesses included the limited number of AMS activities being carried out and poor participation from HCPs within the AMS team.Conclusion. A pre-implementation phase in the building of an ASP can greatly assist in finding gaps for improvement, which can then be addressed in the implementation phase. Furthermore, the pre-implementation phase provides a baseline to measure improvements once the implementation phase has been instituted.

Multidrug-resistant Gram-negative bacterial colonization in patients, carriage by healthcare workers and contamination of hospital environments in Ghana.

BACKGROUND: Patients already colonized with multidrug-resistant (MDR) Gram-negative bacteria (GNB) on admission to critical care units may be an important source of transmission of these bacteria in hospitals. We sought to determine the prevalence of MDR GNB colonization in patients, staff and the ward environment and to assess the risk factors for colonization of patients in wards. METHODS: The study was conducted from April 2021 to July 2021 in a teaching hospital in Ghana. MDR GNB were isolated from rectal, and hand swabs were taken from patients on admission and after 48 h. Swabs from HCW's hands and the ward environment were also taken. Risk factors for colonization with MDR GNB were assessed using univariate and multivariate analysis. RESULTS: MDR GNB rectal colonization rate among patients was 50.62% on admission and 44.44% after 48 h. MDR GNB were isolated from 6 (5.26%) and 24 (11.54%) of HCW's hand swabs and environmental swabs, respectively. Previous hospitalization (p-value = 0.021, OR, 95% CI= 7.170 (1.345-38.214) was significantly associated with colonization by MDR GNB after 48 h of admission. Age (21-30 years) (p-value = 0.022, OR, 95% CI = 0.103 (0.015-0.716) was significantly identified as a protective factor associated with a reduced risk of rectal MDR GNB colonization. CONCLUSION: The high colonization of MDR GNB in patients, the carriage of MDR GNB on HCW's hands, and the contamination of hospital environments highlights the need for patient screening and stringent infection prevention and control practices to prevent the spread of MDR GNB in hospitals.

Antibiotic prescribing amongst South African general practitioners in private practice: an analysis of a health insurance database.

Objectives: To investigate the appropriateness of antibiotic prescribing among GPs in the private primary healthcare sector in South Africa. Methods: An anonymized national database of claims for antibiotic prescriptions was obtained from a large medical insurer. Antibiotic prescriptions were categorized based on International Classification of Diseases (ICD-10) codes as 'appropriate', 'potentially appropriate' and 'inappropriate' using a classification scheme developed by Chua et al. (BMJ 2019; 364: k5092). Further assessments of antibiotic choice, dosage and duration of treatment were carried out to determine the appropriateness of 'appropriate' and 'potentially appropriate' prescriptions in comparison with treatment guidelines. Results: In February 2018, 188 141 antibiotics were prescribed for 174 889 patients who consulted GPs in the private sector. Penicillins were the most frequently prescribed antibiotic class, making up 40.7% of all antibiotics prescribed. Amoxicillin/clavulanic acid was the most frequently prescribed antibiotic, making up 28.6% of all antibiotics prescribed. Diseases of the respiratory system generated the highest number of prescriptions, making up 46.1% of all diagnoses. Of all prescriptions, 8.8% were appropriate, 32.0% were potentially appropriate, 45.4% were inappropriate and 13.8% could not be assessed. Of the appropriately and potentially appropriately prescribed antibiotics, 30.8% were correct antibiotic selections. Of the correctly selected antibiotics for adults, 57.7% had correct doses. Of the antibiotics prescribed with correct doses for adults, 76.7% had correct dosage frequencies and durations of treatment. Conclusions: The study revealed that antibiotics were frequently prescribed inappropriately by GPs in the private primary healthcare sector. There is thus a need to develop stewardship interventions in the sector.

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.

Molecular Epidemiological Characterisation of ESBL- and Plasmid-Mediated AmpC-Producing Escherichia coli and Klebsiella pneumoniae at Kamuzu Central Hospital, Lilongwe, Malawi.

The global rise in infections caused by multidrug resistant (MDR) Enterobacterales poses a public health problem. We have performed a molecular epidemiological characterisation of representative plasmid-mediated AmpC (pAmpC) and ESBL-positive clinical isolates of Escherichia coli (n = 38) and Klebsiella pneumoniae (n = 17) from a tertiary hospital in Malawi collected in 2017. BlaCTX-M-15 was the most prevalent ESBL-determinant in E. coli (n = 30/38) and K. pneumoniae (n = 17/17), whereas blaCMY-2 was detected in nearly all AmpC-phenotype E. coli (n = 15/17). Whole genome sequencing revealed dominant globally disseminated E. coli sequence types (STs); ST410 (n = 16), ST131 (n = 7), and ST617 (n = 6). The ST distribution in K. pneumoniae was more diverse but included ST101 (n = 2), ST14 (n = 2), and ST340 (n = 2), all considered high-risk MDR clones. The isolates expressed an MDR profile, including resistance against commonly used antibiotics, such as fluoroquinolones, aminoglycosides, and/or trimethoprim-sulfamethoxazole, and harboured corresponding resistance determinants. Clonal analyses of the major STs of E. coli revealed closely related genetic clusters within ST410, ST131, and ST617 supporting within-hospital transmission between patients and/or via a common reservoir. The overall findings add to the limited knowledge on the molecular epidemiology of MDR E. coli and K. pneumoniae in Malawi and may help health policy makers to identify areas to target when addressing this major threat of antibiotic resistance.

Genomic Investigation of Carbapenem-Resistant Klebsiella pneumonia Colonization in an Intensive Care Unit in South Africa.

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 ß-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.

Draft genome sequence of a clinical Acinetobacter haemolyticus isolate from South Africa.

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

ß-lactam and fluoroquinolone resistance in Enterobacteriaceae from imported and locally-produced chicken in Mozambique.

INTRODUCTION: Plasmid-mediated resistance to ß-lactam and fluoroquinolone antibiotics was investigated in Enterobacteriaceae isolated from retailed frozen chickens from Brazil, South Africa and Mozambique. METHODOLOGY: Carcass swabs and the liquid thaw of 33 chickens from each of the three countries constituted the total sample size of 198. Isolates were identified by biochemical tests, antibiotic susceptibility was ascertained by the disc diffusion assay and ß-lactamases were detected using the double-disk synergy test. PCR was used to detect the presence of blaCTX-M, blaSHV, blaTEM, blaCMY, blaMOX, blaFOX, blaDHA, qnrB, qnrD, qnrS and qepA genes. A random selection of CTX-M genes was sequenced. RESULTS: The 198 samples yielded 27 (13.6%) putative extended-spectrum ß-lactamase (ESBL)-positive isolates, 15 from carcass swabs and 12 from the liquid thaw from 22 chickens with 19, 5 and 3 isolates from South African, Mozambican and Brazilian chicken, respectively. Isolates exhibited the following resistance: ampicillin 100%, ceftriaxone 89%, trimethoprim-sulfamethoxazole 78%, cefotaxime 74%, ciprofloxacin 70%, ceftazidime 67%, cefoxitin 22% and gentamicin 8%. The predominant putative ESBL gene was blaSHV (85%), followed by blaCTX-M (62.9%) and blaTEM (44.4%) whilst blaMOX and blaDHA were the most common pAmpC genes at 33.3%. The predominant plasmid-mediated fluoroquinolone-resistance gene was qepA (22.2%). DNA sequencing identified blaCTX-M-55/-79/-101/-164. ERIC-PCR profiles did not show strong evidence of clonality. CONCLUSION: The Mozambican population is exposed to a reservoir of plasmid-mediated, and hence mobile ß-lactam and quinolone resistance genes via imported, and to a lesser extent, locally produced poultry. This presents a food safety concern.

Pathogenomics and Evolutionary Epidemiology of Multi-Drug Resistant Clinical Klebsiella pneumoniae Isolated from Pretoria, South Africa.

Antibiotic-resistant Klebsiella pneumoniae is increasingly being implicated in invasive infections worldwide with high mortalities. Forty-two multidrug resistant (MDR) K. pneumoniae isolates were collected over a 4-month period. Antimicrobial susceptibility was determined using Microscan. The evolutionary epidemiology, resistome, virulome and mobilome of the isolates were characterised using whole-genome sequencing and bioinformatics analysis. All isolates contained the blaCTX-M gene, whilst 41/42(97%) contained blaTEM, 36/42(86%) contained blaOXA and 35/42(83%) harboured blaSHV genes. Other resistance genes found included blaLEN, aac(6')-lb-cr, qnrA, qnrB, qnrS, oqxAB, aad, aph, dfr, sul1, sul2, fosA, and cat genes. Fluoroquinolone and colistin resistance-conferring mutations in parC, gyrAB, pmrAB, phoPQ and kpnEF were identified. The blaLEN gene, rarely described worldwide, was identified in four isolates. The isolates comprised diverse sequence types, the most common being ST152 in 7/42(17%) isolates; clone-specific O and K capsule types were identified. Diverse virulence genes that were not clone-specific were identified in all but one isolate. IncF, IncH and IncI plasmid replicons and two novel integrons were present. The blaCTX-M-15 and blaTEM-1 genes were bracketed by Tn3 transposons, ISEc9, a resolvase and IS91 insertion sequence. There were 20 gene cassettes in 14 different cassette arrays, with the dfrA and aadA gene cassettes being the most frequent. Phylogenetic analysis demonstrated that the isolates were evolutionarily associated with strains from both South Africa and abroad. These findings depict the rich resistome, mobilome and virulome repertoire in clinical K. pneumoniae strains, which are mainly transmitted by clonal, multiclonal and horizontal means in South Africa.

Publisher Correction: The Resistome, Mobilome, Virulome and Phylogenomics of Multidrug-Resistant Escherichia coli Clinical Isolates from Pretoria, South Africa.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic analysis of a multidrug-resistant clinical Providencia rettgeri (PR002) strain with the novel integron ln1483 and an A/C plasmid replicon.

Whole-genome sequence analysis was performed on a multidrug-resistant Providencia rettgeri PR002 clinical strain isolated from the urine of a hospitalized patient in Pretoria, South Africa, in 2013. The resistome, mobilome, pathogenicity island(s), as well as virulence and heavy-metal resistance genes of the isolate, were characterized using whole-genome sequencing and bioinformatic analysis. PR002 had a genome assembly size of 4,832,624 bp with a GC content of 40.7%, an A/C2 plasmid replicase gene, four integrons/gene cassettes, 17 resistance genes, and several virulence and heavy metal resistance genes, confirming PR002 as a human pathogen. A novel integron, In1483, harboring the gene blaOXA-2 , was identified, with other uncharacterized class 1 integrons harboring aacA4cr and dfrA1. Aac(3')-IIa and blaSCO-1 , as well as blaPER-7 , sul2, and tet(B), were found bracketed by composite Tn3 transposons, and IS91, IS91, and IS4 family insertion sequences, respectively. PR002 was resistant to all antibiotics tested except amikacin, carbapenems, cefotaxime-clavulanate, ceftazidime-clavulanate, cefoxitin, and fosfomycin. PR002 was closely related to PR1 (USA), PRET_2032 (SPAIN), DSM_1131, and NCTC7477 clinical P. rettgeri strains, but not close enough to suggest it was imported into South Africa from other countries. Multidrug resistance in P. rettgeri is rare, particularly in clinical settings, making this case an important incident requiring urgent attention. This is also the first report of an A/C plasmid in P. rettgeri. The array, multiplicity, and diversity of resistance and virulence genes in this strain are concerning, necessitating stringent infection control, antibiotic stewardship, and periodic resistance surveillance/monitoring policies to preempt further horizontal and vertical spread of these resistance genes.

Bacterial diversity and functional profile of microbial populations on surfaces in public hospital environments in South Africa: A high throughput metagenomic analysis.

With the introduction of the One Health approach to global health advocated by the World Health Organization, the role of the environment as a reservoir and transmission route for diverse microorganisms is increasingly being recognised globally. This study investigated the diversity and functional profiles of bacterial communities using high-throughput metagenomics of the 16S rRNA gene in samples collected from environmental surfaces in different levels of healthcare in South Africa. A total of 150 samples were collected in three public hospitals [District (A), Regional (C) and Central (B)] from intensive care and paediatric wards. Military hospitals were excluded. Swabs were taken from mattresses, drip stands, ward telephones, patient files and sinks. A total of 7,996,346 reads were found, of which 7,319,569 were quality-filtered reads. Unique (and shared) microbial community structures were identified within the different hospital levels, locations and sample source. A total of 11 phyla, 29 classes, 50 orders, 105 families, 190 genera and 288 known species were identified. The primary phyla identified were Proteobacteria, Firmicutes and Actinobacteria. The dominant class identified was Gamma-proteobacteria, followed by Bacilli and Actinobacteria. Acinetobacter (16.08%), Citrobacter (13.64%), Staphylococcus (9.65%) and Corynebacterium (6.15%) were predominant genera. Although the functional profile analysis identified citrate cycle (TCA), signal transduction mechanisms, bisphenol degradation, tyrosine metabolism and transcription-factors as the dominant pathways, human disease functional classes, including involvement in antibiotic resistance, were significantly identified. The drip stands, patient files and ward telephones in all the wards of Hospitals A and C contained a higher number of human diseases functional classes. These findings highlight the potential of different hospital environments to serve as reservoirs and possible sources of bacterial pathogens; thus, the need for better monitoring and hygienic practices within the hospital environment.

Whole Genome Sequencing of Extended Spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae Isolated from Hospitalized Patients in KwaZulu-Natal, South Africa.

Extended spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae remain a critical clinical concern worldwide. The aim of this study was to characterize ESBL-producing K. pneumoniae detected within and between two hospitals in uMgungundlovu district, South Africa, using whole genome sequencing (WGS). An observational period prevalence study on antibiotic-resistant ESKAPE (i.e. Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) bacteria was carried out in hospitalized patients during a two-month period in 2017. Rectal swabs and clinical specimens were collected from patients hospitalized and were screened for ESBL-producing, Gram-negative ESKAPE bacteria using cefotaxime-containing MacConkey agar and ESBL combination disk tests. Nine confirmed ESBL-K. pneumoniae isolated from six patients and two hospitals were whole genome sequenced using an Illumina MiSeq platform. Genome sequences were screened for presence of integrons, insertion sequences, plasmid replicons, CRISPR regions, resistance genes and virulence genes using different software tools. Of the 159 resistant Gram-negative isolates collected, 31 (19.50%) were ESBL-producers, of which, nine (29.03%) were ESBL-K. pneumoniae. The nine K. pneumoniae isolates harboured several ß-lactamase genes, including blaCTX-M-15, blaTEM-1b, blaSHV-1, blaOXA-1 concomitantly with many other resistance genes e.g. acc(6')-lb-cr, aadAI6, oqxA and oqxB that confer resistance to aminoglycosides and/or fluoroquinolones, respectively. Three replicon plasmid types were detected in both clinical and carriage isolates, namely ColRNAI, IncFIB(K), IncF(II). Sequence type ST152 was confirmed in two patients (one carriage isolate detected on admission and one isolate implicated in infection) in one hospital. In contrast, ST983 was confirmed in a clinical and a carriage isolate of two patients in two different hospitals. Our data indicate introduction of ESBL-producing K. pneumoniae isolates into hospitals from the community. We also found evidence of nosocomial transmission within a hospital and transmission between different hospitals. The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-associated cas3 genes were further detected in two of the nine ESBL-KP isolates. This study showed that both district and tertiary hospital in uMgungundlovu District were reservoirs for several resistance determinants and highlighted the necessity to efficiently and routinely screen patients, particularly those receiving extensive antibiotic treatment and long-term hospitalization stay. It also reinforced the importance of infection, prevention and control measures to reduce the dissemination of antibiotic resistance within the hospital referral system in this district.

The Resistome, Mobilome, Virulome and Phylogenomics of Multidrug-Resistant Escherichia coli Clinical Isolates from Pretoria, South Africa.

Antibiotic-resistant Escherichia coli is a common occurrence in food, clinical, community and environmental settings worldwide. The resistome, mobilome, virulome and phylogenomics of 20 multidrug resistant (MDR) clinical E. coli isolates collected in 2013 from Pretoria, South Africa, were characterised. The isolates were all extended-spectrum ß-lactamase producers, harbouring CTX-M (n = 16; 80%), TEM-1B (n = 10; 50%) and OXA (n = 12, 60%) ß-lactamases alongside genes mediating resistance to fluoroquinolones, aminoglycosides, tetracyclines etc. Most resistance determinants were found on contigs containing IncF plasmid replicons and bracketed by composite transposons (Tn3), diverse ISs and class 1 integrons (In13, In54, In369, and In467). Gene cassettes such as blaOXA, dfrA5-psp-aadA2-cmlA1a-aadA1-qac and estX3-psp-aadA2-cmlA1a-aadA1a-qac were encompassed by Tn3 and ISs; several isolates had same or highly similar genomic antibiotic resistance islands. ST131 (n = 10), ST617 (n = 2) and singletons of ST10, ST73, ST95, ST410, ST648, ST665, ST744 and ST998 clones were phylogenetically related to clinical (human and animal) strains from Egypt, Kenya, Niger, Nigeria, Tanzania, and UK. A rich repertoire of virulence genes, including iss, gad and iha were identified. MDR E. coli harbouring chromosomal and plasmid-borne resistance genes in same and multiple clones exist in South Africa, which is very worrying for clinical epidemiology and infectious diseases management.

Genomic analysis of methicillin-resistant Staphylococcus aureus isolated from poultry and occupational farm workers in Umgungundlovu District, South Africa.

This study detected methicillin-resistant Staphylococcus aureus (MRSA) isolates circulating in poultry and farm workers at an intensive poultry production system in uMgungundlovu, South Africa and established the genetic relatedness and characteristics of the isolates using whole genome sequencing (WGS). A total of 145 S. aureus were isolated from poultry (120) and occupational workers (25) in the "farm to fork" continuum (farm, transport, slaughterhouse, and retail points). Twelve MRSA (12/145; 8.3%) isolates were found in the poultry food-chain. MRSA isolates were subjected to antibiotic susceptibility testing against a panel of 20 antibiotics using the broth dilution method and their whole genome was sequenced via the Illumina MiSeq. All the MRSA isolates were multi-drug resistant (MDR) and carried the mecA gene on the SCCmec mobile genetic element (MGE). The majority (11/12) of the MRSA isolates circulating between humans and animals in the continuum belonged to a human-associated clone, ST612-CC8-t1257-SCCmec_IVd (2B), previously reported in South Africa. Other MGEs present in the isolates included: plasmid replicons based on Rep 7 and 20, insertion sequences (IS1182), and prophages (phi2958PVL). Genomic analysis identified a distinct acquired antibiotic resistome in the clone, which accurately predicted the phenotypic antibiograms. Phylogenetic analysis clustered the isolates within the major cluster (I), suggesting the spread of the local dominant multidrug resistance MRSA clone ST612-CC8-t1257-SCCmec_IVd (2B) between humans and animals along the 'farm to fork' continuum. The findings of this study suggest the need to establish appropriate control measures to curb the spread of MDR-MRSA in the food chain.

Genome analysis of methicillin-resistant Staphylococcus aureus isolated from pigs: Detection of the clonal lineage ST398 in Cameroon and South Africa.

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.