Knowledge, attitudes, and practices of veterinarians on antibiotic use and resistance and its containment in South Africa.

The inappropriate use of antibiotics in the veterinary sector has contributed to antibiotic resistance (ABR), which negatively impacts animal health and welfare. Understanding the knowledge, attitudes, and practices (KAP) on antibiotic use, ABR, and its containment amongst veterinarians is critical to optimise antibiotic use and contain resistance. A quantitative questionnaire-based online survey was conducted amongst members of professional veterinary associations. The questionnaire consisted of four sections focusing on socio-demographic characteristics, KAP of participants on antibiotic use, ABR, and its containment in the South African veterinary sector. The Independent t-test, analysis of variance (ANOVA), and chi-square test were used to establish associations among selected socio-demographic variables and selected KAP parameters. A total of 130 responses were received from 2 178 animal health professionals, yielding a response rate of six per cent, with 102 complete responses constituting the final sample size. Self-reported knowledge on antibiotic stewardship, ABR mechanisms, and pharmacology was good at 96 (94.1%), 91 (89.2%), and 70 (68.6%), respectively. Notably, most of the veterinarians (61; 59.8%) lacked an antibiotic stewardship programme at their practice. Place of practice was significantly associated (p = 0.004) with possession of knowledge about ABR. Veterinarians in urban practice were more knowledgeable about ABR than those in rural practice. Antibiotic stewardship programmes need to be implemented in veterinary practice. Such programmes might encourage the frequent use of consensus guidelines for the appropriate use of antibiotics and microbiology-informed therapy.

Clinical management of COVID-19 in hospitals and the community: A snapshot from a medical insurance database in South Africa.

BACKGROUND: Little is known about the clinical care, use of medicines, and risk factors associated with mortality among the population with private health insurance with COVID-19 in South Africa. METHODS: This was a retrospective cross-sectional study using claims data of patients with confirmed COVID-19. Sociodemographics, comorbidities, severity, concurrent/progressive comorbidity, drug treatment, and outcomes were extracted from administrative data. Univariate and multivariate logistic regression models were used to explore the risk factors associated with in-hospital death. RESULTS: This study included 154,519 patients with COVID-19; only 24% were categorized as severe because they received in-hospital care. Antibiotic (42.8%) and steroid (30%) use was high in this population. After adjusting for known comorbidities, concurrent/progressive diagnosis of the following conditions were associated with higher in-hospital death odds: acute respiratory distress syndrome (aOR = 1.55; 95% CI = 1.44-1.68), septic shock (aOR = 1.55; 95% CI = 2.00-4.12), pneumonia (aOR = 1.35; 95% CI = 1.24-1.47), acute renal failure (aOR = 2.30; 95% CI = 2.09-2.5), and stroke (aOR = 2.09; 95% CI = 1.75-2.49). The use of antivirals (aOR = 0.47; 95% CI= 0.40-0.54), and/or steroids (aOR = 0.46; 95% CI = 0.43-0.50) were associated with decreased death odds. The use of antibiotics in-hospital was not associated with increased survival (aOR = 0.97; 95% CI = 0.91-1.04). CONCLUSIONS: Comorbidities remain significant risk factors for death mediated by organ failure. The use of antibiotics did not change the odds of death, suggesting inappropriate use.

Resistome of a carbapenemase-producing novel ST232 Klebsiella michiganensis isolate from urban hospital effluent in South Africa.

OBJECTIVES: Klebsiella michiganensis is an emerging pathogen implicated in nosocomial infections. Here we report on the resistome, virulome and mobilome of a carbapenemase-producing K. michiganensis isolate from urban hospital effluent in Pietermaritzburg, KwaZulu-Natal, South Africa. Klebsiella sp. isolate KP124 was originally isolated from the final effluent of an urban tertiary hospital in Pietermaritzburg, KwaZulu-Natal. METHODS: Following phenotypic characterisation and antibiotic susceptibility testing, the genome of carbapenemase-producing isolate KP124 was sequenced using an Illumina MiSeq platform, de novo assembled and analysed using established bioinformatics tools. RESULTS: The draft genome of strain KP124 was 6 544 586 bp in length, comprising 203 contigs >200 bp. Following confirmation of isolate KP124 as K. michiganensis using reference genomes, the blaOXA-181 carbapenemase gene as well as 11 additional genes encoding resistance against ß-lactams, aminoglycosides, fluoroquinolones and sulfonamides were detected. Virulence factors enabling iron acquisition and cell adherence, capsule locus type and plasmid replicon types were identified. CONCLUSION: This study represents the first report of an OXA-181 carbapenemase-producing K. michiganensis isolate from hospital effluent in South Africa. The presence of such a strain in the environment owing to the absence of hospital effluent treatment presents a potential risk to informal communities that may use contaminated surface water domestically.

Genomic analysis of two drug-resistant clinical Morganella morganii strains isolated from UTI patients in Pretoria, South Africa.

Morganella morganii is an opportunistic bacterial pathogen of the Enterobacteriaceae family that is occasionally isolated from clinical (animal and human) specimens with varying resistance profiles. Detailed genomic analyses of drug-resistant M. morganii strains are relatively limited, particularly in Africa, which is also due to their relatively low isolation rates from clinical settings. Here we report on two multidrug-resistant clinical M. morganii isolates from urine specimens of two hospitalized patients in South Africa who presented with urinary tract infections in 2013. The isolates, M006 and E042, were only susceptible to carbapenems, amikacin and tigecycline. One strain, M006, had a novel class 1 integron, ln1484, associated with aadA7, sul1and gcuD gene cassettes and a Col3M plasmid replicase gene. The ln1484 intI1:aadA7:sul1 genes were bracketed by a TnAs3 composite transposon while a tet(B) gene was found on an IS4 family transposon. The rare blaDHA-4 and blaDHA-1 AmpC ß-lactamase genes were identified on the isolates' chromosome. The isolates were phylogenetically distant and closely related to other international strains, suggesting that they were not obtained from a single epidemiological source. Further molecular surveillance is necessary to establish the prevalence of these MDR strains in the tertiary hospital. Moreover antibiotic stewardship and antibiotic sensitivity testing of all clinical isolates should be undertaken after empirical treatment to inform tailored therapy as well as reduce escalation of resistance and associated morbidities and mortalities. SIGNIFICANCE AND IMPACT OF THE STUDY: We report on the first clinical Morganella morganii draft genomes from Africa. The isolates were found in the urine of patients presenting with urinary tract infections (UTIs). Notably, they were resistant to important clinical antibiotics, including those used to treat UTIs. Due to the common occurrence of UTIs, particularly among pregnant women for whom drug options are limited, the presence of antibiotic-resistant uropathogens such as M. morganii is a serious public health concern. We therefore characterized the resistance mechanisms and epidemiology of these isolates to provide further insights into their dissemination and background data for future studies.

In vitro potentiation of carbapenems with tannic acid against carbapenemase-producing enterobacteriaceae: exploring natural products as potential carbapenemase inhibitors.

AIMS: We hypothesized and confirmed that tannic acid (TA) reverses carbapenem resistance by inhibiting carbapenemases in class A and B carbapenemase-producing Enterobacteriaceae. METHODS AND RESULTS: Minimum inhibitory concentrations of carbapenems in the presence and absence of TA and other efflux pump inhibitors, TA-carbapenemases inhibition assays and computational studies showed that TA had the greatest effect on metallo-ß-lactamases (MBLs) followed by class A serine-ß-lactamases (SBLs). TA completely reversed the MICs of MBL producers from between 32 and ≥512 mg l-1 to susceptible values (<4 mg l-1 ) while substantially reducing the MICs of SBLs from between 16 and >512 mg l-1 to <4 to 16 mg l-1 . Tolerable cytotoxic effect was observed for the concentrations tested (8-1024 mg l-1 ). TA inhibited enzymes with a marked difference of ≈50% inhibition (IC50 ) for NDM-1 (270 µmol l-1 ) and KPC-2 (15  µmol l-1 ). CONCLUSION: TA inhibited both MBLs and SBLs by targeting their hydrophobic sites. Moreover, TA had a stronger binding affinity for MBLs than SBLs as the MBLs, specifically VIM-1 (-43·7220 ± 0·4513 kcal mol-1 ) and NDM-1(-44·2329 ± 0·3806 kcal mol-1 ), interact with a larger number of their catalytic active-site residues than that of OXA-48 (-22·5275 ±  0·1300 kcal mol-1 ) and KPC-2 (-22·1164 ± 0·0111 kcal mol-1 ). SIGNIFICANCE AND IMPACT OF THE STUDY: Tannic acid or its analogues could be developed into carbapenemase-inhibiting adjuvants to restore carbapenem activity in CRE infections, save many lives and reduce healthcare associated costs.

Molecular characterization of methicillin-resistant Staphylococcus aureus isolates from a hospital in Ghana

Background: Methicillin-resistant Staphylococcus aureus (MRSA) are a major cause of hospital- and community-acquired infection. They can colonize humans and cause a wide range of infections including pneumonia, endocarditis and bacteraemia. We investigated the molecular mechanism of resistance and virulence of MRSA isolates from a teaching hospital in Ghana. Methodology: A total of 91 S. aureus isolates constituted the initial bacterial sample. Identification of S. aureus was confirmed by the VITEK 2 system. The cefoxitin screen test was used to detect MRSA and antibiotic susceptibility was determined using the VITEK 2 system. The resistance (mecA, blaZ, aac-aph, ermC, and tetK) and virulence (lukS/F-PV, hla, hld and eta) genes were amplified by polymerase chain reaction (PCR) and positive samples subjected to DNA sequencing. Pulsed field gel electrophoresis (PFGE) was used to ascertain the relatedness of the isolates. Results: Fifty-eight of 91 (63.7%) isolates were putatively methicillin resistant by the phenotypic cefoxitin screen test and oxacillin MICs. However, 43 (47%) of the isolates were genotypically confirmed as MRSA based on PCR detection of the mecA gene. Furthermore, 37.9% of isolates displayed resistance to tetracycline, 19% to trimethoprim-sulphamethoxazole, 15.5% to clindamycin, 12.1% to gentamicin, 13.8% to ciprofloxacin and erythromycin, 6.9% to moxifloxacin and 7.0% to rifampicin. None of the isolates was positive for inducible clindamycin resistance. The prevalence of resistance (mecA, blaZ, aac(6')-aph(2''), tetK, and ermC) and virulence (hla and lukS/F-PV) genes respectively were 74%, 33%, 22%, 19%, 3%, 5% and 3%, with isolates organized in two highly related clades. Conclusion: Results indicate a fairly high occurrence of MRSA, which can complicate the effective therapy of S. aureus infections, necessitating surveillance and stringent infection control programmes to forestall its spread

Faecal colonization of E. coli and Klebsiella spp. producing extended-spectrum beta-lactamases and plasmid-mediated AmpC in Mozambican university students.

BACKGROUND: In recent years, the world has seen a surge in Enterobacteriaceae resistant to broad-spectrum beta-lactam antibiotics due to the production of extended-spectrum beta-lactamases (ESBLs) or plasmid-mediated AmpC (pAmpC) enzymes. Data on the epidemiology of cephalosporin-resistant Enterobacteriaceae in Sub-Saharan Africa are still limited. METHODS: Two hundred seventy-five non-repetitive stool samples were collected from Mozambican university students of both sexes. Samples were cultured on MacConkey agar with and without ceftriaxone (1 mg/L) for selection of third-generation cephalosporin-resistant isolates, which were subjected to antimicrobial susceptibility testing by disc diffusion, characterization of resistance genes by PCR and ERIC-PCR analysis for strain clonality. RESULTS: Among the 275 students, 55 (20%) carried a total of 56 E. coli (n = 35) and Klebsiella spp. (n = 21) isolates resistant to ceftriaxone and phenotypically positive for ESBL- and/or pAmpC-production. Forty-three percent of the isolates (24/56) contained only ESBL genes, 11% (6/56) only pAmpC genes, and 36% (20/56) both ESBL and pAmpC genes. The remaining six isolates were negative for the CTX-M/pAmpC genes included in the test panel. E. coli and Klebsiella spp. combined demonstrated 70% resistance to tetracycline and co-trimoxazole, 63% to ceftazidime and 34% to ciprofloxacin. In total, 89% of ESBL/pAmpC-positive isolates were defined as multi-resistant by being resistant to three or more antibiotic classes. ERIC-PCR fingerprinting demonstrated low similarity among isolates. None of the participants reported recent hospitalization and just 12.5% had taken antibiotics 3 months prior to the study. CONCLUSION: This study demonstrated 20% colonization with multi-resistant E. coli and Klebsiella spp. among Mozambican students with a diversity of ESBL and pAmpC genes. Colonization was not related to prior hospitalization or antimicrobial consumption.

Antimicrobial resistance in the WHO African region: current status and roadmap for action.

The high burden of communicable diseases in African countries engenders extensive antimicrobial use and subsequent resistance with substantial health, financial and societal implications. A desktop analysis to ascertain whether countries in the WHO African region have implemented the WHO Policy Package to combat antimicrobial resistance (AMR) revealed that just two countries (4.3%) have national AMR plans in place, 14.9% (7) have overarching national infection prevention and control (IPC) policies, 93.6% (44) have essential medicines lists and 91.5% (43) have national medicines policies and treatment guidelines intimating rational use. None currently have representative national surveillance systems nor do any incentivize research and development into new medicines and diagnostics. A regional situational analysis to identify scalable good practices within African, resource-constrained country contexts under the auspices of WHO-AFRO is a necessary initial step towards the development of national and regional action plans in concert with incremental progress towards achieving the objectives of the policy package and global action plan. While it is clearly the responsibility of governments to develop, resource and implement plans, regular reporting to and/or monitoring and evaluation by an overarching body such as WHO-AFRO will ensure persistent incremental progress within continuous quality and accountability improvement paradigms.

Colistin and tigecycline resistance in carbapenemase-producing Gram-negative bacteria: emerging resistance mechanisms and detection methods.

A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase-producing Gram-negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans-complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT-PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC-FGH-IJK, mexAB-XY-oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr-1 gene conferring resistance to colistin was identified via WGS, trans-complementation and a murine thigh infection model studies. Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI-TOF MS, micro-array and real-time multiplex PCR hold much promise for the future as new detection tools.

In vitro evaluation of metal chelators as potential metallo- ß -lactamase inhibitors.

AIMS: This study aimed at investigating the use of metal chelators as potential metallo-ß-lactamase inhibitors (MBL). METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of meropenem was ascertained alone and in combination with various concentrations of macrocyclic (1,4,7- triazacyclononane-1-glutaric acid-4,7-diacetic acid = NODAGA) peptide derivatives and acyclic (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine = TPEN and di-(2-picolyl)amine = DPA) metal chelators using the broth microdilution method. MICs of meropenem against carbapenem-resistant enterobacteriaceae (CRE) producing MBLs were decreased to concentrations as low as 0·06 mg l(-1) in the presence of some metal chelators. TPEN at 4 and 8 mg l(-1) showed the best activity by decreasing meropenem MICs to 0·5 and 0·06 mg l(-1) , respectively, for some New Delhi Metallo-beta-lactamase (NDM) and Verona integron-encoded metallo-ß-lactamase (VIM) -producing enterobacteriaceae. DPA at 8 and 16 mg l(-1) was also able to decrease meropenem MICs to 1 and 0·125 mg l(-1) , respectively, for these CREs. NODAGA peptide derivatives showed the least inhibition as 32 mg l(-1) was required for meropenem MICs to be decreased to 0·06 mg l(-1) against an NDM-1 producing isolate. CONCLUSION: The various metal chelators, TPEN, DPA and NODAGA peptide derivatives were able to inhibit the MBLs in decreasing order of activity, rendering CREs susceptible to meropenem. SIGNIFICANCE AND IMPACT OF THE STUDY: In the absence of new antibiotics, this study evaluated metal chelators as potential MBL inhibitors.

Review of established and innovative detection methods for carbapenemase-producing Gram-negative bacteria.

The minimal antibiotic options for carbapenemase-producing Gram-negative bacteria necessitate their rapid detection. A literature review of a variety of phenotypic and genotypic methods is presented. Advances in culture methods and screening media are still subject to long incubation hours. Biochemical methods have shorter turnaround times and higher sensitivities and specificities, but cannot differentiate between various types and variants. Spectrophotometric methods are cheap and efficient, but are uncommon in many clinical settings, while the MALDI-TOF MS is promising for species identification, typing and resistance gene determination. Although next generation sequencing (NGS) technologies provide a better platform to detect, type and characterize carbapenem-resistant bacteria, the different NGS platforms, the large computer memories and space needed to process and store genomic data and the nonuniformity in data analysis platforms are still a challenge. The sensitivities, specificities and turnaround times recorded in the various studies reviewed favours the use of the biochemical tests (Carba NP or Rapid Carb screen tests) for the detection of putative carbapenemase-producing isolates. MALDI-TOF MS and/or molecular methods like microarray, loop-mediated isothermal amplification and real-time multiplex PCR assays could be used for further characterization in a reference laboratory. NGS may be used for advanced epidemiological and molecular studies.

Prevalence of antibiotic resistance in Campylobacter isolates from commercial poultry suppliers in KwaZulu-Natal, South Africa.

OBJECTIVES: Campylobacter jejuni isolated from broiler and layer chickens from registered abattoirs in KwaZulu-Natal, South Africa, were tested for their susceptibility to eight antibiotics. METHODS: Using agar dilution, susceptibility to eight antibiotics was determined for C. jejuni recovered from the caeca. RESULTS: A total of 155 isolates were collected of which 77 were identified as C. jejuni (broilers n = 56 and layers n = 21). Resistance was highest to tetracycline (broilers 98.2% and layers 100%) and ceftriaxone (broilers 96.4% and layers 100%). High susceptibility was found to ciprofloxacin (broilers 91% and layers 76%) and gentamicin (broilers 98% and layers 81%). Susceptibilities to each of the antibiotics for the broilers and layers, respectively, were: 50% and 57% for erythromycin, 45% and 24% for clarithromycin, 68% and 43% for ampicillin and 64% and 48% for nalidixic acid. Statistically significant differences were detected for the MIC(50) of gentamicin, ciprofloxacin and tetracycline between broilers and layers (P < 0.001) with the MIC(90) of gentamicin also of significant difference (P = 0.01). Multiresistance was detected in 23% and 43% of the isolates from broiler and layer chickens, respectively. CONCLUSIONS: Mass therapy procedures used in animal husbandry have a potential impact on antibiotic resistance development in C. jejuni.

CTX-M-37 in Salmonella enterica serotype Isangi from Durban, South Africa.

Beta-lactamase-mediated resistance was investigated in 59 putative extended-spectrum beta-lactamase (ESBL)-positive Salmonella spp. from the paediatric ward of a tertiary hospital in Durban, South Africa. Three Salmonella enterica serotype Isangi cultured from stool samples were multidrug resistant, with susceptibility only to meropenem, piperacillin/tazobactam and cefoxitin. Isoelectric focusing revealed beta-lactamases with isoelectric points of pI 5.8, 6.8 and 7.2. Sequencing identified beta-lactamases CTX-M-37 and TEM-1. To our knowledge, this is the first report of CTX-M-37 from S. enterica serotype Isangi in South Africa.