Molecular epidemiology and antibiotic resistance of staphylococci other than Staphylococcus aureus in children in Cape Town, South Africa.

Introduction: Staphylococci other than Staphylococcus aureus (SOSA) have emerged as significant pathogens in healthcare settings, particularly among patients with indwelling devices and immunocompromised individuals. Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus hominis are the most common commensal SOSA species and are implicated in infections such as endocarditis and bacteremia. SOSA infections in neonates and children have been reported globally. Recent increases in antibiotic resistance and virulence among SOSA strains in clinical settings have highlighted the need to describe the reservoirs of SOSA to enable monitoring of these emerging pathogens. Methods: Stool samples were collected from 150 healthy children from Cape Town communities between 2017 and 2020. Staphylococci were isolated, identified using mass-spectrometry, and antimicrobial susceptibility testing and Illumina whole genome sequencing were performed. Results: Among the participants, 50 (33.3%) were colonized by SOSA, with S. haemolyticus (n = 38; 25.3%) being the most common, followed by S. hominis (n = 5; 3.3%) and Mammalicoccus sciuri (n = 5; 3.3%). Out of the 77 initially isolated S. haemolyticus strains, 23 were identified as Staphylococcus borealis through whole genome sequencing. All S. haemolyticus isolates (n = 49) were methicillin resistant, with 65.3% (n = 32) harbouring mecA. In S. haemolyticus, SCCmec type VIII(4A) was detected in 42.0% of ST9 isolates while non-mecA methicillin resistant S. haemolyticus isolates were mostly ST49 (41.1%). Additionally, 16 (50.0%) S. haemolyticus strains contained non-typeable SCCmec elements. Discussion: High rates of methicillin resistance were identified among colonizing SOSA in Cape Town, increasing the risk of transmission to clinical settings. This study also identified a new species, S. borealis, for the first time in Africa.

Epidemiology of clinically suspected and laboratory-confirmed bloodstream infections at a South African neonatal unit.

INTRODUCTION: Data from Africa reporting the epidemiology of infection in hospitalised neonates are limited. METHODOLOGY: A prospective study with convenience sampling was conducted to characterise neonates investigated with blood culture/s for suspected infection at a 132-bed neonatal unit in Cape Town, South Africa (1 February-31 October 2018). Enrolled neonates were classified as having proven bloodstream infection (BSI) (blood culture-positive with a pathogen) or presumed infection (clinically suspected but blood culture-negative) or as potentially at risk of infection (maternal risk factors at birth). RESULTS: Of 1299 hospitalised neonates with >1 blood culture sampling episode, 712 (55%) were enrolled: 126 (17.7%) had proven BSI; 299 (42%) had presumed infection and 287 (40.3%) were potentially at risk of infection. Neonates with proven BSI had lower birth weight and higher rates of co-existing surgical conditions versus the presumed/potential infection groups (p < 0.001). Median onset of proven BSI versus presumed infection was at 8 (IQR = 5-13) and 1 (IQR = 0-5) days respectively (p < 0.001). Most proven BSI were healthcare-associated (114/126; 90.5%), with Klebsiella pneumoniae (80.6% extended-spectrum ß-lactamase producers) and Staphylococcus aureus (66.7% methicillin-resistant) predominating. Mortality from proven BSI (34/126; 27%) was substantially higher than that observed in presumed (8/299; 2.7%) and potential infections (3/287; 1.0%) (p < 0.001). The odds of death from proven BSI was 3-fold higher for Gram-negatives than for Gram-positive/fungal pathogens (OR = 3.23; 95% CI = 1.17-8.92). CONCLUSIONS: Proven BSI episodes were predominantly healthcare-associated and associated with a high case fatality rate. Most neonates with presumed infection or at potential risk of infection had favourable 30-day outcomes.

Characterisation of mcr-4.3 in a colistin-resistant Acinetobacter nosocomialis clinical isolate from Cape Town, South Africa.

OBJECTIVES: Colistin resistance in Acinetobacter spp. is increasing, resulting in potentially untreatable nosocomial infections. Plasmid-mediated colistin resistance is of particular concern due to its low fitness cost and potential transferability to other bacterial strains and species. This study investigated the colistin resistance mechanism in a clinical Acinetobacter nosocomialis isolate from Cape Town, South Africa. METHODS: A colistin-resistant A. nosocomialis isolate was identified from a blood culture in 2017. PCR and Illumina whole-genome sequencing (WGS) were performed to identify genes and mutations conferring resistance to colistin. Plasmid sequencing was performed on an Oxford Nanopore platform. mcr functionality was assessed by broth microdilution after cloning the mcr gene into pET-48b(+) and expressing it in SHuffle® T7 Escherichia coli and after curing the plasmid using 62.5 mg/L acridine orange. RESULTS: The colistin minimum inhibitory concentration (MIC) of the A. nosocomialis isolate was 16 mg/L. The mcr-4.3 gene was detected by PCR and WGS. No other previously described colistin resistance mechanism was found by WGS. The mcr-4.3 gene was identified on a 24 024-bp RepB plasmid (pCAC13a). Functionality studies showed that recombinant mcr-4.3 did not confer colistin resistance in E. coli. However, plasmid curing of pCAC13a restored colistin susceptibility in A. nosocomialis. CONCLUSION: We describe the first detection of a plasmid-mediated mcr-4.3 gene encoding colistin resistance in A. nosocomialis and the first detection of mcr-4.3 in a clinical isolate in Africa. Recombinant expression of mcr-4.3 did not confer colistin resistance in E. coli, suggesting that its functionality may be RepB plasmid-dependent or species-specific.

Colistin Resistance Mechanisms in Clinical Escherichia coli and Klebsiella spp. Isolates from the Western Cape of South Africa.

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

Clonal expansion of colistin-resistant Acinetobacter baumannii isolates in Cape Town, South Africa.

OBJECTIVES: To describe colistin-resistant Acinetobacter baumannii isolates in Cape Town, South Africa. METHODS: A. baumannii isolates identified on Vitek 2 Advanced Expert System were collected from Tygerberg Hospital referral laboratory between 2016 and 2017. Colistin resistance was confirmed using broth microdilution and SensiTest. mcr-1-5 were detected using PCR and strain typing was performed by rep-PCR. Whole genome sequencing (WGS) was performed on a subset of isolates to identify chromosomal colistin resistance mechanisms and strain diversity using multilocus sequence typing (MLST) and pairwise single nucleotide polymorphism analyses. RESULTS: Twenty-six colistin-resistant and six colistin-susceptible A. baumannii were collected separately based on Vitek susceptibility; 20/26 (77%) were confirmed colistin-resistant by broth microdilution. Four colistin-resistant isolates were isolated in 2016 and 16 in 2017, from five healthcare facilities. Thirteen colistin-resistant isolates and eight colistin-susceptible isolates were identical by rep-PCR and MLST (ST1), all from patients admitted to a tertiary hospital during 2017. The remaining colistin-resistant isolates were unrelated. CONCLUSIONS: An increase in colistin-resistant A. baumannii isolates from a tertiary hospital in 2017 appears to be clonal expansion of an emerging colistin-resistant strain. This strain was not detected in 2016 or from other hospitals. Identical colistin-susceptible isolates were also isolated, suggesting relatively recent acquisition of colistin resistance.

Plasmid-mediated mcr-1 colistin resistance in Escherichia coli and Klebsiella spp. clinical isolates from the Western Cape region of South Africa.

BACKGROUND: Colistin is a last resort antibiotic for the treatment of carbapenem-resistant Gram negative infections. Until recently, mechanisms of colistin resistance were limited to chromosomal mutations which confer a high fitness cost and cannot be transferred between organisms. However, a novel plasmid-mediated colistin resistance mechanism, encoded by the mcr-1 gene, has been identified, and has since been detected worldwide. The mcr-1 colistin resistance mechanism is a major threat due to its lack of fitness cost and ability to be transferred between strains and species. Surveillance of colistin resistance mechanisms is critical to monitor the development and spread of resistance.This study aimed to determine the prevalence of the plasmid-mediated colistin resistance gene, mcr-1, in colistin-resistant E. coli and Klebsiella spp. isolates in the Western Cape of South Africa; and whether colistin resistance is spread through clonal expansion or by acquisition of resistance by diverse strains. METHODS: Colistin resistant E. coli and Klebsiella spp. isolates were collected from the NHLS microbiology laboratory at Tygerberg Hospital. Species identification and antibiotic susceptibility testing was done using the API® 20 E system and the Vitek® 2 Advanced Expert System™. PCR was used to detect the plasmid-mediated mcr-1 colistin resistance gene and REP-PCR was used for strain typing of the isolates. RESULTS: Nineteen colistin resistant isolates, including 12 E. coli, six K. pneumoniae and one K. oxytoca isolate, were detected over 7 months from eight different hospitals in the Western Cape region. The mcr-1 gene was detected in 83% of isolates which were shown to be predominantly unrelated strains. CONCLUSIONS: The plasmid-mediated mcr-1 colistin resistance gene is responsible for the majority of colistin resistance in clinical isolates of E. coli and Klebsiella spp. from the Western Cape of South Africa. Colistin resistance is not clonally disseminated; the mcr-1 gene has been acquired by several unrelated strains of E. coli and K. pneumoniae. Acquisition of mcr-1 by cephalosporin- and carbapenem-resistant Gram negative bacteria may result in untreatable infections and increased mortality. Measures need to be implemented to control the use of colistin in health care facilities and in agriculture to retain its antimicrobial efficacy.