Medical Microbiology

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Association between fluoroquinolone resistance and MRSA genotype in Alexandria, Egypt
(Nature, 2021-02-19) Alseqely, Mustafa; Newton-Foot, Mae; Khalil, Amal; El-Nakeeb, Mostafa; Whitelaw, Andrew; Abouelfetouh, Alaa
Antimicrobial stewardship isn’t strictly observed in most Egyptian hospitals, raising antibiotic resistance. Epidemiology of Egyptian MRSA isolates, or associations with resistance to other antibiotics remain largely unknown. We identified MRSA genotypes in Alexandria Main University Hospital (AMUH) and investigated rates of moxifloxacin resistance, an alternative MRSA treatment, among different genotypes. Antibiotic susceptibility of 72 MRSA clinical isolates collected in 2015 from AMUH was determined by disc diffusion and broth microdilution. spa- and Staphylococcal Cassette Chromosome mec (SCCmec) typing were performed; with multi-locus sequence typing conducted on isolates representing major genotypes. Resistance to moxifloxacin, levofloxacin and ciprofloxacin were 69%, 78% and 96%, respectively. spa type t037 (57%) was commonest, followed by t127 (12.5%), t267 (8%) and t688 (6%). SCCmec III predominated (57%), all of these were moxifloxacin resistant and 97.6% t037 (ST241). SCCmec IV, IV E and V represented 15%, 7% and 11% of the isolates, respectively, 79% of these were moxifloxacin susceptible and of different spa types. t127 (ST-1) was associated with SCCmec V in 56% of the isolates, mostly moxifloxacin susceptible. Moxifloxacin resistance was high, most resistant isolates belonged to t037 and SCCmec III, suggesting local dissemination and antibiotic pressure. We recommend caution in treating MRSA infections with moxifloxacin.
The carriage of antimicrobial resistance in community children – a TB-CHAMP sub-study.
(Stellenbosch : Stellenbosch University., 2019-12) Brand, Chante; Whitelaw, Andrew Christopher; Newton-Foot, Mae; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Microbiology.
Introduction: The world-wide rise in antimicrobial resistance (AMR) is threatening the effectivity of antibiotics and the control of infectious diseases. The challenge of AMR is considerably exacerbated by the presence of mobile genetic elements harbouring various antibiotic resistance genes, which can easily spread to other species by horizontal gene transfer. This poses serious risks for clinical infections, however, reports on the carriage of these plasmid-mediated resistance genes are still rare in South Africa. This study aimed to describe the rates of carriage and mechanisms of antimicrobial resistance at baseline, as well as the effect of levofloxacin exposure on these rates in children in communities in Cape Town. Methods: Stool samples were collected from 100 children enrolled in the Tuberculosis Child Multidrug-resistant Preventive Therapy Trial (TB-CHAMP) at baseline and at 16- and 24-week follow up visits between November 2017 and November 2019. The stool samples were cultured onto MacConkey agar plates with cefpodoxime and ertapenem disks and in some cases, nalidixic acid and ciprofloxacin disks, in order to select for cephalosporin, carbapenem and quinolone resistant and susceptible E. coli and Klebsiella isolates. Kirby Bauer disk diffusion was used to determine the susceptibility profiles of the organisms and PCR and Sanger sequencing were used for subsequent detection of cephalosporin and quinolone resistance mechanisms. DNA was extracted directly from the stools and targeted molecular detection and quantification of plasmidmediated quinolone resistance (PMQR) genes using real-time PCR. Results: High levels of antibiotic resistance were detected at baseline, with 81% of participants carrying an organism resistant to at least one antibiotic and 49% and 33% carrying quinolone and cephalosporin resistant organisms respectively. These rates increased over time, with significant increases in quinolone resistance after 16 weeks (69.8%). The presence of the extended spectrum -lactamase gene, blaCTX-M, in cephalosporin resistant E. coli and Klebsiella spp. remained relatively constant over time, ranging between 19.7 – 26.8% and 33.3 – 37.5% respectively over 24 weeks. However, this gene was observed in higher proportions in Klebsiella spp. compared to E. coli. We saw high rates of carriage of qnrB (53.3%) and aac(6’)-Ib-cr (66.7%) in Klebsiella spp. at baseline and significant increases in qnrS and aac(6’)-Ib-cr, as well as mutations in gyrA and parC after 16 and, in some cases, 24 weeks. The presence of aac(6’)-Ib-cr also increased significantly in E. coli from baseline (3.8%) to 16 weeks (21.3%). qnrS was detected in 86% of stools in the targeted molecular analysis, while qnrB was only detected in 14%, although it was more abundant than qnrS in the stool samples. Conclusions: We report high rates of resistance to various antibiotics, as well as the presence of -lactamase and PMQR genes, in commensal gut bacteria in children in Cape Town communities before and over 24 weeks of levofloxacin treatment. The high rate of quinolone resistance at baseline is especially worrying as roughly half of these children started levofloxacin treatment after the baseline stool samples were collected. The increase in rates of resistance and presence of PMQR genes is interesting, however, the TB-CHAMP trial is still ongoing and we do not yet know which participants are receiving levofloxacin or placebo. Once the TB-CHAMP study has been completed and the blind has been broken, the results can be stratified according to treatment group to determine the impact of levofloxacin on resistance carriage.
The Characterisation of Antibiotic Resistance Plasmids in Escherichia coli and Klebsiella pneumoniae Isolates from Hospital and Community Settings
(Stellenbosch : Stellenbosch University, 2021-03) Stein, Lisa; Newton-Foot, Mae; Whitelaw, Andrew; Pienaar, Colette; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.
ENGLISH ABSTRACT: Antimicrobial resistance has become one of the biggest challenges and threats to public health systems worldwide. Widespread distribution of resistance is commonly due to horizontal gene transfer, which includes mobile genetic elements (MGE) such as plasmids, insertion sequences, transposons, and integrons. This study aimed to characterise plasmids conferring antibiotic resistance in extended-spectrum β-lactamase (ESBL) positive Escherichia coli and Klebsiella pneumoniae isolates from bloodstream infections to determine whether ESBL and plasmid-mediated quinolone resistance (PMQR) genes were mobilised on the same plasmids and whether the same plasmids are disseminated in healthcare and community settings. Methods Illumina MiSeq whole-genome sequencing (WGS) was previously performed on 112 E. coli and 66 K. pneumoniae isolates from blood cultures submitted to the National Health Laboratory Service Microbiology Laboratory at Tygerberg Hospital during 2017 and 2018. Assembled genomes were interrogated for the presence of ESBL and PMQR genes and plasmid replicon types. Based on the results, eight E. coli and nine K. pneumoniae isolates were selected for plasmid sequencing on the Oxford Nanopore Technologies MinION platform. Unicycler assembler was used for hybrid assembly of Illumina short-reads and Nanopore plasmid long-reads. In silico analyses were performed using ResFinder, PlasmidFinder and ISsaga to identify ESBL and PMQR genes, plasmid replicon types, and MGEs. Results Based on Illumina WGS, the ESBL and PMQR containing isolates contained multiple resistance genes and IncF plasmid replicons, individually or in combination with additional plasmid types. The IncF replicons and resistance genes were on separate contigs, therefore associations between different IncF replicons and with resistance genes could not be confirmed. Nanopore sequencing resolved plasmids from several E. coli and K. pneumoniae isolates; however, chromosomal genes could not be visualised and misassembly resulted in fragmented plasmids. Hybrid assembly fully resolved plasmids and chromosomal genes in several E. coli and K. pneumoniae isolates. Amongst the E. coli isolates, three F-type multireplicon plasmids and two single replicon plasmids IncI1-γ and IncB/O/K/Z, which contained resistance genes, were described. Novel multireplicon plasmid FII(FIC)-FIB-X was detected and harboured ESBL blaTEM-135 and PMQR qnrS1. The blaCTX-M genes were confirmed to be chromosomally located in three E. coli isolates and plasmid-mediated on F-type plasmids in two E. coli isolates. K. pneumoniae isolates harboured single replicon F-type plasmids, multireplicon FIB-HI1B fusion plasmids, and a single replicon IncC plasmid. The FIB-HI1B plasmids were associated with blaCTX-M-15, aac(6’)-Ib-cr, and qnrS1. The blaCTX-M-15 was plasmid-mediated in all K. pneumoniae isolates. Conclusion Amongst the E. coli isolates, ESBL and PMQR genes were present both on the same plasmid and on separate plasmids. In K. pneumoniae, ESBLs and PMQRs were found collectively on the same plasmids, and the F-type plasmids harbouring ESBL and PMQR genes differed from those in E. coli. As only two community-acquired K. pneumoniae isolates were selected for Nanopore plasmid sequencing, conclusions regarding the dissemination of K. pneumoniae plasmids in healthcare and community settings could not be made. Plasmids of the same FAB-types were detected amongst E. coli isolates of various sequence types and from both hospital- and community-settings, which is indicative of spread between these settings.
Colistin resistance in gram-negative pathogens in the Western Cape, South Africa
(Stellenbosch : Stellenbosch University, 2021-12) Snyman, Yolandi; Newton-Foot, Mae; Whitelaw, Andrew Christopher; Maloba, Motlatji Reratilwe Bonnie; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Division of Medical Microbiology.
ENGLISH ABSTRACT: Background Antimicrobial resistance is a public health concern and injudicious antibiotic prescribing and inadequate infection control practices have left the global community with untreatable multidrugresistant (MDR) bacteria. Colistin is a last resort antibiotic used to treat infections with MDR Gramnegative bacteria (GNB), especially carbapenem-resistant GNB. Therefore, the emergence of colistin resistance is a serious problem. This study from the Western Cape, South Africa, describes colistin resistance mechanisms in colistin-resistant GNB isolates from clinical specimens from various hospitals, stool samples from healthy children in the community, and river and storm water. Methods Colistin-resistant GNB isolates from clinical specimens from different healthcare facilities were collected from the NHLS microbiology laboratory at Tygerberg Hospital during 2016 and 2017. Fifty stool samples from healthy children (≤ 5 year of age) in the Cape Town metropolitan were collected between November 2017 and August 2018, and three surface water sources and stormwater were collected in 2019 and 2020. Selective media was used to isolate colistin-resistant GNB from the stool and water samples. Colistin resistance was confirmed using broth microdilution (BMD). The mobile colistin resistance genes, mcr-1-9, were detected by PCR and whole-genome sequencing (WGS). In selected mcr-negative isolates chromosomal colistin resistance mutations were identified by WGS. Strain typing was performed by WGS (MLST and SNP analyses) and repPCR. The functionality of mcr genes with unknown colistin resistance profiles was determined by BMD following recombinant expression or plasmid curing. Results mcr-1 was present in 55% (12/22) of Escherichia coli and 71% (5/7) of Klebsiella spp. isolates from patients at various hospitals during 2016-2017. pmrB mutations were identified in 8/10 mcrnegative E. coli and mgrB was disrupted in the two mcr-negative Klebsiella spp. isolates. Most colistin-resistant GNB isolated from hospitalised patients in 2016 and 2017 were unrelated, however, some clonal relatedness was observed in the 2017 E. coli population and a clonal expansion of an emerging colistin-resistant MDR Acinetobacter baumannii strain was noted among isolates from 2017. No previously described colistin resistance mechanism was detected in the A. baumannii isolates, but a possible novel mechanism was described. mcr-4.3 was detected in a Stellenbosch University https://scholar.sun.ac.za iii single Acinetobacter nosocomialis isolate, although recombinant mcr-4.3 did not confer colistin resistance in E. coli, plasmid curing of the mcr-4.3-containing plasmid restored colistin susceptibility. Colistin-resistant E. coli were isolated from the stools of two healthy children from the community (4%, 2/50) during 2017-2018; however, mcr genes were not detected. Colistin-resistant GNB, mainly Aeromonas spp., and mcr-5.1 and/or various mcr-3 variants were detected in the Plankenburg river, Eerste river, and Berg river and stormwater from Muizenberg and Fish Hoek in 2019 and 2020. Of the colistin-resistant Aeromonas spp. isolated from the Berg river, 25% (6/24) contained five novel mcr-3 variants, which were confirmed to confer colistin resistance. Conclusion The emergence of colistin resistance mechanisms in diverse strains obtained from hospital patients, with the limited gastrointestinal carriage of colistin-resistant Enterobacterales in community children and the disparate colistin-resistant species and mechanisms in the environment, suggest that selective pressure, and not community transmission, is the main driver of colistin resistance in clinical settings.
First report of Wohlfahrtiimonas chitiniclastica bacteraemia in South Africa
(Health & Medical Publishing Group, 2016) Hoffman, R.; Fortuin, F.; Newton-Foot, M.; Singh, S.
ENGLISH ABSTRACT: No abstract available
In vitro activity of tigecycline and comparators against Gram-positive and Gram-negative isolates collected from the Middle East and Africa between 2004 and 2011
(Elsevier, 2014) Kanj, Souha; Whitelaw, Andrew; Dowzicky, Michael J.
The Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) was established in 2004 to monitor longitudinal changes in bacterial susceptibility to numerous antimicrobial agents, specifically tigecycline. In this study, susceptibility among Gram-positive and Gram-negative isolates between 2004 and 2011 from the Middle East and Africa was examined. Antimicrobial susceptibilities were determined using Clinical and Laboratory Standards Institute (CLSI) interpretive criteria, and minimum inhibitory concentrations (MICs) were determined by broth microdilution methods. US Food and Drug Administration (FDA)-approved breakpoints were used for tigecycline. In total, 2967 Gram-positive and 6322 Gram-negative isolates were examined from 33 participating centres. All Staphylococcus aureus isolates, including meticillin-resistant S. aureus, were susceptible to tigecycline, linezolid and vancomycin. Vancomycin, linezolid, tigecycline and levofloxacin were highly active (>97.6% susceptibility) against Streptococcus pneumoniae, including penicillin-non-susceptible strains. All Enterococcus faecium isolates were susceptible to tigecycline and linezolid, including 32 vancomycin-resistant isolates. Extended-spectrum β-lactamases were produced by 16.6% of Escherichia coli and 32.9% of Klebsiella pneumoniae. More than 95% of E. coli and Enterobacter spp. were susceptible to amikacin, tigecycline, imipenem and meropenem. The most active agents against Pseudomonas aeruginosa and Acinetobacter baumannii were amikacin (88.0% susceptible) and minocycline (64.2% susceptible), respectively; the MIC90 (MIC required to inhibit 90% of the isolates) of tigecycline against A. baumannii was low at 2 mg/L. Tigecycline and carbapenem agents were highly active against most Gram-negative pathogens. Tigecycline, linezolid and vancomycin showed good activity against most Gram-positive pathogens from the Middle East and Africa.
The prevalence and molecular mechanisms of mupirocin resistance in Staphylococcus aureus isolates from a Hospital in Cape Town, South Africa
(BMC (part of Springer Nature), 2020-03-14) Abdulgader, Shima M.; Lentswe, Tshepiso; Whitelaw, Andrew; Newton-Foot, Mae
Abstract Background: Antimicrobial resistance is an increasingly serious problem in public health globally. Monitoring resistance levels within healthcare and community settings is critical to combat its ongoing increase. This study aimed to describe the rates and molecular mechanisms of mupirocin resistance in clinical Staphylococcus aureus isolates from Tygerberg Hospital, and to describe its association with strain types. Methods: We retrospectively selected 212 S. aureus isolates which were identified from blood samples and pus swabs during the years 2009–2011 and 2015–2017. The isolates were identified using conventional microbiological methods and genotyping was done using spa typing. Cefoxitin (30 μg) disc diffusion and the two disc strategy (5 μg and 200 μg) were used to determine susceptibility to methicillin and mupirocin, respectively. Isolates with high-level resistance were screened for the plasmid mediated genes mupA and mupB by PCR, and sequencing of the ileS gene was done for all isolates exhibiting low-level resistance to describe the mutations associated with this phenotype. Chi-square test was used to assess the associations between mupirocin resistance and S. aureus genotypes. Results: Of 212 S. aureus isolates, 12% (n = 25) were resistant to mupirocin, and 44% (n = 93) were methicillin resistant. Strain typing identified 73 spa types with spa t045 being the most predominant constituting 11% of the isolates. High-level mupirocin resistance was observed in 2% (n = 5), and low-level resistance in 9% (n = 20) of the isolates. The prevalence of high-level mupirocin resistance amongst MRSA and MSSA was 4 and 1% respectively, while the prevalence of low-level mupirocin resistance was significantly higher in MRSA (18%) compared to MSSA (3%), (p = 0.032). mupA was the only resistance determinant for high-level resistance, and the IleS mutation V588F was identified in 95% of the isolates which showed low-level resistance. A significant association was observed between spa type t032 and high-level mupirocin resistance, and types t037 and t012 and low-level resistance (p < 0.0001). Conclusion: The study reported higher rates of low-level mupirocin resistance compared to high-level resistance, and in our setting, mupirocin resistance was driven by certain genotypes. Our study advocates for the continuous screening for mupirocin resistance in S. aureus in clinical settings to better guide treatment and prescribing practices. Background Antimicrobial resistance is an increasingly serious problem in public health globally. Monitoring resistance levels within healthcare and community settings is critical to combat its ongoing increase. This study aimed to describe the rates and molecular mechanisms of mupirocin resistance in clinical Staphylococcus aureus isolates from Tygerberg Hospital, and to describe its association with strain types. Methods We retrospectively selected 212 S. aureus isolates which were identified from blood samples and pus swabs during the years 2009–2011 and 2015–2017. The isolates were identified using conventional microbiological methods and genotyping was done using spa typing. Cefoxitin (30 μg) disc diffusion and the two disc strategy (5 μg and 200 μg) were used to determine susceptibility to methicillin and mupirocin, respectively. Isolates with high-level resistance were screened for the plasmid mediated genes mupA and mupB by PCR, and sequencing of the ileS gene was done for all isolates exhibiting low-level resistance to describe the mutations associated with this phenotype. Chi-square test was used to assess the associations between mupirocin resistance and S. aureus genotypes. Results Of 212 S. aureus isolates, 12% (n = 25) were resistant to mupirocin, and 44% (n = 93) were methicillin resistant. Strain typing identified 73 spa types with spa t045 being the most predominant constituting 11% of the isolates. High-level mupirocin resistance was observed in 2% (n = 5), and low-level resistance in 9% (n = 20) of the isolates. The prevalence of high-level mupirocin resistance amongst MRSA and MSSA was 4 and 1% respectively, while the prevalence of low-level mupirocin resistance was significantly higher in MRSA (18%) compared to MSSA (3%), (p = 0.032). mupA was the only resistance determinant for high-level resistance, and the IleS mutation V588F was identified in 95% of the isolates which showed low-level resistance. A significant association was observed between spa type t032 and high-level mupirocin resistance, and types t037 and t012 and low-level resistance (p < 0.0001). Conclusion The study reported higher rates of low-level mupirocin resistance compared to high-level resistance, and in our setting, mupirocin resistance was driven by certain genotypes. Our study advocates for the continuous screening for mupirocin resistance in S. aureus in clinical settings to better guide treatment and prescribing practices.

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