The carriage of antimicrobial resistance in community children – a TB-CHAMP sub-study.

Brand, Chante (2019-12)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

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.

Inleiding: Die wêreldwye toename in antibiotiese weerstand bedreig die effektiwiteit van antibiotika, asook die beheer van aansteeklike siektes. Die uitdaging van antibiotiese weerstand word aansienlik vererger deur die teenwoordigheid van mobiele genetiese elemente wat verskeie antibiotiese weerstandsgene dra en maklik kan versprei na ander spesies deur horisontale geenoordrag. Dit hou ernstige risikos in vir kliniese infeksies, maar inligting oor die dra van hierdie plasmied-bemiddelde weerstandsgene is egter nog baie skaars in Suid-Afrika. Hierdie studie beskryf die vlakke en meganismes van antibiotiese weerstand by baslislyn, asook die effek van levofloksasien blootstelling hierop in kinders in gemeenskappe in Kaapstad. Metodes: Stoelgang monsters is ingesamel vanaf 100 kinders wat deelneem aan die “Tuberculosis Child Multidrug-resistant Preventive Therapy Trial (TB-CHAMP)” by basislyn en 16-en 24-week opvolg besoeke tussen November 2017 en November 2019. Die stoelgang monsters is gekweek op MacConkey agar met kefpodoksiem en ertapenem skyfies en, in sommige gevalle, nalidiksiese suur and siprofloksasien skyfies, om kefalosporien-, karbapenem- en kinoloon-weerstandige en -vatbare Escherichia coli and Klebsiella spp. isolate te selekteer. Die Kirby Bauer skyfie diffusiemetode is gebruik om die antibiotiese weerstandsprofiele van die organismes te bepaal en polimerasekettingreaksie (PKR) en Sanger DNS-volgordebepaling is gebruik vir die opsporing van kefalosporien- en kinoloon-weerstandsmeganismes. DNS is ook direk vanaf die stoelgang monsters onttrek om geteikende molekulêre opsporing en kwantifisering van plasmied-bemiddelde kinoloon weerstands- (PBKW) gene uit te voer deur middel van ware-tyd PKR. Resultate: Hoë vlakke van antibiotiese weerstand is opgespoor by basislyn, waar 81% van deelnemers ‘n organisme gedra het wat weerstandig is teen ten minste een antibiotika en 49% en 33% kinoloon- en kefalosporien-weerstandige organismes onderskeidelik gedra het. Hierdie vlakke het toegeneem oor tyd, met betekenisvolle toenames in kinoloon-weerstand na 16 weke (69.8%). Die teenwoordigheid van die verlengde-spektrum -laktamase (VSBL) geen, blaCTX-M, in kefalosporienweerstandige E. coli en Klebsiella spp. het redelik konstant gebly met tyd, tussen 19.7 – 26.8% en 33.3 – 37.5% onderskeidelik oor 24 weke. Hierdie geen is egter in hoër vlakke waargeneem in Klebsiella spp. in vergelyking met E. coli. Ons het hoë vlakke van die PBKW-gene qnrB (53.3%) en aac(6’)-Ib-cr (66.7%) in Klebsiella spp. waargeneem by basislyn en betekenisvolle toenames in qnrS en aac(6’)-Ib-cr, asook mutasies in gyrA en parC na 16 en, in sommige gevalle, 24 weke. Die teenwoordigheid van aac(6’)-Ib-cr het ook betekenisvol toegeneem in E. coli vanaf basislyn (3.8%) na 16 weke (21.3%). qnrS is opgespoor in 86% van die stoelgange in die geteikende molekulêre analise, terwyl qnrB slegs in 14% opgespoor is, alhoewel dit meer oorvloedig was in die stoelgange as qnrS. Gevolgtrekkings: Ons het hoë vlakke van antibiotiese weerstand teenoor verskeie antibiotika aangemeld, asook die teenwoordigheid van VSBL en PBKW-gene, in kommensale bakterieë in kinders in Kaapstadse gemeenskappe voor en oor 24 weke van levofloksasien behandeling. Die hoë vlakke van kinoloon-weerstand by basislyn is veral ‘n bekommernis, aangesien omtrent die helfte van hierdie kinders met levofloksasien behandeling begin het nadat die basislyn monsters ingesamel is. Die toenames in die vlakke van weerstand en teenwoordigheid van PBKW-gene is interessant, maar die TB-CHAMP studie is egter nog aan die gang en ons weet nog nie watter deelnemers ontvang levofloksasien of plasebo nie. Sodra die TB-CHAMP studie voltooi is en ontblinding plaasgevind het, kan die resultate geskei word volgens behandelingsgroep om die invloed van levofloksasien op die dra van antibiotiese weerstand te bepaal.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/108372
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