The carriage of antibiotic resistant Gram-negative organisms in children in the Cape Town community and the impact of antibiotic exposure on the development of resistance (a TB-CHAMP sub-study)

Ocloo, Remous (2019-12)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

Introduction Antibiotic resistance has become a major issue across the globe and the situation is worsening in low- and middle-income countries. In sub-Saharan Africa and the world at large, antibiotic resistance research is localized and focused on hospitalized individuals. There is, therefore, little or no data on antibiotic resistance in the community; especially in children. This study described the carriage of resistant isolates in children in Cape Town and investigated the effects of antibiotic exposure on the development of resistance in stool using an in-vitro model. Materials and Methods Stool samples from fifty participants of the Tuberculosis Child Multidrug-resistant Preventive Therapy Trial (TB-CHAMP) were cultured onto McConkey agar (MCC) with the addition of ertapenem and cefpodoxime discs to select for carbapenem and cephalosporin-resistant and susceptible E. coli and Klebsiella isolates. Antibiotic susceptibility testing was performed using Kirby Bauer disk diffusion. Carbapenem-, quinolone- and cephalosporin-resistance genes were detected by PCR and resistance-conferring mutations were detected using Sanger sequencing. Ten stool samples were exposed to two sub-clinical concentrations of amoxicillin, ciprofloxacin and colistin for 48 hours, whereafter they were plated onto MCC with the addition of various antibiotic discs (amoxicillin, ertapenem, ciprofloxacin, colistin, cefotaxime and nalidixic acid). The impact of antibiotic exposure on the development of resistance was assessed by enumeration of presumptive resistant E. coli and Klebsiella colonies within the zones of inhibition around the antibiotic discs. Results Twenty-one (42%) of the participants were colonized by quinolone-resistant isolates and 18 (36%) by cephalosporin-resistant isolates (predicted ESBL-producing organisms). Of the 21 quinolone-resistant E. coli isolates, 5 (24%) harbored qnrS while of the 6 quinolone-resistant Klebsiella isolates, 4 (67%) had qnrB. The most common quinolone resistance mutations were S83L in gyrA and S80I, A141V and S129A in parC. blaCTX-M was the only ESBL gene detected. All of the blaSHV and blaTEM genes detected were β-lactamases without extended spectrum activity. One of the participants was colonized by a carbapenem resistant Klebsiella isolate, which carried the blaNDM carbapenemase gene. Exposure of the stool samples to ciprofloxacin selected for resistant bacteria, however exposure to amoxicillin and colistin did not. Conclusion Children in Cape Town are frequently colonized by resistant bacteria and are at risk of becoming infected by these resistant organisms. The presence of plasmid-mediated resistance genes is concerning because they can be transferred between bacteria of the same and different species. There is also a need to further investigate what might be driving the high prevalence of quinolone resistance in the community. This study is the first to report the carriage of carbapenemase resistant bacteria in healthy children in South Africa. Although the in-vitro antibiotic exposure model was crude, the approach provides some evidence for the development of resistance during exposure to sub-clinical concentrations of antibiotics (especially ciprofloxacin); and notably, to agents other than those to which the sample had been exposed. This highlights the need for further investigations into the impact of sublethal antibiotic concentrations on the selection of resistance.

Inleiding Antibiotiese weerstand is ‘n wêreldwye probleem, en die situasie is besig om in lae- en middelvlakinkomstelande te versleg. In sub-Sahara Afrika, en ook die res van die wêreld, fokus navorsing oor antibiotiese weerstand grootliks op gehospitaliseerde individue en is meestal gelokaliseerd. Daar is dus min tot geen data oor antibiotiese weerstand in die gemeenskap nie; veral in kinders. Hierdie studie beskryf die verspreiding van weerstandbiedende bakteriële isolate in Kaapstad en het die effek van antibiotika-blootstelling op ontwikkeling van weerstand in stoelmonsters met ‘n in-vitro model ondersoek. Metodes Stoelmonsters vanaf vyftig deelnemers van die “Tuberculosis Child Multidrug-resistant Preventive Therapy Trial” (TB-CHAMP) is op McConkey agar (MCC) met ertapenem en cefpodoksiem skyfies geweek, om karbapenem- en kefalosporien-weerstandbiendende en -vatbare E. coli en Klebsiella isolate te selekteer. Antibiotiese vatbaarheidstoetse is deur Kirby Bauer skyfiediffusie uitgevoer. Karbapenem, kinoloon en kefalosporien-weerstandsgewende mutaties is met Sanger DNA-volgordebepaling bespeur. Tien stoelmonsters was vir 48 uur aan twee subkliniese konsentrasies van amoksisillien, siprofloksasien en colistin blootgestel, waarna hulle op MCC gekweek is met verskeie antibiotika skyfies (amoksisillien, ertapenem, siprofloksasien, colistin, kefotaksiem en nalidiksiensuur). Escherichia coli en Klebsiella kolonies binne die inhibisie-zones rondom die antibiotika skyfies is getel om die impak van antibiotika-blootstelling op die ontwikkeling van weerstand te bepaal. Resultate Een-en-twintig (42%) van die deelnemers was met kinoloon-weerstandige isolate gekoloniseer en 18 (36%) deur kefalosporien-weerstandige isolate (voorspel om “Extended spectrum beta-lactamase”-produserend (ESBL) te wees). Van die 21 kinoloon-weerstandige E. coli isolate, het 5 (24%) qnrS gene besit, terwyl 4 (67%) van die kinoloon-weerstandige Klebsiella isolate positief getoets het vir qnrB. Die algemeenste kinoloon weerstandgewende mutaties was gyrA en S80I, A141V en S129A in parC. blaCTX-M was die algemeenste ESBL geen wat geïdentifiseer is. Geen van die geïdentifiseerde blaSHV en blaTEM gene was ESBL-produserend nie. Een deelnemer was met ‘n karbapenem-weerstandbiedende Klebsiella isolaat met ‘n blaNDM karbapenemase geen gekoloniseer. In die stoelmonsters het blootstelling aan siprofloksasien, eerder as amoksisillien en colistin, vir weerstanbiedende bakterieë geselekteer. Gevolgtrekking Kinders in Kaapstad word gereeld deur weerstandbiedende bakterieë gekoloniseer en beloop die risiko om deur hierdie organismes geïnfekteer te word. Die teenwoordigheid van plasmied-bemiddelde weerstandsgene is daarom kommerwekkend, aangesien hulle tussen bakterieë van dieselfde en verskillende spesies oorgedra kan word. Dit is dus nodig om verdere ondersoek te doen om te bepaal wat die hoë vlak van kinoloon-weerstand in die gemeenskap veroorsaak. Hierdie studie bevat die eerste beskrywing van die verspreiding van karbapenem weerstandbiedende bakterieë in gesonde kinders in Suid-Afrika. Alhoewel die in-vitro antibiotika-blootstelling model nie gesofistikeerd was nie, het hierdie benadering bewys gegee van die ontwikkeling van weerstand tydens blootstelling aan subkliniese konsentrasies van antibiotika (veral siprofloksasien); merkwaardig ook aan ander middels waaraan die monsters nie blootgestel was nie. Dit wys dat verdere ondersoek ingestel moet word om die impak van subkliniese antibiotika konsentrasies op die seleksie van weerstand te bepaal.

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