Evaluation of Antimicrobial Resistance Dissemination in the Environment

Abstract

Antimicrobial resistance (AMR) is an emerging threat to public health worldwide. The importance of environmental reservoirs as a source of AMR emergence and transfer is well established. In the first phase of the study, a comparative genomic analysis of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in the gut microbiome of broiler and household chickens (Gallus gallus domesticus) was performed to evaluate the role of small scale poultry slaughter houses in resistance dissemination. An array of 52 qPCR primers targeting the 16S rRNA gene, ARGs and MGEs was used in the experiment to analyse the abundance of selected genes. A total of 22 ARGs and 7 MGEs were detected in all samples. The detection frequency of specific gene classes and diversity of genes was found to be higher in broiler as compared to household chicken. Sul1 was the most abundant ARG with the highest percent relative abundance (2.4%). Total percent relative abundance of ARGs in broiler chicken was found to be significantly (p < 0.05) greater than household chicken. A significant linear correlation (r2=0.89) was found between relative abundances of int1 and total ARGs. The clustering and correlation of selective ARGs with MGEs has implied that small scale poultry slaughterhouses can be a potential source for the dissemination of ARGs to other non-resistant environmental and/or clinical bacteria. Expansion in whole genome sequencing and subsequent increase in antibiotic resistance targets have paved the way of high throughput qPCR (HT-qPCR) for analyzing hundreds of ARGs in a single run. In the second phase of the study, a meta-analysis of 51 selected studies is performed to evaluate the abundance trends of ARGs over the last 7 years. WaferGenTM SmartChip was found to be the most widely used HT-qPCR platform among others for evaluating ARGs. More than 1000 environmental samples (excluding biological replicates) from different parts of the world have been analysed on HT-qPCR until October, 2018. Calculated detection frequency and normalized ARGs abundance (ARGs/ 16S rRNA gene) reported in gut microbiome have shown a trend of low ARGs as compared to other environmental matrices. Disparities in the HT-qPCR data analysis which are causing difficulties to researchers in precise interpretation of results have been highlighted and a possible way forward for resolving them is also suggested. The importance of anaerobic digesters for mitigating the dissemination of antimicrobial resistance genes is well known. The effect of substrate shock on the resistome of anaerobic digester is never evaluated. In the third phase sludge samples from 20 years old lab-scale bioreactors were analyzed by HT-qPCR for evaluating the impact of glucose shock on the community resistome of the reactors categorized as LS and HS. An array of 96 primers targeting antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) was used in the first experiment to analyze the abundance of genes before and after the glucose perturbation. The relative abundance of majority of the genes was increased in response to glucose perturbation. A second more expanded array consisting of 214 primers was also used to analyze the cooccurrence of the genes and to compare the overall resistome of the two reactor types. The resistome of the two reactor types was found to be significantly different from each other (P<0.01). Analysis of the co-occurrence network implied that anaerobic digesters can be a potent site for the acquisition of ARGs by pathogenic bacteria via horizontal gene transfer, further aggravating the already complex issue of AMR. A significant evidence has suggested that the knowledge and attitude trends among the community, pharmacists and physicians can play a critical role in managing the ever increasing threat of AMR. Thus in the final phase of the study, a cross-sectional survey was performed using three specific self-administered questionnaires for community members, pharmacists/ pharmacy owners and physicians on a randomly selected sample population of 473, 424 and 308 respectively. Bivariate and multivariate logistic regression and Pearson chi-square tests was performed during data analysis. A response rate of 81.2% (n=385), 37.7% (n=160) and 53.9% (n=166) was achieved for general community, pharmacists/ pharmacy owners and physicians respectively. More than half of the community participants (55.6%; n=214), 26.6% (n=4) qualified pharmacists and 86.2% (n=125) non-qualified pharmacy owners possess poor knowledge of AMR. Education level was found to be significantly influencing (p<0.05) the AMR knowledge of the community members. In 90.6% (n=145) of the pharmacies included in the survey, a qualified pharmacist was not present at the time of the operations. Our study will provide effective assessment and potential insights in designing tri-faceted interventions for rationalizing antibiotics consumption thus controlling the development and dissemination of AMR.