Ghadigaonkar Devika, Rath Archana
Antimicrobial Research (AMR) Lab, Department of Biotechnology, University of Mumbai.
Antimicrobial Research (AMR) Lab, Department of Biotechnology, University of Mumbai;
J Vis Exp. 2023 Mar 3(193). doi: 10.3791/63934.
The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 × 10-1.2 × 10 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of ≥0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae, and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (β-lactams), aadA, aac (6')-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, β-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the chloramphenicol acetyltransferase gene catB10, and the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.
通过与淡水水体相关的微生物群产生和传播抗生素耐药性（AR）是一个重大的全球健康问题。在本研究中，采集了淡水样本，并使用传统的基于培养的技术和高通量的不依赖培养的宏基因组方法，对总细菌多样性和AR基因（ARGs）进行了分析。本文提出了一种系统方案，用于从淡水样本中计数总可培养细菌和抗生素耐药可培养细菌，并确定可培养分离株的表型和基因型耐药性。此外，我们报告了对从淡水样本中提取的总宏基因组DNA进行全宏基因组分析，以鉴定包括不可培养细菌在内的总体细菌多样性，以及水体中不同ARGs（耐药组）的总库。按照这些详细方案，我们观察到高抗生素耐药细菌载量在9.6×10 - 1.2×10 CFU/mL范围内。大多数分离株对多种测试抗生素耐药，包括头孢噻肟、氨苄西林、左氧氟沙星、氯霉素、头孢曲松、庆大霉素、新霉素、甲氧苄啶和环丙沙星，多重抗生素耐药（MAR）指数≥0.2，表明分离株耐药水平高。16S rRNA测序鉴定出潜在的人类病原体，如肺炎克雷伯菌，以及机会性细菌，如丛毛单胞菌属、微球菌属、节杆菌属和气单胞菌属。分离株的分子特征显示存在各种ARGs，如blaTEM、blaCTX - M（β - 内酰胺类）、aadA、aac(6') - Ib（氨基糖苷类）和dfr1（甲氧苄啶类），全宏基因组DNA分析也证实了这一点。在宏基因组DNA中还检测到编码抗生素外排泵的其他ARGs的高流行率——mtrA、macB、mdtA、acrD、β - 内酰胺酶——SMB - 1、VIM - 20、ccrA、ampC、blaZ、氯霉素乙酰转移酶基因catB10和利福平耐药基因rphB。借助本研究中讨论的方案，我们确认了具有不同AR表型和基因型特征的水传播MAR细菌的存在。因此，全宏基因组DNA分析可作为传统基于培养的技术的补充技术，以确定水体的总体AR状况。
