Comparative Analysis of E-coli MCR1A. Multicentre evaluation of the impact of COVID-19 pandemic on the spread of antimicrobial and biocide resistance from wastewater to the environment

The COVID-19 pandemic arose at the time of great concern about antimicrobial resistance (AMR), one of the actual major health challenges worldwide. Hygiene measures to prevent COVID-19 contamination, coupled to the use of antimicrobials for curative and preventive purposes could have an impact on the evolution of AMR. That is why the COV-AMR consortium (Cameroon, Madagascar and Brazil) has set as its goal in this multi-centre project to evaluate the impact of the COVID-19 pandemic on the spread of antimicrobials and biocides resistance in the environment. To achieve this, from January to June 2022, in each country, monthly water sampling was performed in healthcare facilities receiving COVID-19 patients and in one control site, to screen for multidrug resistant bacteria and investigate diversity and abundance of their antimicrobials/biocide’s resistance determinants, as well as their genetic relationship using whole genome sequencing. In addition, the antimicrobials and biocides residues in the samples loads in the samples were quantified using liquid chromatography coupled with mass spectrometry. The microbioma of the water samples was analysed using the Ion Torrent platform and QIIME2 tool to examine the composition of the bacterial communities. In order to help understand the link between COVID-19 epidemiology and the resistance determinants in the different study sites, the presence of SARV-CoV-2 was investigated by RT-qPCR. Overall, 119 multidrug resistant bacteria (43 E. coli, 51 K. pneumoniae, 01 P. aeruginosa, 09 S. aureus, and 15 Enterococcus spp.) were isolated in all 03 countries, mainly in hospitals sampling sites (with the exception of Cameroon), with a high resistance rate for β-lactams and fluoroquinolones. Regardless of the country, A very wide variety of resistance genes was registered in E. coli and K. pneumoniae isolates, with the most frequent resistance genes belonging to the following families of antimicrobials: 100% of strains for β-lactam (blaSHV and blaCTXM), 46,9% of strains for aminoglycosides (aph, and aac) and 38,3% of strains for quinolones (OqxA/B and qnr). The most common resistance genes in S.aureus and Enterococcus spp. were mecA (57,1% of strains) and VanHAX (77,7% of strains) respectively. Most of these resistance genes were plasmid mediated (mostly belonging to the incF group) or located on other mobile genetic element such as class I integrons. The SNP trees of the different species did not show specific clusters, although some dangerous clones like ST69 (E. coli), ST11 and ST258 (K. pneumoniae) were detected. A very small proportion of chlorhexidine-resistant bacteria was registered during this study among the 03 countries, though the qacE resistance gene was detected in about 50% isolates. Levofloxacin, azithromicin and ceftriaxome residues were detected in wastewater of all the countries, whereas and meropenem,gentamicina, and chlorhexidine residues were found only in Brazil, at concentrations showing a high-risk quotient of resistance selection for bacteria present in these waters. A high diversity of bacterial community was registered throughout the study, with a predominance of Proteobacteria phylum. In all 03 countries, detection of SARS-CoV-2 was not related to the number of infected people, and the presence of multidrug-resistant bacteria, and in Cameroon it was not depending on the type of sampling site. These data highlight the environmental and human health risk associated to the released of hospital sewage containing antimicrobial resistance determinants and antimicrobial residues in surface waters in all the participating countries. However, they need to be compare with data prior the pandemic to enable us to confirm the impact of the COVID-19 pandemic on the spread of antimicrobial and biocide resistance in the environment.

新冠疫情（COVID-19 pandemic）暴发之时，全球正面临抗菌药物耐药性（antimicrobial resistance, AMR）这一全球性重大公共卫生挑战。为预防新冠病毒污染而采取的卫生措施，加之抗菌药物用于治疗与预防的使用行为，可能会对抗菌药物耐药性的演化产生影响。为此，由喀麦隆、马达加斯加和巴西组成的COV-AMR联盟（COV-AMR consortium）在这项多中心研究项目中设定核心目标：评估新冠疫情对环境中抗菌药物与生物杀灭剂耐药性传播的影响。为达成该研究目标，2022年1月至6月期间，各参与国均在收治新冠患者的医疗机构及1个对照点位开展月度水体采样工作，以筛查多重耐药菌，并通过全基因组测序（whole genome sequencing）探究其抗菌药物/生物杀灭剂耐药决定簇的多样性、丰度及其遗传亲缘关系。此外，采用液相色谱-质谱联用法（liquid chromatography coupled with mass spectrometry）对样本中的抗菌药物与生物杀灭剂残留量进行定量分析。利用Ion Torrent测序平台及QIIME2工具对水样的微生物组进行分析，以考察细菌群落组成。为助力阐明新冠流行病学与不同研究点位耐药决定簇之间的关联，通过实时荧光定量PCR（RT-qPCR）检测了新型冠状病毒（SARS-CoV-2）的存在情况。整体而言，3个参与国共分离得到119株多重耐药菌，其中包括43株大肠埃希菌（E. coli）、51株肺炎克雷伯菌（K. pneumoniae）、1株铜绿假单胞菌（P. aeruginosa）、9株金黄色葡萄球菌（S. aureus）以及15株肠球菌属（Enterococcus spp.）；除喀麦隆外，菌株主要分离自医院采样点位，且对β-内酰胺类（β-lactams）与氟喹诺酮类（fluoroquinolones）药物呈现高耐药率。无论研究国为何，大肠埃希菌与肺炎克雷伯菌分离株中均检测到极为多样的耐药基因，其中最常见的耐药基因隶属于以下几类抗菌药物：β-内酰胺类相关基因blaSHV与blaCTXM在所有菌株中均有检出，氨基糖苷类相关基因aph与aac在46.9%的菌株中检出，喹诺酮类相关基因OqxA/B与qnr在38.3%的菌株中检出。金黄色葡萄球菌与肠球菌属中最常见的耐药基因分别为mecA（占菌株总数的57.1%）与VanHAX（占菌株总数的77.7%）。此类耐药基因大多由质粒介导（多隶属于IncF型质粒组），或定位在Ⅰ类整合子（class I integrons）等其他可移动遗传元件上。不同菌种的单核苷酸多态性系统发育树（SNP trees）未呈现特异性聚类，但检测到ST69（大肠埃希菌）、ST11与ST258（肺炎克雷伯菌）等危险克隆株。本次研究在3个国家中仅检出极少量耐氯己定的细菌，但约50%的分离株中检测到qacE耐药基因。左氧氟沙星、阿奇霉素与头孢曲松残留可在所有参与国的废水中检出，而美罗培南、庆大霉素与氯己定残留仅在巴西检出，其浓度对水体中存在的细菌构成了较高的耐药选择风险商值。本次研究中检测到的细菌群落多样性较高，变形菌门（Proteobacteria）为优势菌门。在3个参与国中，新型冠状病毒的检出情况与感染人数、多重耐药菌的存在均无关联；在喀麦隆，该检出情况也与采样点位类型无关。上述数据凸显了所有参与国的医院污水排放携带抗菌药物耐药决定簇与抗菌药物残留进入地表水所带来的环境与人类健康风险。然而，需将本研究数据与疫情暴发前的相关数据进行对比，才能确认新冠疫情对环境中抗菌药物与生物杀灭剂耐药性传播的影响。