Caltagirone Mariasofia, Nucleo Elisabetta, Spalla Melissa, Zara Francesca, Novazzi Federica, Marchetti Vittoria M, Piazza Aurora, Bitar Ibrahim, De Cicco Marica, Paolucci Stefania, Pilla Giorgio, Migliavacca Roberta, Pagani Laura
Unit of Microbiology and Clinical Microbiology, Department of Clinical-Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Pavia, Italy.
Department of Biomedical and Clinical Sciences, Romeo and Enrica Invernizzi Pediatric Research Center, University of Milan, Milan, Italy.
Front Microbiol. 2017 Nov 10;8:2232. doi: 10.3389/fmicb.2017.02232. eCollection 2017.
To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepò Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. were also characterized for the Extended-Spectrum-β-Lactamases (ESBLs), carbapenemases, and genes presence. From December 2014 to November 2015, 246 water samples were filtered, plated on Plate Count Agar, MacConkey Agar, and MacConkey Agar with cefotaxime. Isolates were species identified using AutoSCAN-4-System and ESBLs, carbapenemases, and colistin resistance determinants were characterized by PCR, sequencing, and microarray. Plasmid conjugative transfer experiments, PCR-based Replicon typing, Pulsed-Field Gel Electrophoresis, Multi-Locus-Sequence-Typing, and plasmid characterization were performed. A total of 132 enterobacteria isolates grew on MacConkey agar with cefotaxime: 82 (62.1%) were obtained from streams, 41 (31.1%) from treatment plants, and 9 (6.8%) from wells. Thirty out of 132 (22.7%) isolates, mainly belonging to ( = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of -type was identified in 19/30 isolates. In further two strains, a gene co-existed with a -type ESBL determinant. A gene was detected in two isolates of ( = 1) and ( = 1), while any ESBL determinant was ascertained in seven strains. A -type gene was detected in a cefoxitin resistant from a stream. Interestingly, two strains of ST307 and ST258, collected from a well and a wastewater treatment plant, resulted KPC-2, and KPC-3 producers, respectively. Moreover, we report the first detection of ST10 on a conjugative IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò Pavese (Northern Italy). Both ESBLs and ESBLs/carbapenemase-producing strains showed clonal heterogeneity by Pulsed-Field Gel Electrophoresis and Multi-Locus-Sequence-Typing. During one-year study and taking in account the whole Gram-negative bacterial population, an average percentage of cefotaxime resistance of 69, 32, and 10.3% has been obtained for the wastewater treatment plants, streams, and wells, respectively. These results, of concern for public health, highlight the need to improve hygienic measures to reduce the load of discharged bacteria with emerging resistance mechanisms.
为评估水体中抗生素耐药性污染率，对位于奥尔特雷波帕韦斯地区的11口水井、5条溪流和4个污水处理厂进行了筛查，以检测对第三代头孢菌素耐药的革兰氏阴性菌的存在情况。还对超广谱β-内酰胺酶（ESBLs）、碳青霉烯酶和相关基因的存在情况进行了鉴定。2014年12月至2015年11月，共过滤了246份水样，分别接种在平板计数琼脂、麦康凯琼脂以及含头孢噻肟的麦康凯琼脂上。使用AutoSCAN-4系统对分离菌株进行物种鉴定，并通过PCR、测序和微阵列技术对ESBLs、碳青霉烯酶和黏菌素耐药决定簇进行鉴定。进行了质粒接合转移实验、基于PCR的复制子分型、脉冲场凝胶电泳、多位点序列分型以及质粒特征分析。共有132株肠杆菌在含头孢噻肟的麦康凯琼脂上生长：其中82株（62.1%）来自溪流，41株（31.1%）来自污水处理厂，9株（6.8%）来自水井。132株分离菌株中有30株（22.7%），主要属于15个菌种，对哌拉西林-他唑巴坦表现出协同效应。在19/30株分离菌株中鉴定出单个型ESBL基因。在另外2株菌株中，一个基因与型ESBL决定簇共存。在1株肺炎克雷伯菌和1株阴沟肠杆菌的分离菌株中检测到基因，而在7株大肠杆菌菌株中未确定任何ESBL决定簇。在一株来自溪流的对头孢西丁耐药的大肠杆菌中检测到型基因。有趣的是，从一口水井和一个污水处理厂采集的2株ST307和ST258大肠杆菌菌株，分别产生KPC-2和KPC-3。此外，我们首次在来自意大利北部奥尔特雷波帕韦斯一条溪流的接合型IncX4质粒（大小为33303 bp）上检测到ST10大肠杆菌。通过脉冲场凝胶电泳和多位点序列分型，ESBLs产生菌株和ESBLs/碳青霉烯酶产生菌株均显示出克隆异质性。在为期一年的研究中，考虑到整个革兰氏阴性菌群体，污水处理厂、溪流和水井中头孢噻肟耐药的平均百分比分别为69%、32%和10.3%。这些结果关乎公众健康，凸显了改进卫生措施以减少携带新出现耐药机制的排放细菌负荷的必要性。
