Data_Sheet_1_Occurrence and temporal distribution of extended-spectrum β-lactamase-producing Escherichia coli in clams from the Central Adriatic, Italy.zip

The spread of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a major public health issue. Bivalves are filter-feeder animals capable of bioaccumulating the microorganisms present in water. This physiological characteristic makes them both good indicators of environmental contamination and possible carriers of pathogenic bacteria, including those resistant to antimicrobials. The aim of this study was to investigate the occurrence of ESBL-producing E. coli in clams (n = 308) collected from harvesting areas of the Central Adriatic Sea between 2018 and 2019. ESBL- /class C β-lactamase (AmpC)- producing E. coli and Escherichia spp. were isolated by streaking over the surface of MacConkey agar plates supplemented with cefotaxime enriched broths of the initial shellfish suspension. E. coli and Escherichia spp. resistant to cefotaxime were screened for ESBL production by using the double disk synergy test. Susceptibility to different antimicrobials and confirmation of ESBL-production were determined by the minimum inhibitory concentration (MIC) test. Isolates were further characterized by whole genome sequencing (WGS) and bioinformatic analysis of genomes with different tools. Overall, ESBL-producing E. coli were isolated from 3% of the samples. Of 13 ESBL- and ESBL−/AmpC-producing Escherichia spp. (n = 11 E. coli, n = 1 E. marmotae, n = 1 E. ruysiae) isolates, 13 were resistant to ampicillin and cefotaxime, 9 to sulfamethoxazole, 6 to tetracycline and nalidixic acid, 4 to trimethoprim, and 3 to ceftazidime, cefoxitin, ciprofloxacin, and chloramphenicol. Moreover, the majority (8/11) of the ESBL-producing E. coli isolates were multidrug-resistant. WGS showed that the isolates predominantly carried the blaCTX-M-15 gene (3/11) and blaCTX-M-14 and blaCTX-M-1 (2/11 each). The AmpC β-lactamase CMY-2 was found in two isolates. Phylogroup A was the most prevalent (5/11), followed by phylogroups D (4/11), F (1/11), and B2 (1/11). Ten different sequence types (STs) were identified. Occurrence at sampling sites ranged between 0 and 27%. To identify associations between the occurrence of ESBL-producing E. coli and E. coli levels, samples were divided into two groups, with E. coli at >230 MPN/100 g and E. coli at ≤230 MPN/100 g. ESBL-producing E. coli isolates were significantly more commonly recovered in samples with higher E. coli levels (14%) than in those with lower levels of E. coli (2%). Moreover, the majority (3/4) of the potentially pathogenic strains were isolated in samples with higher E. coli levels. These findings provided evidence for the bacterial indicator of fecal contamination, E. coli, as an index organism for ESBL-producing E. coli isolates in bivalves.

产超广谱β-内酰胺酶（extended-spectrum β-lactamase, ESBL）大肠埃希菌（Escherichia coli）的传播是一项重大公共卫生问题。双壳类动物为滤食性生物，可生物富集水环境中的各类微生物，这一生理特性使其既能作为环境污染的优质指示生物，同时也可能成为包括抗菌药物耐药菌在内的病原菌的潜在携带者。
本研究旨在调查2018至2019年间，从中亚得里亚海捕捞区域采集的308份蛤蜊样品中产ESBL大肠埃希菌的检出情况。
通过将初始贝类悬浮液的增菌液划线接种于添加头孢噻肟的麦康凯琼脂（MacConkey agar）平板，成功分离得到产ESBL/β-内酰胺酶C类（AmpC）大肠埃希菌及埃希菌属（Escherichia spp.）菌株。采用纸片协同扩散试验（double disk synergy test）对头孢噻肟耐药的大肠埃希菌和埃希菌属菌株进行ESBL产生能力初筛；通过最低抑菌浓度（minimum inhibitory concentration, MIC）试验测定菌株对不同抗菌药物的敏感性，并确证ESBL的产生。随后借助全基因组测序（whole genome sequencing, WGS）及多类生物信息学分析工具对分离株进行深入分型鉴定。
研究整体结果显示，3%的样品中分离出产ESBL大肠埃希菌。在13株产ESBL及产ESBL/AmpC埃希菌属菌株中（含11株大肠埃希菌、1株土拨鼠埃希菌（E. marmotae）、1株鲁伊斯埃希菌（E. ruysiae）），所有13株均对氨苄西林与头孢噻肟耐药，9株对复方新诺明耐药，6株对四环素及萘啶酸耐药，4株对甲氧苄啶耐药，另有3株对头孢他啶、头孢西丁、环丙沙星及氯霉素耐药。此外，8/11的产ESBL大肠埃希菌分离株为多重耐药菌株。
全基因组测序结果表明，分离株主要携带blaCTX-M-15基因（3/11），另有blaCTX-M-14与blaCTX-M-1基因各携带于2/11的菌株中。2株分离株携带AmpC β-内酰胺酶CMY-2基因。系统发育群A最为常见（5/11），其次为D群（4/11）、F群（1/11）与B2群（1/11）。共鉴定出10种不同的序列型（sequence types, STs）。各采样点的检出率区间为0%~27%。
为探究产ESBL大肠埃希菌的检出情况与大肠埃希菌水平之间的关联，本研究将样品分为两组：大肠埃希菌含量>230最可能数（most probable number, MPN）/100g组，与大肠埃希菌含量≤230 MPN/100g组。结果显示，大肠埃希菌含量较高的样品中产ESBL大肠埃希菌的检出率（14%）显著高于含量较低的样品（2%）。此外，3/4的潜在致病性菌株均分离自大肠埃希菌含量较高的样品中。本研究证实，粪便污染指示菌大肠埃希菌可作为双壳类动物中产ESBL大肠埃希菌分离株的指示生物。