AUTHOR=Leoni Francesca , Sacchini Luca , Pieralisi Silvia , Angelico Gabriele , Magistrali Chiara Francesca , Cucco Lucilla , Massacci Francesca Romana , Albini Elisa , Duranti Anna , Cammà Cesare , Secondini Barbara , Rinaldi Antonio , Barchiesi Francesca 

TITLE=Occurrence and temporal distribution of extended-spectrum β-lactamase-producing Escherichia coli in clams from the Central Adriatic, Italy

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1219008

DOI=10.3389/fmicb.2023.1219008

ISSN=1664-302X

ABSTRACT=<p>The spread of extended-spectrum β-lactamase (ESBL)-producing <italic>Escherichia coli</italic> 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 <italic>E. coli</italic> in clams (<italic>n</italic> = 308) collected from harvesting areas of the Central Adriatic Sea between 2018 and 2019. ESBL- /class C β-lactamase (AmpC)- producing <italic>E. coli</italic> and <italic>Escherichia</italic> spp. were isolated by streaking over the surface of MacConkey agar plates supplemented with cefotaxime enriched broths of the initial shellfish suspension. <italic>E. coli</italic> and <italic>Escherichia</italic> 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 <italic>E. coli</italic> were isolated from 3% of the samples. Of 13 ESBL- and ESBL−/AmpC-producing <italic>Escherichia</italic> spp. (<italic>n</italic> = 11 <italic>E. coli</italic>, <italic>n</italic> = 1 <italic>E. marmotae</italic>, <italic>n</italic> = 1 <italic>E</italic>. <italic>ruysiae</italic>) 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 <italic>E. coli</italic> isolates were multidrug-resistant. WGS showed that the isolates predominantly carried the <italic>bla</italic><sub>CTX-M-15</sub> gene (3/11) and <italic>bla</italic><sub>CTX-M-14</sub> and <italic>bla</italic><sub>CTX-M-1</sub> (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 <italic>E. coli</italic> and <italic>E. coli</italic> levels, samples were divided into two groups, with <italic>E. coli</italic> at &gt;230 MPN/100 g and <italic>E. coli</italic> at ≤230 MPN/100 g. ESBL-producing <italic>E. coli</italic> isolates were significantly more commonly recovered in samples with higher <italic>E. coli</italic> levels (14%) than in those with lower levels of <italic>E. coli</italic> (2%). Moreover, the majority (3/4) of the potentially pathogenic strains were isolated in samples with higher <italic>E. coli</italic> levels. These findings provided evidence for the bacterial indicator of fecal contamination, <italic>E. coli</italic>, as an index organism for ESBL-producing <italic>E. coli</italic> isolates in bivalves.</p>