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

Abstract

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.

Technical specifications for a EU‐wide baseline survey of antimicrobial resistance in bacteria from aquaculture animals

“…All plates were incubated at 44 ± 0.5°C for 20 ± 2 h. Additionally, to detect carbapenem‐resistant E. coli , a loop‐full (10 μL) from the enrichment was transferred to CHROMID CARBA and CHROMID OXA‐48 and incubated at 37 ± 0.5°C for 20 ± 2 h BMD EUCAST ECOFF 261 (67) 391 Non‐selective screening: Full susceptibility, 92 Ampicillin, 5 Cephalosporins, 0 Quinolone, 0.8 Sulfamethoxazole, 3 Tetracycline, 6 ESBL‐selective screening: ESBL‐EC, 13 Quinolone selective screening: QR‐EC, 12 Leoni et al. ( 2023 ) Clam (Venus gallina) Flesh and intravalvular liquid 308 Italy 2018–2019 Bivalve molluscs were externally cleaned with running potable water; then, the flesh and liquor of the bivalve molluscs were aseptically collected, diluted, homogenised, and further diluted in a 0.1% sterile peptone water to achieve a final suspension of 1:10. Subsequent decimal dilutions were prepared in a 0.1% sterile peptone solution.…”