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

The spread of extended-spectrum ß-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 ß-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 ß-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.

Vibrio parahaemolyticus control in mussels by a Halobacteriovorax isolated from the Adriatic sea, Italy.

This study evaluated the application of a Halobacteriovorax isolated from water of the Adriatic Sea (Italy) in controlling V. parahaemolyticus in mussels (Mytilus galloprovincialis). Two 72 h laboratory-scale V. parahaemolyticus decontamination experiments of mussels were performed. The test microcosm of experiment 1 was prepared using predator/prey free mussels experimentally contaminated with Halobacteriovorax/V. parahaemolyticus at a ratio of 103 PFU/105 CFU per ml, while that of experiment 2 using mussels naturally harbouring Halobacteriovorax that were experimentally contaminated with 105 CFU per ml of V. parahaemolyticus. For experiment 1, was also tested a control microcosm only contaminated with 105 CFU per ml of V. parahaemolyticus.. Double layer agar plating and pour plate techniques were used to enumerate Halobacteriovorax and V. parahaemolyticus, respectively. 16 S rRNA analysis was used to identify Halobacteriovorax. For both experiments in the test microcosm the concentration of prey remained at the same level as that experimentally added, i.e. 5 log for the entire analysis period. In experiment 1, V. parahaemolyticus counts in mussels were significantly lower in the test microcosm than the control with the maximum difference of 2.2 log at 24 h. Results demonstrate that Halobacteriovorax can modulate V. parahaemolyticus level in the mussels. The public impact of V. parahaemolyticus in bivalves is relevant and current decontamination processes are not always effective. Halobacteriovorax is a suitable candidate in the development of a biological approach to the purification of V. parahaemolyticus in mussels.

Vibrio parahaemolyticus-specific Halobacteriovorax From Seawater of a Mussel Harvesting Area in the Adriatic Sea: Abundance, Diversity, Efficiency and Relationship With the Prey Natural Level.

This research aimed to study the abundance and molecular diversity of Vibrio parahaemolyticus-specific Halobacteriovorax strains isolated from seawater of the Adriatic Sea and the relationship between predator and prey abundances. Moreover, predator efficiency of the Halobacteriovorax isolates toward V. parahaemolyticus and Vibrio cholerae non-O1/O139 strains was tested. V. parahaemolyticus NCTC 10885 was used as primary host for the isolation of Halobacteriovorax from seawater by the plaque assay. Molecular identification was performed by PCR detection of a fragment of the 16S rRNA gene of the Halobacteriovoraceae family members. Moreover, 700 bp PCR products were sequenced and compared between them and to clones described for other sampling sites. Vibrio counts were performed on TCBS agar from 100 ml of filtered water samples and presumptive colonies were confirmed by standard methods. Predatory efficiency of Halobacteriovorax isolates was tested by monitoring abilities of 3-day enrichments to form clear lytic halos on a lawn of Vibrio preys, by the plaque assay. Out of 12 seawater samples monthly collected from June 2017 to May 2018, 10 were positive for V. parahaemolyticus specific Halobacteriovorax with counts ranging from 4 to 1.4 × 103 PFU per 7.5 ml. No significant relationship was found between Halobacteriovorax and Vibrio abundances. The 16SrRNA sequences of our Halobacteriovorax strains, one for each positive sample, were divided into three lineages. Within the lineages, some sequences had 100% similarity. Sequence similarity between lineages was always <94.5% suggesting that they may therefore well belong to three different species. All Halobacteriovorax isolates had the ability to prey all tested Vibrio strains. Additional research is necessary to assess whether stable strains of Halobacteriovorax are present in the Adriatic Sea and to understand the mechanisms by which Halobacteriovorax may modulate the abundance of V. parahaemolyticus and other vibrios in a complex marine ecosystem.