Gabriele Angelico

Background/Objectives: Due to the rising problem of antimicrobial resistance, there is increasing attention in the scientific community towards alternative approaches to combat Antimicrobial-Resistant (AMR) pathogens that do not involve the use of antibiotics. In this regard, the European Medicines Agency (EMA) and the European Food Safety Authority (EFSA) have promoted experimentation with predatory bacteria to fight antibiotic resistance. With the aim of identifying predatory bacteria suitable for the control of antibiotic-resistant bacteria, in this work we isolated a strain of Halobacteriovorax from an estuarine aquatic environment using a CTX-M-producing E. coli strain as prey and characterized it with respect to optimal physico-chemical parameters for growth and predation. Furthermore, we studied its predatory capacity against other E. coli strains and Multi-Drug-Resistant (MDR) Salmonella. Finally, we conducted challenge experiments to evaluate the growth of predator and prey over time. Methods: The Halobacteriovorax strain, designated HE7, was identified by 16S rRNA analysis. To isolate Halobacteriovorax and to evaluate its predatory ability towards different preys, the double-layer agar plating technique was applied. Results: HE7 showed in vitro predatory activity against all MDR strains of E. coli and Salmonella tested. In the 10⁷ predator/10³ prey and 10⁷ predator/10⁷ prey challenges, HE7 after 6 h achieved the total killing and a reduction of about 6 logs in the prey, respectively, maintaining this effect for up to 24 h. Conclusions: The results of this study highlight that HE7, but more generally Halobacteriovorax, could find application both alone and in an integrated context of antimicrobial strategies as an alternative to antibiotics.

It is known that the consumption of edible lamellibranch mollusks (ELMs), undercooked or raw, is a vehicle for the oral transmission of viral and bacterial pathogens. The ELMs most frequently fished/cultivated and consumed in Italy are Mytilus galloprovincialis and Chamelea gallina. The aim of this project was to standardize the cooking conditions of M. galloprovincialis and C. gallina, reproducible in a domestic environment, to obtain food in which the microbiological risk was eliminated but, at the same time, preserved the original organoleptic characteristics. The results showed that for C. gallina and M. galloprovincialis, cooking for 2 minutes after opening all shells, according to a method suitable for home application, is sufficient to eliminate the microbiological risk.

In the present study, we searched Halobacteriovorax strain preying upon Salmonella from the seawater of the Adriatic Sea. The Halobacteriovorax strain, named M7, was identified using 16S rRNA analysis. The M7 strain predation efficiency was tested against different Salmonella and non-Salmonella strains, all isolated from food matrices obtained from the Adriatic Sea. Finally, the M7 strain was exposed to Salmonella enterica subsp. enterica serovar Napoli in challenge tests to evaluate the killing of this specific prey over time. Double -layer agar plating technique was used to enumerate Halobacteriovorax and to evaluate its host specificity and predation efficiency. In the 103 predator/103 prey challenge test, M7 caused a decrease of Salmonella by about 2 log at 24 h compared to the control. In the 107 predator/104 prey challenge test, M7 caused a decrease of Salmonella by about 5 log at 24 h compared to the control, and good levels of decrease were obtained even at shorter times. Halobacteriovorax strains active against Salmonella are rarely present in the Adriatic Sea, Italy seawater. However, the isolate M7 showed high predatory efficiency towards a wide range of Salmonella strains. The presence of Salmonella in bivalves affects food safety since current decontamination processes are not always effective. M7 may represent a potential candidate for reducing and controlling Salmonella contamination in bivalves from harvesting to trade.

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 bla_CTX-M-15 gene (3/11) and bla_CTX-M-14 and bla_CTX-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.

We tested the predation of B. bacteriovorus against Escherichia coli in milk samples in three different experiments. In Experiment 1, the growth and predatory activity of B. bacteriovorus against E. coli in milk stored at 4°C were evaluated. In Experiment 2, the predatory activity of B. bacteriovorus against E. coli in the milk matrix was compared to the optimal one in the medium of choice. In Experiment 3, the influence of the native milk microbial community on the predation of B. bacteriovorus against E. coli experimentally added or indigenous grown at 4°C was tested. The predator increased at 4°C by about 1 Log in the first 48 hours and caused E. coli decrease by about 2 Log after 24 hours. The predator at 30°C reduced E. coli faster (3 Log after 6 hours) than at 4°C (2 Log after 24 hours). B. bacteriovorus at 30°C preyed on E. coli more in the nutrient broth than in the milk, with the most significant difference by about 4 Log after 48 hours. In raw milk contaminated only by the predator, it increased by about 1 Log after 48 hours at 4°C, suggesting that it preyed on indigenous microorganisms. B. bacteriovorus could find application in raw milk used as food or raw material during storage at 4°C to reduce the microbial load of spoilage and Shigatoxin-producing E. coli (STEC) strains of E. coli and therefore increase its shelf-life and healthiness.

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 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.

Summary: Bdellovibrio bacteriovorus is a predator micro‐organism towards other Gram‐negative bacteria. We tested B. bacteriovorus to control Escherichia coli growth on chicken slices and canned beef. Moreover, we analysed B. bacteriovorus's lytic ability on eight toxigenic or multidrug‐resistant E. coli strains. In chicken slices, the predator induced the highest prey reduction (4.3 log) respect to control at 6 h. In canned beef, the predator induced the highest prey reduction (2.1 log) respect to control at 6 h. Moreover, B. bacteriovorus showed lytic ability towards all tested E. coli strains. B. bacteriovorus could control E. coli and other pathogenic and spoilage bacteria in those meat‐based foods that have a shelf life B. bacteriovorus could also minimise the use of additives.