Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment

none13sìListeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.openGiuditta Fiorella Schiavano * , Collins Njie Ateba , Annalisa Petruzzelli , Veronica Mele , Giulia Amagliani , Fabrizia Guidi , Mauro De Santi , Francesco Pomilio , Giuliana Blasi , Antonietta Gattuso , Stefania Di Lullo , Elena Rocchegiani, Giorgio BrandiSchiavano, GIUDITTA FIORELLA; Njie Ateba, Collins; Petruzzelli, Annalisa; Mele, Veronica; Amagliani, Giulia; Guidi, Fabrizia; DE SANTI, Mauro; Pomilio, Francesco; Blasi, Giuliana; Gattuso, Antonietta; Di Lullo, Stefania; Rocchegiani, Elena; Brandi, Giorgi

Bioaccumulation Experiments in Mussels Contaminated with the Food-Borne Pathogen Arcobacter butzleri: Preliminary Data for Risk Assessment

The aim of this study was to evaluate, at a laboratory scale, the ability of this microorganism to grow in seawater and bioaccumulate in mussels (Mytilus galloprovincialis) maintained in constantly aerated tanks, containing twenty litres of artificial seawater. Three concentrations of A. butzleri LMG 10828T were tested (about 5×106 CFU/mL, 5×104 CFU/mL, and 5×102 CFU/mL). Following contamination, enumeration of A. butzleri was performed from water and mussels each day, for up to 96 h. Three contamination experiments with artificial seawater in absence of mussels were also performed in the same manner. In the experiments with mussels, A. butzleri declined in water of approximately 1 log every 24 h from the contamination. In artificial seawater without mussels the concentration of A. butzleri remained on the same logarithmic level in the first 48 h and then decreased of about 1 log every 24 hours. In mussels, the concentration was approximately 2 log lower than the exposition level after 24 h from the contamination, and then it decreased exponentially of 1 log every 24 h. Our findings suggest that in the experimental conditions tested A. butzleri is neither able to effectively grow in seawater nor bioaccumulate in mussels, at least in the free and cultivable form

Halobacteriovorax isolated from the Adriatic Sea to challenge Salmonella

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 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

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

The Emerging Nosocomial Pathogen Klebsiella michiganensis: Genetic Analysis of a KPC-3 Producing Strain Isolated from Venus Clam

The recovery and characterization of a multidrug-resistant, KPC-3-producing Klebsiella michiganensis that was obtained from Venus clam samples is reported in this study. A whole-genome sequencing (WGS) analysis using Illumina and Nanopore technologies of the K. michiganensis 23999A2 isolate revealed that the strain belonged to the new sequence type 382 (ST382) and carried seven plasmid replicon sequences, including four IncF type plasmids (FII, FIIY, FIIk, and FIB), one IncHI1 plasmid, and two Col plasmids. The FIB and FIIk plasmids showed high homology to each other and to multireplicon pKpQIL-like plasmids that are found in epidemic KPC-K. pneumoniae clones worldwide. The strain carried multiple β-lactamase genes on the IncF plasmids: blaOXA-9 and blaTEM-1A on FIB, blaKPC-3 inserted in a Tn4401a on FIIK, and blaSHV-12 on FIIY. The IncHI1-ST11 harbored no resistance gene. The curing of the strain caused the loss of all of the bla genes and a rearrangement of the IncF plasmids. Conjugal transfer of the blaOXA-9, blaTEM-1A and blaKPC-3 genes occurred at a frequency of 5 × 10-7, using K. quasipneumoniae as a recipient, and all of the bla genes were transferred through a pKpQIL that originated from the recombination of the FIB and FIIk plasmids of the donor. A comparison with 31 K. michiganensis genomes that are available in the NCBI database showed that the closest phylogenetic relatives of K. michiganensis 23999A2 are an environmental isolate from soil in South Korea and a clinical isolate from human sputum in Japan. Finally, a pan-genome analysis showed a large accessory genome of the strain as well as the great genomic plasticity of the K. michiganensis species. IMPORTANCE Klebsiella michiganensis is an emerging nosocomial pathogen, and, so far, few studies describe isolates of clinical origin in the environment. This study contributes to the understanding of how the dissemination of carbapenem-resistance outside the hospital setting may be related to the circulation of pKpQIL-like plasmids that are derived from epidemic Klebsiella pneumoniae strains. The recovery of a carbapenem-resistant isolate in clams is of great concern, as bivalves could represent vehicles of transmission of pathogens and resistance genes to humans via the food chain. The study demonstrates the plasticity of K. michiganensis genome, which is probably useful to multiple environment adaptation and to the evolution of the species

An Extensive investigation into the prevalence and the genetic and serological diversity of toxigenic <i>Vibrio parahaemolyticus</i> in Italian marine coastal waters

The relationship between Vibrio parahaemolyticus strains isolated from the aquatic environment and those isolated from cases of infection in humans is poorly understood due to the low prevalence of tdh- and/or trh-positive strains in the environment. To address this concern, it would be useful to analyse the genetic relationships among environmental and food strains and with reference to clinical isolates, also applying molecular typing methods. The aim of this study was to evaluate the prevalence of toxigenic V. parahaemolyticus in Italian coastal waters and seafood, to examine intra-species variability and to identify, using serotyping and pulsed-field gel electrophoresis (PFGE), relationships among strains from different sources, geographical origin and period of isolation. Of the 192 V. parahaemolyticus strains isolated in different Italian areas and examined in this study, 25 (13.0%) proved to carry the trh gene while none of the strains proved positive to the search by PCR for tdh and Group-Specific-toxRS genes. The prevalence of toxigenic strains in the Tyrrhenian Sea was significantly lower than that calculated for the Ligurian coasts. Regarding the sources of isolation, the higher prevalence of trh-positive V. parahaemolyticus was revealed in fish, followed by clams, plankton, oysters, mussels and lastly seawater. Within the toxigenic strains, 16 serotypes and 20 distinct PFGE patterns were identified. Two clusters, which included a total of 8 V. parahaemolyticus strains, were specifically associated with the North Adriatic Sea area and were stable over time. Our results demonstrate that trh-positive V. parahaemolyticus strains circulated in Italy in the period 2002–2009 with a prevalence higher than that reported from other European and extra-European countries, confirming that toxigenic V. parahaemolyticus is an emerging public health concern in Italy, regardless of its pandemic potential

Bdellovibrio bacteriovorus to control Escherichia coli on meat matrices

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 &lt;10 days. It could integrate modified atmosphere packaging (MAP) to prolong the shelf life and improve the safety of prepacked fresh meat, meat preparations and meat products. In future applications on meat-based foods, B. bacteriovorus could also minimise the use of additives

A Strong Evidence Outbreak of <em>Salmonella</em> Enteritidis in Central Italy Linked to the Consumption of Contaminated Raw Sheep Milk Cheese

Salmonellosis is the second most commonly reported gastrointestinal infection in humans after campylobacteriosis, and an important cause of foodborne outbreaks in the EU/EEA. The vast majority (72.4%) of the salmonellosis foodborne outbreaks reported in EU in 2019 were caused by Salmonella Enteritidis, even if their total number due to this serovar decreased. In spring 2020, a foodborne outbreak of S. Enteritidis occurred in the Marche region (Central Italy), involving 85 people. The common exposure source was a cheese, pecorino “primo sale”, produced with raw sheep milk. The cheese batches were produced by two local dairies, with a livestock production facility, also including a sheep farm, being part of one dairy. Bacteriological analysis of samples collected allowed the detection of S. Enteritidis in animal faeces, environmental samples, raw-milk bulk tanks and milk taken from single animals. These data confirm that, despite the scarce scientific evidence, S. Enteritidis can infect sheep and be shed into the animals’ milk. Hence, this is a real risk for public health when unpasteurized milk is used in production of such cheese. The present paper describes the results of the investigations conducted to clarify this outbreak