Listeria monocytogenes (Lm) can persist in food processing environments (FPEs), surviving environmental stresses and disinfectants. We described an intensive environmental monitoring plan performed in Central Italy and involving food producing plants (FPPs) and retail grocery stores (RSs). The aim of the study was to provide a snapshot of the Lm circulation in different FPEs during a severe listeriosis outbreak, using whole genome sequencing (WGS) to investigate the genetic diversity of the Lm isolated, evaluating their virulence and stress resistance profiles. A total of 1217 samples were collected in 86 FPEs with 12.0% of positive surfaces at FPPs level and 7.5% at RSs level; 133 Lm isolates were typed by multilocus sequencing typing (MLST) and core genome MLST (cgMLST). Clonal complex (CC) 121 (25.6%), CC9 (22.6%), CC1 (11.3%), CC3 (10.5%), CC191 (4.5%), CC7 (4.5%) and CC31 (3.8%) were the most frequent MLST clones. Among the 26 cgMLST clusters obtained, 5 of them persisted after sanitization and were re-isolated during the follow-up sampling. All the CC121 harboured the Tn6188_qac gene for tolerance to benzalkonium chloride and the stress survival islet SSI-2. The CC3, CC7, CC9, CC31 and CC191 carried the SSI-1. All the CC9 and CC121 strains presented a premature stop codon in the inlA gene. In addition to the Lm Pathogenicity Island 1 (LIPI-1), CC1, CC3 and CC191 harboured the LIPI-3. The application of intensive environmental sampling plans for the detection and WGS analysis of Lm isolates could improve surveillance and early detection of outbreaks.

Introduction. In May–June 2018, an outbreak of campylobacteriosis involved students and school staff from kindergartens and primary schools in Pescara, southern Italy. Aim. We present details of the epidemiological and microbiological investigation, and the findings of the analytical study, as well as the implemented control measures. Methodology. To identify possible risk factors associated with the observed outbreak, a case control study was conducted using a questionnaire to collect information on the date of symptoms onset, type and duration of symptoms, type of healthcare contact, school attendance, and food items consumed at school lunches during the presumed days of exposure. Attack rates were calculated for each date and school. Logistic regression models were used to estimate the odds ratios of being a case and the odds of illness by food items consumed, respectively. Moreover, we carried out a comparative genomic analysis using whole genome multilocus sequence typing (wgMLST) of Campylobacter jejuni strains isolated during the outbreak investigation to identify the source of the outbreak. Results. Overall, 222 probable cases from 21 schools were identified, and C. jejuni was successfully isolated from 60 patients. The meals in the schools involved were provided by two cooking centres managed by a joint venture between two food companies. Environmental and food sampling, epidemiological and microbiological analyses, as well as a case control study with 176 cases and 62 controls from the same schools were performed to identify the source of the outbreak. The highest attack rate was recorded among those having lunch at school on 29 May (7.8 %), and the most likely exposure was ‘caciotta’ cheese (odds ratio 2.40, 95 % confidence interval 1.10–5.26, P=0.028). C. jejuni was isolated from the cheese, and wgMLST showed that the human and cheese isolates belonged to the same genomic cluster, confirming that the cheese was the vehicle of the infection. Conclusion. It is plausible that a failure of the pasteurization process contributed to the contamination of the cheese batches. Timely suspension of the catering service and summer closure of the schools prevented further spread.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes the foodborne illness, listeriosis. Clonal complexes (CC), such as CC121, are overrepresented in the food production industry, and are rarely reported in animals and the environment. Working within a European-wide project, we investigated the routes by which strains are transmitted from environments and animals to food and the food production environment (FPE). In this context, we report, for the first time, the occurrence of a ST121 (CC121) strain isolated from a dolphin brain. The genome was compared with the genomes of 376 CC121 strains. Genomic comparisons showed that 16 strains isolated from food were the closest to the dolphin strain. Like most of the food strains analyzed here, the dolphin strain included genomic features (transposon Tn6188, plasmid pLM6179), both described as being associated with the strain’s adaptation to the FPE. Like all 376 strains, the dolphin strain contained a truncated actA gene and inlA gene, both described as being associated with attenuated virulence. Despite this fact, the strain was able to cross blood-brain barrier in immunosuppressed dolphin exposed polychlorinated biphenyl and invaded by parasites. Our data suggest that the dolphin was infected by a food-related strain released into the Mediterranean Sea.

Listeria monocytogenes (Lm) is a foodborne pathogen causing listeriosis. Invasive forms of the disease mainly manifest as septicaemia, meningitis and maternal-neonatal infections. Lm-associated respiratory infections are very rare and little known. We reported two Lm respiratory infection cases occurred in Central Italy during the summer of 2020, in the midst of the SARS-CoV2 pandemic. In addition to collect the epidemiological and clinical characteristics of the patients, we used Whole Genome Sequencing to study the genomes of the Lm isolates investigating their virulence and antimicrobial profiles and the presence of genetic mobile elements. Both the strains belonged to hypervirulent MLST clonal complexes (CC). In addition to the Listeria Pathogenicity Island 1 (LIPI-1), the CC1 strain also carried LIPI-3 and the CC4 both LIPI-3 and LIPI-4. Genetic determinants for antimicrobial and disinfectants resistance were found. The CC1 genome presented prophage sequences but they did not interrupt the comK gene, involved in the phagosomal escape of Lm. None of the strains carried plasmids. Lm is an important, although rare, opportunistic pathogen for respiratory tract and lung infections. To avoid dangerous diagnostic delays of these severe clinical forms, it is important to sensitize hospital laboratories to this rare manifestation of listeriosis considering Lm in the differential diagnosis of respiratory infections.

Despite Klebsiella pneumoniae being widely recognized as a nosocomial pathogen, there is a critical lack in defining its reservoirs and sources of infections. Most studies on risk factors have focused on multidrug-resistant (MDR) isolates and clinically-oriented questions. Over a two-year period, we sampled 131 wild animals including mammal and bird species from three regions of Central Italy. All typical colonies isolated from the analytical portions were confirmed by real-time PCR and identified by MALDI-TOF mass spectrometry (MALDI-TOF MS). All confirmed K. pneumoniae isolates were tested for antimicrobial susceptibility to 29 antimicrobials and subjected to whole genome sequencing. Typical colonies were detected in 17 samples (13%), which were identified as K. pneumoniae (n = 16) and as K. quasipneumoniae (n = 1) by MALDI-TOF MS. The antimicrobial susceptibility profile showed that all the isolates were resistant to β-lactams (ceftobiprole, cloxacillin, cefazolin) and tetracycline; resistance to ertapenem and trimethoprim was observed and nine out of 16 K. pneumoniae isolates (56.2%) were classified as MDR. Genomic characterization allowed the detection of fluoroquinolone resistance-associated efflux pumps, fosfomycin and β-lactamase resistance genes, and virulence genes in the overall dataset. The cluster analysis of two isolates detected from wild boar with available clinical genomes showed the closest similarity. This study highlights the link between humans, domestic animals, and wildlife, showing that the current knowledge on this ecological context is lacking and that the potential health risks are underestimated.