Caratterizzazione molecolare di ceppi di Listeria monocytogenes isolati da matrici alimentari e da fonti umane

Scopo del lavoro è stato la caratterizzazione molecolare di ceppi di L. monocytogenes (L.m.) isolati da alimenti e dall’uomo nel triennio 2019-2021. Un totale di 67 ceppi di L.m. sono stati isolati (norma UNI EN ISO 11290-1:2017) presso i laboratori di Microbiologia degli alimenti dell’IZS Palermo da diverse matrici alimentari con prevalenza di alimenti pronti quali prodotti lattiero-caseari, a base di carne, ittici, vegetali, prelevati nell’ambito dei campionamenti dei Servizi Veterinari delle ASP. Gli isolati batterici sono stati conservati a –20 °C in Microbank ed in seguito inviati al LNR di Teramo per la determinazione del sierogruppo (PCR-Multiplex) e successiva indagine molecolare (MLST e Whole Genome Sequencing -WGS). Il sequenziamento è stato effettuato su piattaforma NextSeq 500 Illumina e l’analisi dei dati eseguita su una piattaforma bioinformatica e di raccolta dati del LNR L.m., denominata GenPat. Presso i laboratori ospedalieri di Palermo (Ospedale Civico, Ospedali Riuniti Villa Sofia-Cervello e Azienda Ospedaliera Universitaria Policlinico, quest’ultimo anche Laboratorio di Riferimento Regionale per la listeriosi) sono stati isolati n. 39 ceppi clinici di L.m. I ceppi, insieme alle schede per la raccolta dei dati epidemiologici, sono stati inviati all’Istituto Superiore di Sanità (ISS-Operational Contact Point microbiologico per L.m.) per la caratterizzazione molecolare mediante WGS. Il sequenziamento è stato effettuato su una piattaforma IonTorrent S5 e l’analisi è stata eseguita automaticamente su una piattaforma bioinformatica e di raccolta dati, denominata IRIDA-ARIES. I risultati sulla caratterizzazione molecolare dei ceppi isolati dagli alimenti hanno mostrato una prevalenza del sierogruppo IVb (70%), seguito dal IIa (24%), dal IIb (4.5%) e dal IIc (1.5%). Il 46.3% dei ceppi, tutti appartenenti al sierogruppo IVb, sono stati isolati da mozzarella e formaggio a pasta filata, prelevati presso una stessa azienda produttrice, durante controlli ufficiali, nel periodo agosto-settembre 2020, a seguito di positività rilevata in autocontrollo. I risultati del sequenziamento effettuato dal LNR hanno evidenziato l’esistenza di cluster di ceppi appartenenti al Clonal Complex (CC) 2, CC199 e CC1. In particolare, tutti i ceppi isolati nello stesso caseificio, sia nel periodo considerato che in un successivo campionamento a gennaio 2022, appartenevano al CC2 e Sequence Type 2 (ST2). Riguardo i ceppi clinici, l’analisi filogenetica dei genomi dei 39 isolati di L.m. ha consentito l’identificazione di un Cluster_90 composto da 25 ceppi dei quali 6 isolati nel 2019, 16 nel 2020 e 1 nel 2021. I 25 ceppi sono risultati appartenere al sierogruppo IVb, ST2 e CC2 presentando tra loro una differenza allelica al core genome MLST compresa tra 0 e 12. Da sottolineare la presenza di 8 ceppi, isolati tra ottobre 2019 e agosto 2021, associati a casi materno neonatali. Le sequenze genomiche dei 39 ceppi clinici di L.m. sono state confrontate con le sequenze depositate nel database IRIDA-ARIES. Dall’analisi è emerso che al Cluster_90, ST2 appartenevano anche 3 ceppi isolati in Lombardia (2 nel 2019, 1 nel 2020), 2 in Piemonte, 1 nel Lazio, 1 in Toscana nel 2020 e 2 ceppi isolati in Sicilia (1 nel 2019 da Siracusa e 1 nel 2020 dalla provincia di Palermo). Il presente studio ha permesso di avviare la costruzione di una rete integrata medico/veterinaria tra laboratori per potenziare il sistema di sorveglianza della listeriosi nella regione Sicilia

Hypo- and Hyper-Virulent Listeria monocytogenes Clones Persisting in Two Different Food Processing Plants of Central Italy

A total of 66 Listeria monocytogenes (Lm) isolated from 2013 to 2018 in a small-scale meat processing plant and a dairy facility of Central Italy were studied. Whole Genome Sequencing and bioinformatics analysis were used to assess the genetic relationships between the strains and investigate persistence and virulence abilities. The biofilm forming-ability was assessed in vitro. Cluster analysis grouped the Lm from the meat plant into three main clusters: two of them, both belonging to CC9, persisted for years in the plant and one (CC121) was isolated in the last year of sampling. In the dairy facility, all the strains grouped in a CC2 four-year persistent cluster. All the studied strains carried multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA). CC121 also harbored the Tn6188 specific for tolerance to Benzalkonium Chloride. Only CC9 and CC121 carried a Stress Survival Islet and presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids. They showed a greater biofilm production when compared with CC2. All the CC2 carried a full-length inlA while CC9 and CC121 presented a Premature Stop Codon mutation correlated with less virulence. The hypo-virulent clones CC9 and CC121 appeared the most adapted to food-processing environments; however, even the hyper-virulent clone CC2 warningly persisted for a long time. The identification of the main mechanisms promoting Lm persistence in a specific food processing plant is important to provide recommendations to Food Business Operators (FBOs) in order to remove or reduce resident Lm

Detection of Vibrio splendidus and related species in Chamelea gallina sampled in the Adriatic along the Abruzzi coastline

Vibrio species are an important and widespread component of marine microbial communities. Some Vibrio strains are potentially pathogenic to marine vertebrates and invertebrates. The aim of this study was to identify vibrios, in particular Vibrio splendidus and related species, isolated from clams (Chamelea gallina) collected along the coasts of the Abruzzi region from May to October 2007. The isolates obtained were phenotyped and classified as belonging to the genus Vibrio. The strains underwent biochemical testing in accordance with Alsina’s scheme for V. splendidus identification. Molecular analysis of the 16S-23S intergenic space region and recA gene was used to identify V. splendidus and related species. All the samples examined were found to contain halophylic Vibrio species, with V. alginolyticus, V. splendidus-related species and V. mediterranei most commonly found. A polymerase chain reaction of the 16S-23S intergenic space region and sequencing of the recA gene from isolates confirmed that phenotyping of Vibrio species is not sufficient to distinguish between different species. Differentiation of the highly related species among V. splendidus-related clusters remains an important issue. In this regard, our data suggests sequencing the recA genes was far more discriminatory than sequencing 16S rDNA for this purpose

Characterisation of Listeria monocytogenes strains isolated from soft and semi-soft cheeses sampled in a region of Italy

A total of 47 Listeria monocytogenes strains isolated in a survey of cheeses sampled from retail outlets were characterised. Five cheeses (Gorgonzola, Taleggio, Asiago, Crescenza and Brie) were chosen from the most popular soft and semi-soft cheeses consumed in Italy and most commonly contaminated with L. monocytogenes. The serotype and antibiotic resistance pattern were determined for each strain and their macrorestriction profile was analysed with pulsed-field gel electrophoresis (PFGE). The main serotypes detected were 1/2a (76.6%) and 1/2c (21.3%). Serotype 1/2b was found in only one sample. A total of 97.9% of strains were resistant to oxacillin (OX), 80.9% to lincomycin (L) and 78.7% to clindamycin (CC). Of these strains, 17% were found to be resistant to two antibiotics (OX-CC or OX-L) while 70.2% were resistant to three antibiotics (OX-CC-L). No strains were susceptible to all the compounds tested. A combined analysis of the macrorestriction profiles AscI and ApaI identified eleven pulsotypes divided into three clusters. Two pulsotypes predominated, accounting for 57.4% and 21.3% of the isolated strains. Analysis of the PFGE profiles did not reveal any correlation between pulsotype and type of cheese, producer or retail outlet. A temporal analysis revealed that one pulsotype was persistent throughout the study period, with the exception of August and September, in which time a different pulsotype was detected. This variability suggests the influence of factors affecting the dynamics of the contamination of these products. Large-scale studies could help clarify this phenomenon

Caratterizzazione di ceppi Listeria monocytogenes isolati da formaggi a pasta molle e semi-molle prelevati nella regione Abruzzo

Sono stati caratterizzati 47 ceppi di Listeria monocytogenes isolati in un’indagine condotta su formaggi prelevati al dettaglio. Cinque formaggi (gorgonzola, taleggio, asiago, crescenza e brie) sono stati selezionati tra quelli a pasta semi molle e molle di maggior consumo su scala nazionale e più frequentemente contaminati da Listeria monocytogenes. Per ogni ceppo è stato determinato il sierotipo, il pattern di resistenza agli antibiotici e il profilo di macrorestrizione con la Pulsed Field Gel Electrophoresis (PFGE). I principali sierotipi rilevati sono risultati l’1/2a (76,6%) e l’1/2c (21,3%) mentre il sierotipo 1/2b è stato rilevato solo in 1 campione. Il 97,9% dei ceppi è risultato resistente all’oxicillina, l’80,9% alla lincomicina e il 78,7% alla clindamicina. La resistenza a 2 antibiotici è stata rilevata nel 17% dei ceppi con due diversi pattern (OXCC, OXL) mentre a 3 nel 70,2% con un unico profilo (OXCCL). Non sono stati rilevati ceppi sensibili a tutte le molecole testate. Con l’analisi combinata dei profili di macrorestrizione di AscI e ApaI, sono stati identificati 11 pulsotipi suddivisi in 3 cluster. Due pulsotipi sono risultati prevalenti raggruppando rispettivamente il 57,4% e il 21,3% dei ceppi isolati. L’analisi dei profili PFGE non ha rilevato relazioni tra pulsotipo e tipo di formaggio, ditta produttrice o punto vendita. La valutazione della distribuzione temporale dei pulsotipi ha messo in evidenza la presenza di un profilo persistente durante tutto il periodo di studio, ad eccezione dei mesi di agosto e settembre in cui è stato rilevato un pulsotipo diverso. Pertanto l’indagine ha permesso di rilevare la variabilità nel tempo della prevalenza di alcuni specifici pulsotipi che ha fatto ipotizzare l’intervento di fattori che hanno influenzato le dinamiche di contaminazione di questi prodotti. Studi su larga scala potrebbero contribuire al chiarimento di tale fenomeno

Línguas em conflito: modelos de acesso lexical a partir do input ortográfico em bilíngues e multilíngues e o efeito do multilinguismo sobre as funções executivas

The phenomenon of bilingualism is the essential premise for translation. This paper presents contemporary cognitive models for written word recognition, based on previous experimental work with interlingual homographs. We will discuss studies on language conflict between two or more languages, and how these findings provide support for the language non-selective hypothesis for lexical access, which elucidates the impact of bilingualism in the processes of reading and translation. As a secondary goal and with a neuroscientific perspective, we will also describe the changes in the brain and mental processes that underlie the experience of bilingualism and multilingualism. Studies discussed here demonstrate an advantage in executive function (updating, inhibition and mental flexibility) as well as structural and functional changes in circuits, especially involving the prefrontal cortices bilaterally. These changes have been indicated as important protective factors in different situations of neural loss.O fenômeno do bilinguismo é a premissa essencial da tradução. Este trabalho apresenta modelos cognitivos contemporâneos que explicam o acesso lexical de bilíngues e multilíngues para o reconhecimento de palavras escritas, a partir de experimentos com homógrafos interlinguais. São abordados os trabalhos sobre conflito entre duas ou mais línguas,e como seus achados convergem para a hipótese de acesso não-seletivo ao léxico, o que elucida o impacto do bilinguismo nos processos de leitura etradução. Como objetivo secundário, e a partir de uma perspectiva neurocientífica, também descrevemos as mudanças no cérebro e nos processos mentais decorrentes à experiência do bilinguismo e do multilinguismo. Os estudos mostram vantagens nas funções executivas (atualização, inibição e flexibilidade mental) e mudanças estruturais e funcionais em circuitos, especialmente envolvendo os córtices pré-frontais bilateralmente. Estas mudanças têm sido apontadas como fatores importantes de proteção (reserva cognitiva e cerebral) frente a diferentes situações de perdaneural

Listeria monocytogenes persistence in food processing environments: Whole Genome Sequencing and in vitro assessment of disinfectants resistance and biofilm forming ability

Listeria monocytogenes (Lm) is the causative agent of listeriosis, an invasive disease primarily affecting immunocompromised people, the elderly, children and pregnant women, with high hospitalization (98.6%) and fatality rates (13.8%) 1. The disease is most commonly caused by eating contaminated food, in particular ready-to-eat. The ability of some strains to persist, even for years, in food processing environments can increase the risk of food contamination. Persistence can results from Lm survival after disinfection, thanks to protective biofilm formation, disinfectants and stresses resistance mechanisms or from the repeated reintroduction from raw materials 2 3. The identification of recurring highly genetically related isolates (Whole Genome Sequencing, WGS and core genome MLST, cgMLST) is necessary to define a strain persistent in a plant 4. The aim of this study was to evaluate persistence and resistance to commercial sanitizers commonly used in food processing environments, in Lm strains isolated within the laboratory activity of IZSUM (Istituto Zooprofilattico Sperimentale Umbria e Marche). Our approach was based on both WGS and in vitro assays

Molecular characterization of Listeria monocytogenes strains isolated from food and human sources

Introduction: The aim of the work was the molecular characterization of L. monocytogenes (L.m.) isolated from food and humans in the period 2019-2021. Materials and methods: A total of 67 L.m. isolates have been collected (UNI EN ISO 11290-1: 2017) at the Food Microbiology laboratories of the IZS Palermo from different food matrices with a prevalence of ready-to-eat food products such as dairy, fresh cheese, meat, fish sampled by Veterinary Services. The bacterial isolates were stored at -20 ° C in Microbank and sent to the LNR of Teramo for the determination of the serogroup (PCR-Multiplex) and molecular investigation (MLST and Whole Genome Sequencing -WGS). The sequencing was carried out on the Illumina NextSeq 500 platform and the data analysis was performed on a bioinformatics and data collection platform of the LNR L.m, called GenPat. N. 39 clinical strains of L.m. were collected at Palermo hospital laboratories (Regional Reference Laboratory for listeriosis). The strains, together with the sheets for the collection of epidemiological data, were sent to the Istituto Superiore di Sanità (ISS- Operational Microbiological Contact Point for L.m.) for molecular characterization by WGS. The sequencing was carried out on an IonTorrent S5 platform and the analysis was performed automatically on a bioinformatics and data collection platform, called IRIDA-ARIES. Results: The results on the molecular characterization of the strains isolated from food showed a prevalence of serogroup IVb (70%), followed by IIa (24%), IIb (4.5%) and IIc (1.5%). The 46.3% of the strains, all belonging to serogroup IVb, have been isolated from milk products (mozzarella and string cheese) taken from the same producer during official controls, in the period August- September 2020, following a positivity detected in self-control. The results of the sequencing carried out by the LNR highlighted the existence of clusters of strains belonging to Clonal Complex (CC) 2, CC199 and CC1. In particular, all the strains isolated in the same cheese factory, both in the period considered and in a subsequent sampling in January 2022, belonged to CC2 and Sequence Type 2 (ST2). Regarding the 39 clinical L.m. strains, the phylogenetic analysis of the genomes allowed the identification of a Cluster_90 composed of 25 strains of which 6 were isolated in 2019, 16 in 2020 and 1 in 2021. The 25 strains were found to belong to serogroup IVb, ST2 and CC2, presenting between them an allelic difference in the MLST core genome between 0 and 12. It has been noted the presence of 8 strains, isolated between October 2019 and August 2021, associated with maternal and neonatal cases. The genomic sequences of the 39 clinical strains of L.m. were compared with the sequences deposited in the IRIDA-ARIES database. From the analysis, it emerged that the Cluster_90, ST2 also included 3 isolated strains in Lombardy (2 in 2019, 1 in 2020), 2 in Piedmont, 1 in Lazio, 1 in Tuscany in 2020 and 2 isolated strains in Sicily (1 in 2019 from Syracuse and 1 in 2020 from the province of Palermo). Conclusion: This study allowed to start the construction of an integrated medical/veterinary network between laboratories to enhance the listeriosis surveillance system in the Sicily region

The Slaughterhouse as Hotspot of CC1 and CC6 Listeria monocytogenes Strains with Hypervirulent Profiles in an Integrated Poultry Chain of Italy

In Europe, very few studies are available regarding the diversity of Listeria monocytogenes (L. monocytogenes) clonal complexes (CCs) and sequence types (ST) in poultry and on the related typing of isolates using whole genome sequencing (WGS). In this study, we used a WGS approach to type 122 L. monocytogenes strains isolated from chicken neck skin samples collected in two different slaughterhouses of an integrated Italian poultry company. The studied strains were classified into five CCs: CC1-ST1 (21.3%), CC6-ST6 (22.9%), CC9-ST9 (44.2%), CC121-ST121 (10.6%) and CC193-ST193 (0.8%). CC1 and CC6 strains presented a virulence gene profile composed of 60 virulence genes and including the Listeria Pathogenicity Island 3, aut_IVb, gltA and gltB. According to cgMLST and SNPs analysis, long-term persistent clusters belonging to CC1 and CC6 were found in one of the two slaughterhouses. The reasons mediating the persistence of these CCs (up to 20 months) remain to be elucidated, and may involve the presence and the expression of stress response and environmental adaptation genes including heavy metals resistance genes (cadAC, arsBC, CsoR-copA-copZ), multidrug efflux pumps (mrpABCEF, EmrB, mepA, bmrA, bmr3, norm), coldshock tolerance (cspD) and biofilm-formation determinants (lmo0673, lmo2504, luxS, recO). These findings indicated a serious risk of poultry finished products contamination with hypervirulent L. monocytogenes clones and raised concern for the consumer health. In addition to the AMR genes norB, mprF, lin and fosX, ubiquitous in L. monocytogenes strains, we also identified parC for quinolones, msrA for macrolides and tetA for tetracyclines. Although the phenotypical expression of these AMR genes was not tested, none of them is known to confer resistance to the primary antibiotics used to treat listeriosis The obtained results increase the data on the L. monocytogenes clones circulating in Italy and in particular in the poultry chain