Distribution of epidemic clonal genetic markers among Listeria monocytogenes 4b isolates.

Recent genome sequencing of isolates of Listeria monocytogenes serotype 4b implicated in some major outbreaks of foodborne listeriosis has revealed unique genetic markers in these isolates. The isolates were grouped into two distinct epidemic clones, ECI and ECII. In the present study, selected ECI- and ECII-specific genetic markers were detected in 16 and 15 of 89 L. monocytogenes 4b isolates, respectively. The ECI markers were found in 6 of 34 clinical isolates, 9 of 50 food isolates, and 1 of 5 environmental isolates, and the ECII markers were detected in 7 of 34 clinical isolates, 7 of 50 food isolates, and 1 of 5 environmental isolates. Hence, of the isolates with the epidemic clonal genetic markers, 38% (13 of 34) were of clinical origin, 32% (16 of 50) were of food origin, and 40% (2 of 5) were of environmental origin. The predominance of the epidemic clonal markers among the clinical and environmental isolates supports the hypothesis that these markers are correlated with the pathogenic potential of strains and with their environmental persistence. Several isolates had only one epidemic clonal marker, either the ECI-specific marker 133 or the ECII-specific marker 4bSF18. Pulsed-field gel electrophoresis analysis revealed higher genomic diversity among the strains with ECII-like characteristics than among those strains carrying the ECI-specific genetic markers

Listeria monocytogenes in Ready-to-Eat Seafood and Potential Hazards for the Consumers

The risk of exposure to Listeria monocytogenes (L. monocytogenes) when consuming Ready-to-Eat (RTE) seafood was assessed in the Veneto Region (Italy). Thirty-eight samples were analyzed, each sample consisted of three subunits belonging to the same batches. The first of the three units was examined immediately, the second was stored at +4°C (for all of its shelf-life) and the third at +10°C (for the latter third of its shelf-life) before the analysis. Chemical-physical and microbiological parameters were tested simultaneously. Culture results showed the presence of viable L. monocytogenes in 9 (23,68%) of the 38 samples analysed, 3 (33,33%) of which with a concentration >100 cfu/g. PCR tests yielded 12 L. monocytogenes positive samples. Semipreserves with aw (water activity) and pH values that favour L. monocytogenes growth were the only ones to result positive to microbiological and PCR tests. Temperature proved to be an important factor as it limits the growth of L. monocytogenes, including products with potentially high competitive microbial charges. Four different serotypes were recovered and ribotyping has helped to highlight the genomic variability of L. monocytogenes strains in food. This supports the hypothesis that L. monocytogenes continues to evolve genetically to the detriment of phenotypic conservation

Survival of Listeria monocytogenes in uncooked Italian dry sausage (salami).

This study was undertaken to supplement existing information on the survival of Listeria monocytogenes in Italian salami. The fact that Italian salami is frequently consumed by a large number of people poses some serious health implications. Some raw materials have been found to be microbiologically contaminated, for their production occurs without any thermic treatment, and these are in circulation throughout Italy all year round. We selected the product for its microbiological, technological, and commercial characteristics. We analyzed 1,020 samples taken during the autumn and winter 2002 and spring and summer 2003 periods and immediately before selling. The samples were collected from 17 plants with an annual production of between 1 and 2,000 metric tons and with a distribution of products in over 80% of Italy in geographic terms. To detect and enumerate L. monocytogenes, we followed International Organization for Standardization (ISO) 11290 part 1 and 2: 1996 (modified using chromogenic medium Agar Listeria according to Ottarviani and Agosti [ALOA]). L. monocytogenes was found in 22.7% of samples, but the contamination level was less than 10 CFU/g. Contamination prevalence ranged from 1.6 to 58.3% and was lower than 10% in 5 of the 17 plants checked. The most frequently isolated serotypes were 1/2c, 1/2a, 1/2b, and 4b. Additional studies are necessary to establish if the exposure to a small number of L. monocytogenes cells through the consumption of salami represents a significant health risk and, in light of the future introduction of the SANCO/4198/2001 revision 21 "Commission Regulation on Microbiological Criteria for Foodstuffs," is a necessary investigation

Listeria monocytogenes serotypes in human infections (Italy, 2000-2010)

BACKGROUND: In developed countries invasive listeriosis is an infection of great concern to public health to due its clinical severity and high fatality rate, despite its low incidence. In Europe, statistically significant increasing trends in listeriosis notification rates from 2005 to 2009 were noted in Austria, Denmark, Hungary, Italy, Spain and Sweden. MATERIALS AND METHODS: The standardized techniques based on phenotype to typing Listeria monocytogenes is the serotyping. In Europe, as elsewhere in the world, about 95% of L. monocytogenes strains isolated from clinical and food samples belongs to serovars 1/2a, 1/2b, 1/2c and 4b. RESULTS: The target of this work is to draw attention to this important and atypical foodborne disease, reporting epidemiological data and serotypes distribution of 251 human L. monocytogenes isolates reported during 2000-2010 to Veterinary Public Health and Food Safety Department of Istituto Superiore di Sanità, focusing on epidemiological trend of invasive listeriosis in Lombardia, a North Italian Region. The serotypes most frequently identified are 1/2a, 4b, 1/2b (in total 92%), but the detection of uncommon serotypes is not missing (1/2c, 3a, 3b, 4d). CONCLUSIONS: In Italy the surveillance laboratory network, as well as the foodborne disease network (ENTER-NET), has revealed in the last 11 years an increase trend of listeriosis cases reported likewise with results of Notificable National Infectious Disease surveillance System. This is probably due to a real increase of listeriosis, even if there is a greater sensitivity of the network in some regions

Clostridium botulinum spores and toxin in mascarpone cheese and other milk products

A total of 1,017 mascarpone cheese samples, collected at retail, were analyzed for Clostridium botulinum spores and toxin, aerobic mesophilic spore counts, as well as pH, a(w) (water activity), and Eh (oxidation-reduction potential). In addition 260 samples from other dairy products were also analyzed for spores and botulinum toxin. Experiments were carried out on naturally and artificially contaminated mascarpone to investigate the influence of different temperature conditions on toxin production by C. botulinum. Three hundred and thirty-one samples (32.5%) of mascarpone were positive for botulinal spores, and 7 (0.8%) of the 878 samples produced at the plant involved in an outbreak of foodborne botulism also contained toxin type A. The chemical-physical parameters (pH, a(w), Eh) of all samples were compatible with C. botulinum growth and toxinogenesis. Of the other milk products, 2.7% were positive for C. botulinum spores. Growth and toxin formation occurred in naturally and experimentally contaminated mascarpone samples after 3 and 4 days of incubation at 28 degrees C, respectively

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

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

Application of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of <i>Listeria monocytogenes</i> in Cooked Ham

Changing eating habits and rising demand of food have increased the incidence of foodborne diseases, particularly in industrialized countries. In this context, contaminated ready-to-eat food (RTE) may be a vehicle for the transmission of Listeria monocytogenes (L. monocytogenes), a foodborne pathogen responsible of listeriosis, a severe infectious disease involving humans and animals. It would be useful to have rapid detection methods to screen the presence of L. monocytogenes in food. In this study, a colorimetric Loop-mediated isothermal amplification (LAMP) assay was applied to the detection of L. monocytogenes in 37 experimentally contaminated RTE meat samples. The LAMP primers consisted of a set of six primers targeting eight regions on the hlyA gene; the assay was carried out in 30 min at 65 °C in a water bath. Amplification products were visualized by color change assessment. The results of colorimetric LAMP assays based on the hly gene obtained in this study were compared to microbiological cultural methods, real-time PCR and real-time LAMP PCR, which show 100% specificity and sensitivity. These data suggest that colorimetric LAMP assays can be used as a screen to detect L. monocytogenes in ready-to-eat meat food

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