15 research outputs found
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
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
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