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

Indagine su un focolaio di tossinfezione da Salmonella enterica subsp. enterica serovar Hadar nella regione Abruzzo

E’ stato eseguito uno studio comparativo tra 22 ceppi di Salmonella Hadar isolati da soggetti coinvolti in un focolaio di tossinfezione alimentare in Abruzzo nel 2000 e 21 ceppi dello stesso sierotipo isolati da carni avicole e da feci umane in Abruzzo e Molise nel periodo 2000 e 2001. L’indagine aveva come obiettivo di fornire una interpretazione epidemiologica del focolaio di tossinfezione alimentare determinando il grado di similarità tra i ceppi di Salmonella Hadar isolati dai soggetti coinvolti nel focolaio, quelli isolati da carne avicola, identificata ma non confermata come possibile fonte di infezione, e da altri campioni umani pervenuti in laboratorio. A tal fine sono state impiegate tecniche di caratterizzazione genotipica come pulsed-field gel electrophoresis (PFGE) e random amplified polymorphic DNA (RAPD) e sono stati determinati i pattern di resistenza agli antimicrobici. Dall’analisi in PFGE dei profili di restrizione ottenuti con XbaI e BlnI sono stati identificati 12 pulsotipi suddivisi in 3 gruppi. La RAPD non ha fornito indicazioni non riuscendo a discriminare i ceppi isolati dai soggetti con gastroenterite appartenenti al focolaio tossinfettivo. Il test di resistenza agli antimicrobici ha evidenziato pattern di resistenza multipla ma non sono stati identificati ceppi resistenti al Ciprofloxacin o altri Chinoloni testati. I ceppi aviari sono risultati resistenti all’acido nalidixico mentre solo il 31,8% di quelli umani ha presentato tale profilo. Da un’analisi combinata dei pattern di resistenza e dei pulsotipi sono stati identificati 4 profili di cui quello associato al focolaio è risultato non correlato agli altri presenti nello stesso periodo. E’ stata confermata la necessità di applicare un set di metodi di analisi differenti per garantire una migliore caratterizzazione e una maggiore capacità discriminante nell’identificazione delle possibili origini della contaminazione e stabilire correlazioni fra gli isolati

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

Co-Infection of L. monocytogenes and Toxoplasma gondii in a Sheep Flock Causing Abortion and Lamb Deaths

Abortion in livestock is a public health burden, and the cause of economic losses for farmers. Abortion can be multifactorial, and a deep diagnostic investigation is important to reduce the spread of zoonotic disease and public health prevention. In our study, a multidisciplinary investigation was conducted to address the cause of increased abortion and lamb mortality on a farm, which detected a co-infection of Listeria monocytogenes and Toxoplasma gondii. Hence, it was possible to conclude that this was the reason for a reduced flock health status and the cause of an increased abortion rate. Furthermore, the investigation work and identification of the L. monocytogenes infection root allowed the reduction of economic loss

A Real-Time PCR Screening Assay for Rapid Detection of Listeria Monocytogenes Outbreak Strains

From January 2015 to March 2016, an outbreak of 23 human cases of listeriosis in the Marche region and one human case in the Umbria region of Italy was caused by Listeria monocytogenes strains showing a new pulsotype never described before in Italy. A total of 37 clinical strains isolated from patients exhibiting listeriosis symptoms and 1374 strains correlated to the outbreak were received by the Italian National Reference Laboratory for L. monocytogenes (It NRL Lm) of Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM) for outbreak investigation. A real-time PCR assay was purposely designed for a rapid screening of the strains related to the outbreak. PCR-positive strains were successively typed through molecular serogrouping, pulsed field gel electrophoresis (PFGE), and Next Generation Sequencing (NGS). Applying the described strategy, based on real-time PCR screening, we were able to considerably reduce time and costs during the outbreak investigation activities