Genetic diversity of Listeria monocytogenes strains contaminating food and food producing environment as single based sample in Italy (retrospective study).

Human listeriosis outbreaks are often associated with food products, which could be contaminated, at the same time, also by different clones of Listeria monocytogenes. This emphasize the need to type more than one L.monocytogenes isolate found in a single food or environmental sample. The purpose of the present study was to evaluate the presence of different L.monocytogenes strains in food and food production environment in order to understand if there is need to type more isolates from the same sample in case of presence of L.monocytogenes. Between 2011 and 2015, at the Italian National Reference Laboratory for L.monocytogenes, for each positive sample, from two to twenty-three isolates of L.monocytogenes were collected. All the isolates were characterized by conventional serotyping and pulsed field gel electrophoresis (PFGE). Moreover, isolates from the same sample, having indistinguishable PFGE profile, were subjected to whole genome sequencing in order to perform core genome Multi Locus Sequence Typing (cgMLST). Within each sample, more than one serotype and one pulsotype were found in 11.9% and 27.5%, respectively. For indistinguishable PFGE patterns the cgMLST analysis showed 96.2% of concordance demonstrating the added value of new sequencing technologies. This study has demonstrated the need to select and type more than one L.monocytogenes colony in one food or food environmental sample to detect the diversity of L.monocytogenes strains and facilitate downstream investigations and effective source attribution in foodborne outbreak inquiry.

Phylogenetic Analysis and Genome-Wide Association Study Applied to an Italian Listeria monocytogenes Outbreak.

From May 2015 to March 2016, a severe outbreak due to Listeria monocytogenes ST7 strain occurred in Central Italy and caused 24 confirmed clinical cases. The epidemic strain was deeply investigated using whole-genome sequencing (WGS) analysis. In the interested area, the foodborne outbreak investigation identified a meat food-producing plant contaminated by the outbreak strain, carried by pork-ready-to-eat products. In the same region, in March 2018, the epidemic strain reemerged causing one listeriosis case in a 10-month-old child. The aim of this study was to investigate the phylogeny of the epidemic and reemergent strains over time and to compare them with a closer ST7 clone, detected during the outbreak and with different pulsed-field gel electrophoresis (PFGE) profiles, in order to identify genomic features linked to the persistence and the reemergence of the outbreak. An approach combining phylogenetic analysis and genome-wide association study (GWAS) revealed that the epidemic and reemergent clones were genetically closer to the ST7 clone with different PFGE profiles and strictly associated with the pork production chain. The repeated detection of both clones was probably correlated with (i) the presence of truly persistent clones and the repeated introduction of new ones and (ii) the contribution of prophage genes in promoting the persistence of the epidemic clones. Despite that no significant genomic differences were detected between the outbreak and the reemergent strain, the two related clones detected during the outbreak can be differentiated by transcriptional factor and phage genes associated with the phage LP-114.

A large food-borne outbreak of campylobacteriosis in kindergartens and primary schools in Pescara, Italy, May-June 2018.

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.

Application of the Micro Biological Survey analytical method for the determination of bacterial load in cow raw milk.

The aim of this study was to evaluate the performance of "Micro Biological Survey - MBS Test" in the enumeration of bacterial load in cow raw milk. The MBS test is based on a colorimetric method recently developed and patented by "Roma Tre" University, Italy. The evaluation of the performance of the MBS method was carried out by comparison with plate count at 30°C (gold standard) and flow cytometry. Thirteen independent set of experiments were performed analyzing a total of 104 samples of cow raw milk with the selected methods. Results obtained using the MBS method are comparable with those obtained with the plate count method at 30°C (CFU/mL) and flow cytometry technology; in particular, the results obtained with the MBS method are very close to plate count's at 30°C. On the other hand, there are statistically significant differences between these two methods' and flow cytometry technology's results that could be due to the different experimental conditions.

Listeria monocytogenes in poultry: Detection and strain characterization along an integrated production chain in Italy.

In this study, thirteen batches of broiler chicken from an integrated Italian poultry company were investigated for the detection of Listeria monocytogenes. The prevalence was evaluated in faeces samples at farm level and after transport, caecal contents and carcass neck skin from 2 slaughterhouses (M1 and M2), for a total of 2080 samples, throughout a 27-month period. No positive results were recorded in faeces, while the overall prevalence of contamination in carcass neck skin was 26.7%. Then, 123 isolates out of 139 positive skin samples, with the prevalent serotypes 4b (76%) and 1/2b (94%) from slaughterhouses M1 and M2 respectively, were PFGE characterized, showing the presence of 18 different pulsotypes and 8 genetic clusters. The same pulsotypes were found in carcasses from different farms, but slaughtered in the same abattoir, highlighting the environmental origin of contamination. The persistence of the pathogen over long time seemed to be very likely, considering that undistinguishable pulsotypes were found in carcasses slaughtered in the same slaughterhouse after periods up to 18 months long. The implementation of cleaning and sanitation at slaughterhouse level could represent the main factor for the control of such pathogen in the poultry meat processing line.

Development and comparative study of a pat/bar real-time PCR assay for integrating the screening strategy of a GMO testing laboratory.

BACKGROUND: The number and variety of genetically modified organisms (GMOs) used globally for the production of food and feed, and potentially circulating in the European Union (EU), is constantly increasing. This implies an additional effort for the EU enforcement laboratories to optimize available resources, to contain costs and time. A well established approach for streamlining the analytical workflow is the introduction of a screening step, typically based on a smart set of real-time polymerase chain reaction (PCR) screening methods. The multiplexing strategy, allowing the detection of several screening elements simultaneously, is a further optimization of this step. RESULTS: In this study, we present the validation of a real-time PCR duplex assay for the pat and bar screening elements to be easily incorporated in the GMO diagnostic routine. We also provide a comparison between this method and the related singleplex and pre-spotted assays. CONCLUSION: Our results fully respect all the validation parameters suggested by the Minimum Performance Criteria of the European Network of GMO Laboratories. Furthermore, the duplex assay is equivalent in terms of performance compared to the other two methods, but it shows a higher overall flexibility and cost effectiveness. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Thermotolerant Campylobacter spp. in chicken and bovine meat in Italy: Prevalence, level of contamination and molecular characterization of isolates.

Campylobacter species are common foodborne pathogens associated with cases of human gastroenteritis worldwide. A detailed understanding of the prevalence, contamination levels and molecular characteristics of Campylobacter spp. in cattle and chicken, which are likely the most important sources of human contamination, is imperative. A collection of 1243 poultry meat samples (665 chicken breasts and 578 chicken thighs) and 1203 bovine meat samples (689 hamburgers and 514 knife-cut meat preparations) were collected at retail outlets, in randomly selected supermarkets located in different Italian regions during one year. Of these samples, 17.38% of the poultry meat and 0.58% of the bovine meat samples tested positive for Campylobacter, of which 131 were Campylobacter jejuni (57.96%) and 95 were Campylobacter coli (42.03%). Campylobacter isolates were genotyped with the aim of assessing the genetic diversity, population structure, source distribution and Campylobacter transmission route to humans. All isolates were molecularly characterized by pulse field gel electrophoresis (PFGE), and further genotyped using multilocus sequence typing (MLST) and fla-SVR sequencing to gain better insight into the population structure. Antibiotic resistance was also investigate. The highest levels of resistance among chicken strains were observed for ciprofloxacin (88.25%), nalidixic acid (81.45%) and tetracycline (75.6%). PFGE analysis revealed 73 pulsotypes for C. jejuni and 54 pulsotypes for C. coli, demonstrating the existance of different and specific clones circulating in Italy. MLST of C.jejuni isolates mainly clustered in the CC353, CC354, CC21, CC206 and CC443; while C.coli isolates clustered only in CC828. The most common flaA alleles were 287 for C. jejuni and 66 for C. coli. Our study confirms that poultry meat is the main source of Campylobacteriosis, whereas red meat had a low level of contamination suggesting a minor role in transmission. The high presence of Campylobacter in retail chicken meat, paired with its increased resistance to antimicrobials with several multidrug resistance profiles detected, is alarming and represents a persistent threat to public health.

A severe outbreak of listeriosis in central Italy with a rare pulsotype associated with processed pork products.

PURPOSE: From May 2015 to March 2016, an outbreak due to Listeria monocytogenes serotype 1/2a and clinical pulsotype never previously isolated in Europe occurred in central Italy, involving 24 confirmed clinical cases. The article provides a description of the outbreak and the investigation carried out by a multidisciplinary network. METHODOLOGY: Epidemiological and microbiological surveillance was conducted to confirm the outbreak and to detect the food vehicle of infection. The origin and destination of the implicated food and its ingredients were investigated by tracing-back and -forward investigation. RESULTS: Next-generation sequencing confirmed the unique outbreak strain. On 4 January 2016, a L. monocytogenes strain with pulsotype indistinguishable from that isolated from clinical cases in the outbreak was detected in a sample of hog head cheese purchased from a retail supermarket by one of the patients. The hog head cheese was produced by a small meat processing plant in the Marche region, where microbiological investigation confirmed environmental and food contamination by the outbreak strain. Plant production was suspended and all contaminated batches of the hog head cheese were withdrawn from the market by 19 February by local health authority. We subsequently observed a sharp decline in clinical cases, the last being reported on 11 March 2016. CONCLUSION: The key factor in the timely conclusion of this investigation was intersectoral collaboration among epidemiologists, microbiologists, veterinarians, statisticians and health and food safety authorities at national, regional and local levels.

Identification and characterization of Yersinia enterocolitica strains isolated from pig tonsils at slaughterhouse in Central Italy.

Yersinia enterocolitica causes foodborne disease in humans and infections are usually acquired from contaminated raw or undercooked pork. Pigs are considered the primary reservoir of human pathogenic bio-serotypes. A total of 376 tonsil tissue samples collected after evisceration and cutting from pig carcasses were tested for Yersinia enterocolitica. Animals came from an abattoir located in the Abruzzo region, Italy. Yersinia enterocolitica was isolated from 35 out of 376 (9.31%) samples. A total of 47 strains were isolated, the prevalent bio-serotype was 4÷O:3 (95.74%), followed by bio-serotype 4÷O:9 (2.13%), and 3÷O:9 (2.13%). When characterized by DNA microarray, all strains clustered into 2 main groups. The bigger group was characterised by the presence of plasmid genes of the secretion apparatus as well as by the genes for the agellum transport machinery, while the smaller group was characterised only by genes for the agellum transport machinery. The high frequency of the pathogenic biotype 4÷O:3 able to infect humans and considered an emerging zoonotic pathogen con rms the role of pigs as natural reservoir. Since there is no official data on Yersinia enterocolitica, it is difficult to assess the implications of this food pathogen for public health. A monitoring program should be implemented for contamination in food in order to assess the risk for the consumer linked to raw or undercooked pork products.

Outbreak of unusual Salmonella enterica serovar Typhimurium monophasic variant 1,4 [5],12:i:-, Italy, June 2013 to September 2014.

Monophasic variant of Salmonella enterica subspecies enterica serovar Typhimurium (monophasic S. Typhimurium), with antigenic structure 1,4,[5],12:i:-, appears to be of increasing importance in Europe. In Italy, monophasic S. Typhimurium represented the third most frequent Salmonella serovar isolated from human cases between 2004 and 2008. From June 2013 to October 2014, a total of 206 human cases of salmonellosis were identified in Abruzzo region (Central Italy). Obtained clinical isolates characterised showed S. Typhimurium 1,4,[5],12:i:- with sole resistance to nalidixic acid, which had never been observed in Italy in monophasic S. Typhimurium, neither in humans nor in animals or foods. Epidemiological, microbiological and environmental investigations were conducted to try to identify the outbreak source. Cases were interviewed using a standardised questionnaire and microbiological tests were performed on human as well as environmental samples, including samples from fruit and vegetables, pigs, and surface water. Investigation results did not identify the final vehicle of human infection, although a link between the human cases and the contamination of irrigation water channels was suggested.

Characterisation of Staphylococcus aureus strains isolated from food for human consumption.

An investigation was conducted to evaluate Staphylococcus aureus contamination in various types of food of animal origin. Of the 350 samples examined, 14.0% were found to be contaminated with S. aureus. Prevalence rates varied according to type, namely: 19.3% for fresh meat products, 13.3% for fresh cheeses, 3.6% for bakery products and 7.7% for deli products. The isolated S. aureus strains then underwent 16S rDNA polymerase chain reaction (PCR) followed by reverse latex agglutination tests to identify enterotoxigenic strains. The results were compared with data obtained by subjecting the same strains to tests for the genes coding for the S. aureus enterotoxins (SEs) sea, seb, sec, sed, see, seg, seh, sei and toxic shock syndrome toxin 1 (TSST 1). Reversed passive latex agglutination (RPLA) testing revealed that 16.3% of strains (8/49) produced enterotoxins, while on PCR, 48.97% (24/49) were found to carry one or more genes for the production of SEs, and were therefore potentially enterotoxigenic.

Gastroenteritis outbreak at holiday resort, central Italy.

During the summer of 2003, a gastroenteritis outbreak spread throughout a holiday resort in central Italy. Fecally contaminated groundwater and seawater were leaking into the non-drinking-water system, which was found to be connected to the drinking-water system of a large resort. This contamination had a primary role in the onset of the outbreak and spread of the infection.

Investigation of an outbreak of Salmonella enterica subsp. enterica serovar Hadar food illness in the Abruzzi region of Italy.

A comparative study was made of 22 strains of Salmonella Hadar isolated from victims of an outbreak of food illness in the Abruzzi region of Italy in 2000 and 21 strains of the same serotype isolated from poultry meat and human stool samples in the Abruzzi and Molise regions between 2000 and 2001. The aim of the investigation was to provide an epidemiological interpretation of the food illness outbreak to establish the degree of similarity between the S. Hadar strains isolated from victims of the outbreak and those isolated from poultry meat (identified but unconfirmed as the possible source of infection) and from other human samples received in the laboratory. Pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) were used to identify the genotypes and antimicrobial resistance patterns were determined. PFGE analysis of the restriction patterns obtained with XbaI and BlnI led to the identification of 12 pulsotypes in three groups. RAPD did not provide any information, as it was unable to differentiate between the strains isolated from food illness victims with gastroenteritis. Antimicrobial resistance tests revealed multiple resistance patterns and no strains were found to be resistant to ciprofloxacin or to the other quinolones tested. The poultry strains were found to be resistant to nalidixic acid, while the resistance in human strains was 31.8%. A combined analysis of resistance patterns and pulsotypes revealed four resistance patterns; the pattern associated with the outbreak was not correlated with the others present in the same period. This work suggests that a study of the relationship between different strains of the same serovar requires the implementation of different analytical methods.

An outbreak of gastroenteritis in a holiday resort in Italy: epidemiological survey, implementation and application of preventive measures.

A major gastroenteritis outbreak was reported in a vacation resort in Central Italy in 2003. A total of 183 cases were identified. The case-control study identified a statistically significant correlation between the disease and sea bathing, use of sanitary facilities in bungalows and of common showers. Stool samples taken from people affected were found positive for Norovirus (68%, 13 of 19 samples), Rotavirus (38%, 1 of 14 samples) and Campylobacter (7%, 3 of 8 samples). Environmental investigations revealed serious faecal contamination of the groundwater and the presence of Norovirus in the seawater near the resort. The mixing of groundwater and seawater with the non-drinking water system - which was also found to be connected to the drinking water system - had a primary role in the onset and spread of infection within the village. The complete absence of any gastroenteritis epidemics among the site guests since 2006 demonstrates the effectiveness of the environmental corrective measures taken.