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.

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.

The role of staff and contaminated environmental surfaces in spreading of norovirus infection in a long-term health care facility in Italy.

Some residents and people from the staff of a geriatric health care facility in Teramo province, developed acute gastroenteritis from March 8th to March 21st 2017. A prompt epidemiological investigation was conducted to identify the etiological agent, the trace back the potential ways of transmission and control the infection. Information on the outbreak was collected through an epidemiological questionnaire. Faecal samples from all human cases (n = 50) and swabs from environmental surfaces were collected and analysed by RT-PCR for the presence of Norovirus (NoV). Among faecal samples, 34 out of 50 were positive for NoV with no other pathogen detected. In particular, 2 (2/34) were positive to NoV genogroup I (GI), 31 (31/34) to NoV genogroup II (GII), and one sample (1/34) was positive to both NoV GI and GII. Moreover, faecal samples of people from the canteen (n = 8) were also tested resulting negative to NoV detection. Norovirus was also detected in 28 of the 122 swabs from environmental surfaces collected. Among the positive samples, 12 NoV strains were subtyped as NoV GII.4 Sydney_2012 variant. Person-to-person close contact and contaminated environmental surfaces were the probable transmission route among the people of the health care facility. The members of the staff were considered to play an important role in transmission of NoV. A proper disinfection procedure applied during the outbreak could have been critically important to limit the dissemination of the viral infection.

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.

Validation via challenge test of a dynamic growth-death model for the prediction of Listeria monocytogenes kinetics in Pecorino di Farindola cheese.

Pecorino di Farindola is a typical cheese produced in the area surrounding the village of Farindola, located in the Abruzzo Region (central Italy), unique among Italian cheese because only raw ewe milk and pig rennet are used for its production. In the literature it is well documented that raw milk is able to support the growth of pathogenic microorganisms such as Listeria monocytogenes. Predictive microbiology can be useful in order to predict growth-death kinetics of pathogenic bacteria, on the basis of known environmental conditions. Aim of this study was to compare predictions obtained from a model, originally designed to predict the kinetics of L. monocytogenes in the dynamic growth-death environment of drying fresh sausage, with the results of challenge tests performed during the ripening of Pecorino di Farindola produced from artificially contaminated raw ewe milk. A challenge test was carried out using ewe raw milk inoculated with L. monocytogenes, in order to produce Pecorino di Farindola cheese stored at 18 °C for 149 days of ripening. During the ripening period, pH and aw values decreased in all samples analysed; lactic acid bacteria become the prevailing microbial population, while for L. monocytogenes a period of stability (neither growth nor death) followed the initial situation. The growth inhibition and the following inactivation may mostly be due to competition with the autochthonous microbiota and to the reduction of water activity. Mathematical modelling was used in order to predict microbial kinetics in the dynamic ripening environment, joining growth and death patterns in a continuous way, and including the highly uncertain growth/no growth range separating the two regions. The effect of lactic acid bacteria on the growth of pathogens was also included. Predicted microbial kinetics were satisfactory, as confirmed by the absence of statistically significant difference between observed and predicted values (p > 0.05). The present study proved, via challenge tests, that a dynamic growth/death model, previously used for a meat product, can be fruitfully used in cheese characterized by active competitive microbiota and progressive drying during ripening.

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.

Predicting the kinetics of Listeria monocytogenes and Yersinia enterocolitica under dynamic growth/death-inducing conditions, in Italian style fresh sausage.

Traditional Italian pork products can be consumed after variable drying periods, where the temporal decrease of water activity spans from optimal to inactivating values. This makes it necessary to A) consider the bias factor when applying culture-medium-based predictive models to sausage; B) apply the dynamic version (described by differential equations) of those models; C) combine growth and death models in a continuous way, including the highly uncertain growth/no growth range separating the two regions. This paper tests the applicability of published predictive models on the responses of Listeria monocytogenes and Yersinia enterocolitica to dynamic conditions in traditional Italian pork sausage, where the environment changes from growth-supporting to inhibitory conditions, so the growth and death models need to be combined. The effect of indigenous lactic acid bacteria was also taken into account in the predictions. Challenge tests were carried out using such sausages, inoculated separately with L. monocytogenes and Y. enterocolitica, stored for 480h at 8, 12, 18 and 20°C. The pH was fairly constant, while the water activity changed dynamically. The effects of the environment on the specific growth and death rate of the studied organisms were predicted using previously published predictive models and parameters. Microbial kinetics in many products with a long shelf-life and dynamic internal environment, could result in both growth and inactivation, making it difficult to estimate the bacterial concentration at the time of consumption by means of commonly available predictive software tools. Our prediction of the effect of the storage environment, where the water activity gradually decreases during a drying period, is designed to overcome these difficulties. The methodology can be used generally to predict and visualise bacterial kinetics under temporal variation of environments, which is vital when assessing the safety of many similar products.