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We recently described the genetic antimicrobial resistance and virulence profile of a collection of 279 commensal E. coli of food-producing animal (FPA), pet, wildlife and human origin. Phenotypic antimicrobial resistance (AMR) and the role of commensal E. coli as reservoir of extra-intestinal pathogenic Escherichia coli (ExPEC) virulence-associated genes (VAGs) or as potential ExPEC pathogens were evaluated. The most common phenotypic resistance was to tetracycline (76/279, 27.24%), sulfamethoxazole/trimethoprim (73/279, 26.16%), streptomycin and sulfisoxazole (71/279, 25.45% both) among the overall collection. Poultry and rabbit were the sources mostly associated to AMR, with a significant resistance rate (p > 0.01) to quinolones, streptomycin, sulphonamides, tetracycline and, only for poultry, to ampicillin and chloramphenicol. Finally, rabbit was the source mostly associated to colistin resistance. Different pandemic (ST69/69*, ST95, ST131) and emerging (ST10/ST10*, ST23, ST58, ST117, ST405, ST648) ExPEC sequence types (STs) were identified among the collection, especially in poultry source. Both ST groups carried high number of ExPEC VAGs (pandemic ExPEC STs, mean = 8.92; emerging ExPEC STs, mean = 6.43) and showed phenotypic resistance to different antimicrobials (pandemic ExPEC STs, mean = 2.23; emerging ExPEC STs, mean = 2.43), suggesting their role as potential ExPEC pathogens. Variable phenotypic resistance and ExPEC VAG distribution was also observed in uncommon ExPEC lineages, suggesting commensal flora as a potential reservoir of virulence (mean = 3.80) and antimicrobial resistance (mean = 1.69) determinants.

Avian botulism is a paralytic disease due to the ingestion of botulinum neurotoxins (BoNT) produced by anaerobic, sporigenic bacteria (notably, Clostridium botulinum). Wild waterbirds worldwide are affected with variable recurrence and severity, and organic material decaying in wetland habitats may constitute a suitable substrate for the replication of clostridia strains producing BoNT in conditions of high temperatures and the absence of oxygen. Here, we describe a large outbreak of avian botulism that occurred in the Valle Mandriole protected area of northeastern Italy (VM). After the recovery in late summer of a few duck carcasses that molecularly tested positive for BoNT-producing clostridia, in October 2019, the avian botulism escalation led to a total of 2367 birds being recovered (2158 carcasses and 209 sick birds). Among these, 2365/2367 were waterbirds, with ducks accounting for 91.8% of the total (2173/2367) and green-winged teals representing 93.5% of the ducks. After the quick collection of dead and sick birds (from 4 to 11 October 2019) and the flooding of the VM wetland (from 5 to 12 October 2019), the 2019 botulism emergency apparently ended. Following two water inputs in May and July 2020, only one pooled sample obtained from 16 bird carcasses found that year in VM tested positive for clostridia type C by real-time PCR, whereas, after to the implementation of measures deterring the bird’s presence, new avian botulism cases—due to clostridia type C and C/D, according to molecular and animal-model tests of confirmation—led to the collection of 176 waterbirds (82 carcasses and 94 sick ducks) and 16 waterbirds (9 carcasses and 7 sick ducks) in the summers 2021 and 2022, respectively. In conclusion, the prevention, management, and control of the disease rely on habitat management, the quick and careful collection/removal of animal carcasses, and the regular monitoring and surveillance of live and dead birds.

The interest in certified game meat chains highlights the need for the evaluation and the management of factors affecting carcass hygiene along the peculiar steps of the production. The effects of time and temperature before chilling were specifically evaluated on aerobic colony count and Enterobacteriaceae count in hunted wild boar carcasses. Thirty wild boars were considered in two process steps where the hunted animal are still not chilled: after evisceration and just before chilling. Environmental temperature, carcass temperature and the elapse time between the two-step considered were registered. Furthermore, surface microbial loads were analyzed on the inner part of the carcasses. The mean time between the two sampling steps was 6 hours with an average environmental temperature of 20.49°C. A carcass temperature 9.6°C drop was observed during this period. In this lap of time aerobic colony count and Enterobacteriaceae count increased of 0.68 Log CFU/cm2 and 1.01 Log CFU/cm2 respectively, with a moderate correlation with the time but not with the temperature delta. The results reveal that the temperature conditions in central Italy hunting areas were not able to quickly reduce the carcass temperature and therefore the time between carcass evisceration and chilling should not exceed 6 hours.

In this study the effect of the application of High Pressure Treatment (HPP) combined with four different manufacturing processes on the inactivation of Listeria innocua, used as a surrogate for L. monocytogenes, in artificially contaminated coppa samples was evaluated in order to verify the most suitable strategy to meet the Listeria inactivation requirements needed for the exportation of dry-cured meat in the U.S. Fresh anatomical cuts intended for coppa production were supplied by four different delicatessen factories located in Northern Italy. Raw meat underwent experimental contamination with Listeria innocua using a mixture of 5 strains. Surface contamination of the fresh anatomical cuts was carried out by immersion into inoculum containing Listeria spp. The conditions of the HPP treatment were: pressure 593 MPa, time 290 seconds, water treatment temperature 14°C. Listeria innocua was enumerated on surface and deep samples post contamination, resting, ripening and HPP treatment. The results of this study show how the reduction of the microbial load on coppa during the production process did not vary among three companies (P>0.05) ranging from 3.73 to 4.30 log CFU/g, while it was significantly different (P<0.01) for the fourth company (0.92 log CFU/g). HPP treatment resulted in a significant (P<0.01) deep decrease of L.innocua count with values ranging between 1.63-3.54 log CFU/g with no significant differences between companies. Regarding superficial contamination, HPP treatment resulted significant (P<0.01) only in Coppa produced by two companies. The results highlight that there were processes less effective to inhibit the pathogen; in particular for company D an increase of L. innocua count was shown during processing and HPP alone cannot be able to in reaching the Listeria inactivation requirements needed for exportation of dry-cured meat in the U.S. According to the data reported in this paper, HPP treatment increases the ability of the manufacturing process of coppa in reducing Listeria count with the objective of a lethality treatment

The aim of the study was to investigate the combined effect of the manufacturing process followed by HPP treatment on the inactivation of Salmonella spp. in artificially contaminated coppa samples, in order to verify the ability of the combined processes to achieve the objective of a 5-log reduction of Salmonella spp. needed for exportation to the U.S. Fresh anatomical cuts intended for coppa production were supplied by four different delicatessen factories located in Northern Italy. Raw meat underwent experimental contamination with Salmonella spp. using a mixture of 3 strains. Surface contamination of the fresh anatomical cuts was carried out by immersion into inoculum containing Salmonella spp. The conditions of the HPP treatment were: pressure 593 MPa, time 290 seconds, water treatment temperature 14°C. Surface and deep samples were performed post contamination (T0), end of the cold phase (T1), end of process (Tend), and after HPP treatment (postHPP) and Salmonella spp. Enumerated. The results of this study show a significant reduction of Salmonella spp. all through the production process (P<0.01) for all companies, followed by an additional reduction of bacterial counts due to HPP treatment (P<0.01), both in superficial and deep contaminations (P<0.01). The superficial overall reduction resulted of 1.58 to 5.04 log CFU/g during the production process. HPP treatment resulted in a significant (P<0.01) superficial and deep decrease in Salmonella spp. enumeration varying from 0.61 to 4.01 log and from 1.49 to 4.13 log. According to the data presented in this study, only the combined approach of coppa manufacturing process followed by HPP treatment always led to a 5-log reduction of Salmonella spp. required by USDA/FSIS guidelines.

In 2012-2013 Emilia-Romagna Region introduced a monitoring plan for Shiga toxin-producing Escherichia coli (STEC) in foodstuff. Six hundred eighty-nine meat samples and 273 fruit and vegetable products were analyzed according to ISOTS13136. Pre-enriched samples were tested by multiplex real time PCR targeting the virulence genes eae, stx1 and stx2. Stx2 positive samples were investigated for the presence of serogroup O104 associated gene. O103, O111, O145, O157, O26 associated genes were tested on samples positive for stx in association with eae gene. Isolation of E. coli strains was attempted from samples positive for serogroup-associated genes. Thirtyfour meat products (4.9%) resulted positive for stx1 and/or stx2 genes and 46 (6.7%) for stx1 and/or stx2 genes in association with eae gene. Forty-five (6.5%) samples resulted positive at least at one serogroup. Serogroup O103, O104, O111, O145, O157 and O26 genes were detected respectively in 1.3, 0.3, 0.1, 3.9, 2.9 and 2.5% samples; 0.6% samples resulted positive for STEC isolation (2 E. coli O103 and 2 E. coli O157). It is worth noting that STEC virulence genes were detected at high frequency (19%) in fresh pork meat sausages. Four (1.5%) vegetable samples were positive for stx1 and/or stx2 genes and 1 (0.4%) for stx1 and/or stx2 genes in association with eae gene; none resulted positive for the tested serogroups. Only a low number of samples positive by molecular methods were confirmed by cultural isolation. It is therefore of the uttermost importance for appropriate risk management, to be fully aware of the meaning of the analytical result.

During the years 2015-2016, 83 faecal samples were collected at slaughter from pigs reared in farms located in Central- Northern Italy. During the years 2014-2016 a total of 562 pork products [465 not-ready-to-eat (NRTE) and 97 ready-to-eat (RTE) products] were collected from retail outlets, large retailers and processing plants. The samples were analysed according to ISO TS 13136:2012. Out of 83 swine faecal samples, 77 (92.8%) resulted stx-positive by real time polymerase chain reaction (PCR), 5 stx2+ and 1 stx1+ Shiga toxin-producing Escherichia coli (STEC) strains were isolated. Among the 465 NRTE samples, 65 (14.0%) resulted stx-positive by real time PCR and 7 stx2+ STEC strains were isolated. The stx2 gene was detected more frequently than the stx1 gene both in faecal samples (90.4 vs 8.4%) and in NRTE pork products (13.3 vs 1.3%). All the RTE samples included in the analysis resulted stxnegative. Among the samples resulted positive for stx and eae genes, serogroup-associated genes were detected at high frequency: O26 resulted the most frequent in faecal samples (81.3%) and O145 in pork products (88.1%). The O157 serogroup resulted positive in 83.3 and 78.1% of pork products and faecal samples, respectively. Despite the frequent detection by real time PCR of genes indicating the possible presence of STEC strains belonging to the six serogroups, the bacteriological step did not confirm the isolation of any such strains.

Foodstuffs should not contain microorganisms or their toxins or metabolites in quantities suggesting an unacceptable risk for human health. The detection of food hazards in foods is performed by several tests that produce results dependent on the analytical method used: an analytical reference method, defined as standard, is associated with each microbiological criterion laid down in Regulation 2073/2005, but, analytical methods other than the reference ones, in particular more rapid methods, could be used. Combined screening methods performed by real time PCR are currently validated as alternative methods according to the ISO 16140:2003 and certified by the Association Française de Normalisation. However, the positive results obtained with these alternative methods, the investigated molecular relations that resulted positive have to be confirmed with cultural methods using the same enrichment media in which the molecular screening was performed. Since it is necessary to assess if these testing schemes provide equivalent guarantees of food safety, the aim of this retrospective study is to analyze the data collected, from 2012 to 2014, by Emilia Romagna Region in the field of Piano Regionale Alimenti (Food Regional Plan), during official controls monitoring food samples, of animal and other than animal origin. Records performed by combined methods of molecular screening of Salmonella spp., Listeria monocytogenes and thermophilic Campylobacter and cultural confirmation results were gathered together and the results were compared in order to assess the sensitivity of the methods. A total of 10.604 food samples were considered in this study: the comparison of the data revealed that the RT-PCR method detected Salmonella, L. monocytogenes, and thermophilic Campylobacter in 2.18, 3.85 and 3.73% of the samples, respectively, whereas by using cultural method these pathogens were isolated in 0.43, 1.57 and 1.57 % of samples, respectively. In spite of the use of the same enrichment broth, the real time PCR method disclosed a percentage of positive samples that were negative to cultural examination ranging between 20 and 43%, with a PCR/culture ratio between 2.37 to 5.00. In conclusion, the results of this study pose a doubt about the sensitivity of the official cultural methods regarding the isolation of the three investigated foodborne pathogens. Moreover this study may be a useful tool for Veterinary Authorities to assess appropriate sampling plans to control the risk relating to the consumption of contaminated foods.

Chemical treatments are commonly adopted for poultry house sanitation. In fact, ordinary floor disinfection is needed to deplete the pathogenic population (i.e. various species of bacteria and fungi) and reduce the risk of meat contamination. The increasing focus on the health of consumers and operators, as well as on food quality, has led farmers to consider alternative environmentally friendly methods. Research was carried out to set up a new machine for floor disinfection of poultry houses by open flame. The trials were run in controlled conditions in the laboratory of the University of Pisa, Italy, and on a private farm. The first experiment consisted of a series of test bench trials carried out to evaluate the efficacy and the adjustment of liquefied petroleum gas (LPG)-fed open flame burners on pre-inoculated steel plates. In the second experiment, the operative parameters of a custom-built 1.5 m wide mounted flaming machine were determined and the biological effects of the treatment were compared to ordinary chemical treatments. The results obtained were very promising. Test bench trials showed a 4-log reduction in E. coli, and microbial determinations carried out on-farm did not show any difference between thermal and chemical treatment. In addition, the cost estimation showed that thermal disinfection is approximately 4-fold cheaper than chemical sanitation methods. The effective working capacity of the machine was approximately 1700 m2 h–1, and the LPG consumption was approximately 16 kg per 1000 m2. Flame disinfection of poultry grow-out facilities could represent a valid alternative to chemical disinfection.

The objective of this study was to alyse over time the evolution of E. coli O157:H7 faecal shedding in a dairy herd producing raw milk for direct human consumption. The study was performed between October 2012 and September 2013 in an average size Italian dairy farm where animals are housed inside the barn all over the year. The farm housed about 140 animals during the study – 70 cows and 70 calves and heifers. Twenty-six animals were randomly selected from both the cows and young animals group, and faecal sampling was performed rectally six times two months apart in each animal. Eleven animals were culled during the study and a total of 285 faecal samples were collected. At each faecal sampling, three trough water samples and two trough feed samples were also collected for a total of 36 water samples and 24 feed samples. Samples were alysed by real time polymerase chain reaction (RT-PCR) and culture. Overall, 16 (5.6%) faecal samples were positive for E. coli O157 by RT-PCR. Cultural examition found 9 (3.1%) samples positive for E. coli O157; all the isolates were positive for stx1, stx 2 and eae genes. One (4.1%) feed sample was positive for E. coli O157 by RT-PCR; none of the water samples was positive for E. coli O157. The model highlighted a general significant reduction of the number of positive samples observed during the study from the first to the sixth sampling (P=0.000) and a positive relation between the presence of positive samples and average environmental temperature (P=0.003). The results of the study showed that in an Italian dairy farm housing animals all year, faecal shedding of E. coli O157 followed the same temporal trend reported for other types of farming. The enhanced faecal shedding during warmer months may have a significant impact on environmental contamition and the safety of raw milk and its byproducts.