Genomic and Antimicrobial Surveillance of Campylobacter Population in Italian Poultry.

Campylobacter is one of the most common foodborne diseases worldwide with increasing rates of antibiotic resistance. Most cases of campylobacteriosis can be traced back to the consumption of poultry meat. Despite many efforts to reduce contamination in farms and in slaughterhouses, the persistence of this pathogen in poultry products remains a problem. This study aimed to evaluate the genetic diversity and antibiotic resistance of 542 C. jejuni and C. coli in Italian poultry, in the framework of two National Monitoring Programs. Genomes were screened for antibiotic resistance, virulence determinants and contextualized within a global collection of C. jejuni. ST2116, ST2863 and ST 832 were the most prevalent and significantly associated with Italian poultry. A worrying increase in resistance to quinolones, fluoroquinolones and tetracycline was observed in C. jejuni, while an increased occurrence of multidrug resistant (MDR) strains and strains resistant to macrolides was detected in C. coli. Low resistance rates were found for aminoglycosides. Molecular resistance determinants were consistent with the phenotypic resistance for tetracycline and quinolones. In silico analysis revealed 119 genes associated with virulence factors, with a notably higher prevalence of some genes in ST2863 genomes. This study highlights the increased resistance to macrolides and the emergence of MDR strains for C. coli, the genetic basis of AMR and the predominance of two genotypes among Campylobacter strains isolated from the Italian poultry farms.

Assessing pig farm biosecurity measures for the control of Salmonella on European farms.

Salmonella spp. is a common zoonotic pathogen, causing gastrointestinal infections in people. Pigs and pig meat are a major source of infection. Although farm biosecurity is believed to be important for controlling Salmonella transmission, robust evidence is lacking on which measures are most effective. This study enrolled 250 pig farms across nine European countries. From each farm, 20 pooled faecal samples (or similar information) were collected and analysed for Salmonella presence. Based on the proportion of positive results, farms were categorised as at higher or lower Salmonella risk, and associations with variables from a comprehensive questionnaire investigated. Multivariable analysis indicated that farms were less likely to be in the higher-risk category if they had '<400 sows'; used rodent baits close to pig enclosures; isolated stay-behind (sick) pigs; did not answer that the hygiene lock/ anteroom was easy to clean; did not have a full perimeter fence; did apply downtime of at least 3 days between farrowing batches; and had fully slatted flooring in all fattener buildings. A principal components analysis assessed the sources of variation between farms, and correlation between variables. The study results suggest simple control measures that could be prioritised on European pig farms to control Salmonella.

Cross-sectional study of hepatitis E virus (HEV) circulation in Italian pig farms.

Foodborne transmission is considered the main way of spreading zoonotic hepatitis E virus (HEV) infection in Europe. In recent years, the human cases of hepatitis E in subjects without history of travel in endemic areas have raised, suggesting that domestic HEV transmission is increasing. Pork products with or without liver, are often indicated as the source of many human foodborne HEV cases as well as small outbreaks. Pigs are recognized as the main reservoir of the zoonotic HEV-3 genotype, the most frequently detected in human cases in the EU. In the absence of a harmonized surveillance of HEV circulation, data on prevalence are heterogeneous but confirm a widespread circulation of HEV-3 in pig herds across EU. HEV-3 can pass through the food chain from farm to fork when infected animals are slaughtered. In Italy, several studies reported the circulation of HEV-3 in pig farms, but results are heterogeneous due to different methodologies applied. In the present study, we performed a survey over 51 pig herds belonging to three main types of farms: breeding, fattening and farrow-to-finish. HEV-RNA was analyzed by broad range Real-time RT-PCR on 20 samples for each farm, obtained by pooling together feces from 10 individuals. Overall, HEV RNA was confirmed on 150 fecal pooled samples out of 1,032 (14.5%). At least one positive pooled sample was detected from 18 farms out of 51 tested (35.3%). By lowering the number of infected pigs at primary production, the risk of HEV-3 entering into the food chain can be reduced. Hence, information on HEV circulation in herds is highly relevant for choosing preventive measures and deserves development of a monitoring program and further investigations.

Antibiotic resistance, plasmids, and virulence-associated markers in human strains of Campylobacter jejuni and Campylobacter coli isolated in Italy.

Campylobacteriosis, a prevalent foodborne gastrointestinal infection in Europe, is primarily caused by Campylobacter jejuni and Campylobacter coli, with rising global concerns over antimicrobial resistance in these species. This study comprehensively investigates 133 human-origin Campylobacter spp. strains (102 C. jejuni and 31 C. coli) collected in Italy from 2013 to 2021. The predominant Multilocus Sequence Typing Clonal complexes (CCs) were ST-21 CC and ST-206 CC in C. jejuni and ST-828 CC in C. coli. Ciprofloxacin and tetracycline resistance, mainly attributed to GyrA (T86I) mutation and tet(O) presence, were prevalent, while erythromycin resistance was associated with 23S rRNA gene mutation (A2075G), particularly in C. coli exhibiting multidrug-resistant pattern CipTE. Notable disparities in virulence factors among strains were observed, with C. jejuni exhibiting a higher abundance compared to C. coli. Notably, specific C. jejuni sequence types, including ST-21, ST-5018, and ST-1263, demonstrated significantly elevated counts of virulence genes. This finding underscores the significance of considering both the species and strain-level variations in virulence factor profiles, shedding light on potential differences in the pathogenicity and clinical outcomes associated with distinct C. jejuni lineages. Campylobacter spp. plasmids were classified into three groups comprising pVir-like and pTet-like plasmids families, exhibiting diversity among Campylobacter spp. The study underscores the importance of early detection through Whole Genome Sequencing to identify potential emergent virulence, resistance/virulence plasmids, and new antimicrobial resistance markers. This approach provides actionable public health data, supporting the development of robust surveillance programs in Italy.

The Current Landscape of Antibiotic Resistance of Salmonella Infantis in Italy: The Expansion of Extended-Spectrum Beta-Lactamase Producers on a Local Scale.

Salmonella enterica serovar Infantis is one of the five main causes of human salmonellosis in the European Union (EU) and in recent years, has been increasingly reported to carry multiple antimicrobial resistance determinants, including extended-spectrum beta-lactamase (ESBL) genes. In our study, we used WGS-based tools to characterize S. Infantis strains circulating in the Abruzzo and Molise regions of Italy between 2017 and 2020 and compared this local dataset to the S. Infantis population present in Italy over the last two decades. Phylogenetic analyses demonstrated that the majority of strains isolated from poultry and turkeys from Abruzzo and Molise were closely related and belonged to one of the two main genetic clusters present in Italy, which were grouped predominantly as ESBL-producing strains that harbored pESI-like plasmid. We showed that 60% of the local strains carried multiple antibiotic resistance genes, including ESBL gene bla CTX-M-1 as well as aadA1, dfrA1, dfrA14, sul1, and tet(A) genes present on the pESI-like megaplasmid. The analysis of strains from Abruzzo and Molise and the publicly available Italian S. Infantis sequences revealed a dramatic increase in the number of identified AMR genes in the strains isolated after 2011. Moreover, the number of strains resistant to five or more antibiotic classes increased from 20-80% in the last decade likely due to the acquisition of the megaplasmid. The persistence of the ESBL-producing and the multidrug-resistant (MDR) clone of S. Infantis in poultry populations in Italy and in Europe requires rapid and efficient intervention strategies to prevent further expansion of the clone.

Distribution of Salmonella spp. Serotypes Isolated from Poultry in Abruzzo and Molise Regions (Italy) during a 6-Year Period.

Human salmonellosis incidence is increasing in the European Union (EU). Salmonellaenterica subsp. enterica serovar Enteriditis, Salmonellaenterica subsp. enterica serovar Typhimurium (including its monophasic variant) and Salmonellaenterica subsp. enterica serovar Infantis represent targets in control programs due to their frequent association with human cases. This study aimed to detect the most prevalent serotypes circulating in Abruzzo and Molise Regions between 2015 and 2020 in the framework of the Italian National Control Program for Salmonellosis in Poultry (PNCS)]. A total of 332 flocks of Abruzzo and Molise Regions were sampled by veterinary services in the period considered, and 2791 samples were taken. Samples were represented by faeces and dust from different categories of poultry flocks: laying hens (n = 284), broilers (n = 998), breeding chickens (n = 1353) and breeding or fattening turkeys (n = 156). Breeding and fattening turkeys had the highest rate of samples positive for Salmonella spp. (52.6%; C.I. 44.8%-60.3%). Faeces recovered through boot socks represented the greatest number of positive samples (18.2%). Salmonellaenterica subsp. enterica serovar Infantis was the prevalent serotype in breeding and fattening turkeys (32.7%; C.I. 25.8%-40.4%) and in broiler flocks (16.5%; C.I. 14.4%-19.0%). Salmonellaenterica subsp. enterica serovar Typhimurium was detected at low levels in laying hens (0.7%; C.I. 0.2%-2.5%) followed by breeding and fattening turkeys (0.6%; C.I. 0.2%-2.5%). Salmonellaenterica subsp. enterica serovar Enteriditis was also detected at low levels in laying hens (2.5%; C.I. 1.2%-5.0%). These findings highlight the role of broilers and breeding/fattening turkeys as reservoirs of Salmonella spp. and, as a consequence, in the diffusion of dangerous serotypes as Salmonellaenterica subsp. enterica serovar Infantis. This information could help veterinary services to analyze local trends and to take decisions not only based on indications from national control programs, but also based on real situations at farms in their own competence areas.

Characterization of Salmonella Typhimurium and monophasic Salmonella Typhimurium isolated in Abruzzo and Molise regions, Italy, from 2012 to 2017.

Salmonellosis is currently the second most common zoonosis in European Union but in the 6-years periods, between 2012 and 2017, there has been a significant decrease trend in the yearly number of infections caused by Salmonella. In Italy, S. Typhimurium and monophasic S. Typhimurium represent the most prevalent serotypes. In this paper, we investigated these two serovars isolated from 2012 to 2017 in Abruzzo and Molise regions. A set of 345 strains isolated from human sporadic cases, surface water, food and animals were collected and analyzed. Monophasic S. Typhimurium strains were found to be resistant to streptomycin, sulfisoxazole, ampicillin, tetracycline and nalidixic acid, while S. Typhimurium isolates showed high levels of resistance to tetracycline, sulfisoxazole and ampicillin. The 5-loci Multilocus Variable-Number Tandem Repeat Analysis (MLVA) identified 88 genotypes (GT), six of which were common for the two serovars. Several MLVA profiles were shared by human and non-human isolates. MLVA had sufficient typing resolution for epidemiological studies of S. Typhimurium but demonstrated poor discriminatory in trace-back study of monophasic S. Typhimurium.

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.

Genomic Comparison of Salmonella Enteritidis Strains Isolated from Laying Hens and Humans in the Abruzzi Region during 2018.

Salmonellosis is a major cause of bacterial foodborne infection. Since 2016, an increased number of cases of gastroenteritis caused by Salmonella enterica serovar Enteritidis linked to eggs produced in Poland has been reported in Europe. In Italy, S. Enteritidis is one of the three most commonly reported serotypes, associated mainly with the consumption of contaminated eggs and derived products. In our work, we analysed 61 strains of S. Enteritidis obtained from humans and farms in the Abruzzi region, Italy, in 2018. We used Multiple-Loci Variable-Number Tandem Repeat (VNTR) analysis (MLVA)-based typing and Whole-Genome Sequencing (WGS) tools to identify closely related strains and perform cluster analysis. We found two clusters of genetically similar strains. The first one was present in the local farms and isolated from human cases and had single-linkage distance of no more than two core genes and less than five Single-Nucleotide Polymorphisms (SNPs). The second cluster contained strains isolated from humans and from a dessert (tiramisù) sample that shared identical core genome and were assigned the same SNP address. Cluster 2 isolates were found to be genetically similar to an S. Enteritidis strain from a multi-country outbreak linked to Polish eggs.

Correction: Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry.

[This corrects the article DOI: 10.1371/journal.pone.0223804.].

Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry.

Campylobacter jejuni, a common foodborne zoonotic pathogen, causes gastroenteritis worldwide and is increasingly resistant to antibiotics. We aimed to investigate the antimicrobial resistance (AMR) genotypes of C. jejuni isolated from humans, poultry and birds from wild and urban Italian habitats to identify correlations between phenotypic and genotypic AMR in the isolates. Altogether, 644 C. jejuni isolates from humans (51), poultry (526) and wild- and urban-habitat birds (67) were analysed. The resistance phenotypes of the isolates were determined using the microdilution method with EUCAST breakpoints, and AMR-associated genes and single nucleotide polymorphisms were obtained from a publicly available database. Antimicrobial susceptibility testing showed that C. jejuni isolates from poultry and humans were highly resistant to ciprofloxacin (85.55% and 76.47%, respectively), nalidixic acid (75.48% and 74.51%, respectively) and tetracycline (67.87% and 49.02%, respectively). Fewer isolates from the wild- and urban-habitat birds were resistant to tetracycline (19.40%), fluoroquinolones (13.43%), and quinolone and streptomycin (10.45%). We retrieved seven AMR genes (tet (O), cmeA, cmeB, cmeC, cmeR, blaOXA-61 and blaOXA-184) and gyrA-associated point mutations. Two major B-lactam genes called blaOXA-61 and blaOXA-184 were prevalent at 62.93% and 82.08% in the poultry and the other bird groups, respectively. Strong correlations between genotypic and phenotypic resistance were found for fluoroquinolones and tetracycline. Compared with the farmed chickens, the incidence of AMR in the C. jejuni isolates from the other bird groups was low, confirming that the food-production birds are much more exposed to antimicrobials. The improper and overuse of antibiotics in the human population and in animal husbandry has resulted in an increase in antibiotic-resistant infections, particularly fluoroquinolone resistant ones. Better understanding of the AMR mechanisms in C. jejuni is necessary to develop new strategies for improving AMR programs and provide the most appropriate therapies to human and veterinary populations.

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.

Definition of a Standard Protocol to Determine the Growth Potential of Listeria Monotgenes and Yersinia Enterocolitica in Pork Sausage Produced in Abruzzo Region, Italy.

Pork meat products consumed raw or after a short period of fermentation can be considered at risk for food safety. Sausages (fresh sausage made from pork meat) are produced in several Italian regions, with variation in ingredients. In some Italian Regions, including Abruzzo, these products are frequently consumed raw or undercooked, after a variable period of fermentation. The European Community food regulation promotes the use of challenge tests to determine safety levels. This study is aimed to ensure safety of Abruzzo's sausages, compared with growth potential (δ) of Listeria monocytogenes and Yersinia enterocolitica, and also aims to define an experimental standard protocol document to carry out challenge tests. Guidelines classify ready-to-eat foods in categories that are able to support (δ>0.5 log10 ufc/g) and not support (δ≤0.5 log10 ufc/g) the growth of Listeria monocytogenes. The products were manufactured according to traditional recipes and were contaminated in laboratory. Results from the experiment yielded information useful to assess the ability of these products to support the growth of pathogenic microorganisms. The batches of sausages were stored at 8, 12, 18 and 20°C to get statistical evaluation. The results showed that, despite the conditioning of the storage temperature and the level of water activity, both organisms remain in the product in concentrations similar to those leading or being able to increase its charge. In particular, the period of greatest consumption of this product (7/8 days of preparation) corresponds to the period of greatest growth of pathogenic microorganisms studied, except for those stored at a temperature of 8°C, which are safer for the consumer.