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Paratuberculosis of ruminants is characterised by chronic enteritis but, at advanced stages of the disease, a systemic dissemination of Mycobacterium avium subsp. paratuberculosis (MAP) in tissues and organs can occur. MAP has been recovered from lymph nodes and muscles of clinical and sub-clinical cows. In most countries, dairy and beef cattle infected with paratuberculosis are routinely sent to slaughter and the consumption of their meat could be a possible route of human exposure to MAP. However, few studies on MAP in ground beef are currently available. During the period November 2013–March 2014 we carried out a survey on the ground beef produced in an industrial meat processing plant. One-hundred and forty samples of ground meat were analysed by IS900-qPCR and culture (VersaTrek System®). The limit of detection (LOD) of qPCR was 630 MAP cells/g (107 CFU/g) while the LOD for culture was 170–230 MAP cells/g (62–115 CFU/g). No samples were positive by direct IS900 qPCR, while two samples were positive by liquid culture. Our data suggest that the presence of live MAP in raw minced meat is possible. In order to avoid exposure for humans through the consumption of contaminated meat, proper cooking of meat is recommended. [ABSTRACT FROM AUTHOR]

• Phylogenetic analysis disclosed the within-herd population structure of MAP. • Bayesian phylodynamic inference was feasible despite a low substitution rate. • Multiple isolates recovered from a single animal originate from different donors. Mycobacterium avium subsp. paratuberculosis (MAP) is characterized by a low genomic rate of mutation. Current subtyping tools, such as Mini-Micro-satellite analyses, do to have not sufficient discriminatory power to disclose MAP's evolution on small spatial and temporal scales. The aim of the study was to investigate the population structure of MAP inside a single dairy herd using whole genome sequencing (WGS) approaches. For this purpose, the genomes of 43 field isolates, recovered from the faeces of 36 cows of the same dairy herd from 2012 to 2016, were sequenced by WGS. The isolates' genomes showed a low number (43) of polymorphic sites (SNPs), confirming the clonal origin of the herd infection. However, despite the limited genomic diversity found in WGS, the phylogenetic analysis was discriminatory enough to detect the presence of different genomic clades and sub-clades inside the herd population. In addition, the phylodynamic reconstruction showed the existence of an ancestor clade from which the other clades and sub-clades originated. Moreover, by reconstructing the putative within-herd transmission networks using WGS data, we demonstrated that: (i) in a herd where MAP is endemic, multiple isolates recovered from a single animal and differing from each other by few (three/four) SNPs can originate from different transmission or passive shedding events and not from intra-host evolution; and (ii) variability of minisatellites coupled with a few microsatellites does not represent reliable tracers of within-herd infection chains. Our findings show that WGS, coupled with relevant epidemiological information, represents a valuable tool to work out fine epidemiological and micro-evolutionary relationships such as those at herd-level scale. [ABSTRACT FROM AUTHOR]

Objectives: We described the molecular characterization of an Enterobacter cloacae strain resistant to cefiderocol, co-harbouring bla IMP-19 and mcr-4.3 resistance genes METHODS: The strain was isolated from the rectal swab of a 77-year-old woman during the screening for the detection of patients colonized by carbapenemase-producing Enterobacterales (CPE), in Bologna (Italy). The strain was identified at the species level by MALDI-TOF mass spectrometry and the presence of IMP carbapenemase was confirmed both by a phenotypic immunochromatographic method, and by a rapid commercial NAAT. The strain underwent an antimicrobial susceptibility testing and its genome was fully characterized to assess (i) the Multi Locus Sequence Type (MLST), (ii) the presence of antimicrobial resistance genes (AMR), (iii) the presence of virulence genes coupled with Mobile Genetic Elements., Results: MLST identified the strain as ST837. A plasmid of 60'691 bp, very similar to MW574937 plasmid of Escherichia coli was identified and carried the following AMR genes: aac(6')-II,bla BEL-1 , bla IMP-19 , msr(E), mph(E), sul1. This plasmid harboured several tra gene (traB, traC, traD, traG, traI, traJ, traKtraL, traM) of the transfer operon of E. coli conjugative F plasmid. Other AMR genes were located in the chromosome (bla CMH-3 and fosA) or in other plasmids (bla SHV-12 and mcr-4.3). In vitro the strain showed resistance to many beta-lactams, including cefiderocol, whereas, despite the presence of mcr-4.3 gene, it was susceptible to colistin., Conclusions: Further studies are needed to better evaluate the origin of this strain, monitoring the presence of gram-negative bacilli with a similar molecular structure in our area., Competing Interests: Declaration of competing interest None declared., (Copyright © 2025. Published by Elsevier Ltd.)

Objectives: Infections by carbapenem-resistant Enterobacterales (CRE) in hospitals represent a severe threat but little is known on outbreaks in rehabilitation wards caused by Klebsiella pneumoniae producing Klebsiella pneumoniae Carbapenemase (KPC-Kp). We report an outbreak by KPC-Kp, in a neurorehabilitation unit in Italy, analysed through whole-genome sequencing (WGS) for transmission routes reconstruction to improve management of KPC-Kp infections in rehabilitation units., Methods: We investigated cases and KPC-Kp isolates collected from February to October 2022 from hospital surveillance. Carbapenem resistance was identified with disk diffusion and minimum inhibitory concentration tests, carbapenemase production through immunochromatographic lateral flow assays. All isolates were genotyped through WGS to highlight possible phylogenetic relationships. The most likely transmission networks of KPC-Kp were reconstructed with Bayesian discrete-time stochastic models., Results: Nineteen patients were colonized by KPC-Kp. Two isolates belonged to sporadic sequence types (STs; ST348 and ST219) whereas 9 of 19 and 8 of 19 isolates belonged to ST307 and ST716, respectively. Among the ST307 isolates, we identified two genomically distinct clusters of five and two cases. All ST716 isolates were highly similar. Genotyping and the reconstruction of transmission networks of KPC-Kp based on genomic data identified seven independent introductions into the neurorehabilitation unit rather than a single outbreak as initially hypothesized before genomic investigation. Three of the seven introductions generated chains of secondary infections, whereas the remaining four remained single cases., Conclusions: The outbreak root-causes were identified in temporary staff shortage and insufficient microbiological surveillance. Measures were adopted accordingly and the outbreak ended. The study highlights the critical role of genomic epidemiology in hospital outbreaks. © 2025 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy., (Copyright © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Salmonella is diffused worldwide, and Salmonella enterica subsp. enterica is spread worldwide with many serovars associated with the infection of domestic bovines. The most spread are S . Dublin, S . Typhimurium and S . Infantis. S. Napoli is, however very rarely reported in domestic ruminants. Here, we report an outbreak of S . Napoli on a dairy cow farm in Northern Italy (Piacenza). A total of 18 S . Napoli isolates were recovered from aborted fetuses, feces, tissues and environmental samples. Whole genome sequencing suggested that all isolates belonged to the same cluster. After the application of stringent biocontainment and biosecurity measures, no further cases were reported. However, four months after the first case, the serovar was still isolated in environmental samples, underlying the importance of adopting the correct biosecurity and biocontainment measures in order to prevent the circulation and transmission of Salmonella within the farm.

Human salmonellosis is a high-priority foodborne disease worldwide. The main reservoir of Salmonella is livestock, mainly swine and poultry that are infected both by generalist serovars and serovars adapted to them. The most widespread livestock-adapted serovars are attenuated in both their primary hosts and humans. We previously identified a lineage of the swine-adapted Salmonella Derby carrying mutations in Salmonella Pathogenicity Island-1(SPI-1) giving attenuation. To evaluate if SPI-1 decay is a general feature of swine-adapted serovars, we analyzed the Enterobase global population of Salmonella serovars most frequently isolated from swine. We found that deleterious mutations in SPI-1 are accumulated more in swine-adapted ( S . Derby and Salmonella Rissen) than in generalist ( Salmonella Typhimurium, I 1,4,[5],12:i:-, and Salmonella Infantis) serovars. The genomes carrying such mutations are widespread in the population of S . Derby and S . Rissen, indicating that the occurrence and fixation of deleterious mutations in SPI-1 are frequent. Similarly, also poultry-adapted Salmonella Kentucky has accumulated deleterious mutations in SPI-1, leading to over 70% of mutated genomes of this serovar compared with negligible proportions of mutated genomes in the generalist serovars from poultry. Although in vitro attenuation of S . Derby carrying mutated SPI-1 had been previously demonstrated, we showed the same phenotype for mutated S . Rissen. Considering also the known attenuation of S . Kentucky, our population-scale findings provide evidence of progressive evolution toward the attenuation of serovars adapted to the main animal reservoirs of human salmonellosis through the food chain. Intensive farming with a high density of animals could be a possible driver of this evolution.IMPORTANCEThis study shows at the global population level of Salmonella that the main attenuated serovars adapted to food-producing animals are undergoing convergent evolution toward further attenuation through the decay of SPI-1, considered critically important for the intestinal phase of Salmonella infection. The drivers of this evolution are unknown, but they could be attributed, at least in part, to the intensive farming of livestock with its high densities. On one side, our results contribute to the knowledge of the interaction between livestock populations and their host-adapted serovars of Salmonella . On the other side, the study provides scientific background for reconsidering the microbiological criteria adopted by the food safety legislation of many countries that ask for the absence of Salmonella in foods, regardless of any virulence evaluation of the detected strain. In this respect, the study provides molecular bases to investigate the virulence of different lineages within these host-adapted serovars.

Modular supramolecular complexes, where different proteins are assembled to gather targeting capability and photofunctional properties within the same structures, are of special interest for bacterial photodynamic inactivation, given their inherent biocompatibility and flexibility. We have recently proposed one such structure, exploiting the tetrameric bacterial protein streptavidin as the main building block, to target S. aureus protein A. To expand the palette of targets, we have linked biotinylated Concanavalin A, a sugar-binding protein, to a methylene blue-labelled streptavidin. By applying a combination of spectroscopy and microscopy, we demonstrate the binding of Concanavalin A to the walls of Gram-positive S. aureus and Gram-negative E. coli . Photoinactivation is observed for both bacterial strains in the low micromolar range, although the moderate affinity for the molecular targets and the low singlet oxygen yields limit the overall efficiency. Finally, we apply a maximum entropy method to the analysis of autocorrelation traces, which proves particularly useful when interpreting signals measured for diffusing systems heterogeneous in size, such as fluorescent species bound to bacteria.

The study assessed the role of equids at slaughter as faecal carriers of Salmonella enterica and the occurrence of contaminated equid carcasses during the slaughter process in Northern Italy (Emilia-Romagna Region). From June to November 2021, 152 equids (146 horses, 5 donkeys and 1 mule) were tested for Salmonella both in caecal contents and through carcass swabs. Antimicrobial resistance (AMR) of recovered strains was tested against 15 antimicrobials. Salmonella was detected in 3/152 of the caecal contents (2.0 %), while all carcass samples were negative. S. enterica serovars Enteriditis, Typhimurium and Stanleyville were identified. The only AMR isolate was S. Typhimurium with AMR profile AmCStxT. Considering the consumption of raw horse meat (i.e., minced raw meat named "pesto di cavallo" and dried and smoked strips named "sfilacci di cavallo") in different areas of Northern Italy, we also investigated the possible link between horse meat eating and salmonellosis cases in the human population in the same area. Specifically, we compared the Salmonella strains collected during the study with those routinely processed in the laboratory surveillance system for human salmonellosis in Emilia-Romagna (a region with about 4.5 million inhabitants). The comparison was based on whole genome sequencing data through core genome multi-locus sequence typing (cgMLST) used in routine surveillance. A genomic match in cgMLST was found between the strain of S. enterica serovar Enteritidis isolated from a horse caecal content and an enduring outbreak of 17 human cases in Emilia-Romagna during the study period. The consequent epidemiological investigation highlighted that a number of cases with known food history reported the consumption of horse meat and traced different batches of the consumed meat, released weeks apart from each other, to the slaughter investigated in the study. The results of the epidemiological investigation suggested the role of horses in the S. enterica serovar Enteritidis outbreak affecting raw horse meat consumers. This study shows that, despite the low prevalence on equid carcasses, S. enterica in horse meat can represent a risk to consumers. From the perspective of the slaughter activities, this highlights the need to maintain a high level of hygiene during the entire process, starting from the hygiene at lairage up to the slaughtering phase and dressing of carcasses., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Importance: This study integrated population data with in vitro assessment of virulence phenotypes to unveil that a considerable part of the global population of Salmonella Derby is evolving to enhance its host adaptation to the swine host and that this evolution is simultaneously increasing its attenuation for humans. The study shows that the fixation of deleterious mutations in SPI-1 has a role in this process. This evidence indicates that SPI-1 has a key role for S . Derby virulence in humans but not for its circulation in swine. The results show that genes generally considered essential for Salmonella pathogenesis do not play the same key role for all Salmonella serovars or lineages and/or all hosts. The study helps in understanding the molecular mechanisms underlying the ecology and host adaptation of Salmonella showing that the adaptation process can vary for different types of Salmonella and hosts., Competing Interests: The authors declare no conflict of interest.

We report Listeria monocytogenes infection in a patient in Italy who was transfused with pooled platelet concentrate. Genomic analysis revealed that L. monocytogenes isolates from the donor blood unit, the transfused platelets, and the patient's blood culture were genetically closely related, confirming transfusion transmission. Additional surveillance and secondary bacterial screening could improve transfusion safety.

Rare cases of Pseudomonas aeruginosa community-acquired pneumonia (PA-CAP) were reported in non-immunocompromised patients. We describe a case of Pseudomonas aeruginosa (PA) necrotizing cavitary CAP with a fatal outcome in a 53-year-old man previously infected with SARS-CoV-2, who was admitted for dyspnea, fever, cough, hemoptysis, acute respiratory failure and a right upper lobe opacification. Six hours after admission, despite effective antibiotic therapy, he experienced multi-organ failure and died. Autopsy confirmed necrotizing pneumonia with alveolar hemorrhage. Blood and bronchoalveolar lavage cultures were positive for PA serotype O:9 belonging to ST1184. The strain shares the same virulence factor profile with reference genome PA01. With the aim to better investigate the clinical and molecular characteristics of PA-CAP, we considered the literature of the last 13 years concerning this topic. The prevalence of hospitalized PA-CAP is about 4% and has a mortality rate of 33-66%. Smoking, alcohol abuse and contaminated fluid exposure were the recognized risk factors; most cases presented the same symptoms described above and needed intensive care. Co-infection of PA-influenza A is described, which is possibly caused by influenza-inducing respiratory epithelial cell dysfunction: the same pathophysiological mechanism could be assumed with SARS-CoV-2 infection. Considering the high rate of fatal outcomes, additional studies are needed to identify sources of infections and new risk factors, along with genetic and immunological features. Current CAP guidelines should be revised in light of these results.

A clinical strain of Klebsiella pneumoniae typed as sequence type 307 carrying three different alleles of the flu gene encoding the Escherichia coli virulence factor antigen 43 associated with biofilm formation was detected and characterized. The flu alleles are located in the chromosome inside putative integrative conjugative elements. The strain displays the phenotypes associated with Ag43, i.e. bi-phasic colony morphology and enhanced biofilm production. Furthermore, the strain produces low amount of capsule known to affect Ag43 function. Analysis of 1431 worldwide deposited genomes revealed that 3.7% Klebsiella pneumoniae carry one or two flu alleles., (© 2023. The Author(s).)

Wild boar (Sus scrofa) is the most widely distributed large wildlife mammal worldwide. To investigate the transmission of Salmonella enterica amongst wild boars (Sus scrofa), humans, and livestock, we compared via pulsed-field gel electrophoresis and whole genome sequences the isolates of S. enterica serovar Typhimurium (biphasic and monophasic variants) and Enteritidis collected from wild boars, food-producing animals, and human patients in Emilia-Romagna region (Northern Italy) between 2017 and 2020. Specifically, we analysed 2175 isolates originated from human (1832), swine (117), bovine (128), poultry (76), and wild boar (22). The genomic analyses showed that wild boars shared most of their lineages of biphasic Typhimurium with bovines and most of Enteritidis with poultry, whilst we did not find any lineage shared with swine. Moreover, almost 17% of human biphasic Typhimurium and Enteritidis belonged to genomic clusters including wild boar isolates, but the inclusion of bovine and poultry isolates in the same clusters and the peculiar spatial distribution of the isolates suggested that human cases (and wild boar infections) likely originated from bovines and poultry. Consequently, wild boars appear not to play a significant role in infecting humans with these serovars, but seem to get infected themselves from livestock, probably through the environment., (© 2023. EcoHealth Alliance.)

Background: Salmonella enterica is among the major burdens for public health at global level. Typing of salmonellae below the species level is fundamental for different purposes, but traditional methods are expensive, technically demanding, and time-consuming, and therefore limited to reference centers. Fourier transform infrared (FTIR) spectroscopy is an alternative method for bacterial typing, successfully applied for classification at different infra-species levels., Aim: This study aimed to address the challenge of subtyping Salmonella enterica at O-serogroup level by using FTIR spectroscopy. We applied machine learning to develop a novel approach for S. enterica typing, using the FTIR-based IR Biotyper® system (IRBT; Bruker Daltonics GmbH & Co. KG, Germany). We investigated a multicentric collection of isolates, and we compared the novel approach with classical serotyping-based and molecular methods., Methods: A total of 958 well characterized Salmonella isolates (25 serogroups, 138 serovars), collected in 11 different centers (in Europe and Japan), from clinical, environmental and food samples were included in this study and analyzed by IRBT. Infrared absorption spectra were acquired from water-ethanol bacterial suspensions, from culture isolates grown on seven different agar media. In the first part of the study, the discriminatory potential of the IRBT system was evaluated by comparison with reference typing method/s. In the second part of the study, the artificial intelligence capabilities of the IRBT software were applied to develop a classifier for Salmonella isolates at serogroup level. Different machine learning algorithms were investigated (artificial neural networks and support vector machine). A subset of 88 pre-characterized isolates (corresponding to 25 serogroups and 53 serovars) were included in the training set. The remaining 870 samples were used as validation set. The classifiers were evaluated in terms of accuracy, error rate and failed classification rate., Results: The classifier that provided the highest accuracy in the cross-validation was selected to be tested with four external testing sets. Considering all the testing sites, accuracy ranged from 97.0% to 99.2% for non-selective media, and from 94.7% to 96.4% for selective media., Conclusions: The IRBT system proved to be a very promising, user-friendly, and cost-effective tool for Salmonella typing at serogroup level. The application of machine learning algorithms proved to enable a novel approach for typing, which relies on automated analysis and result interpretation, and it is therefore free of potential human biases. The system demonstrated a high robustness and adaptability to routine workflows, without the need of highly trained personnel, and proving to be suitable to be applied with isolates grown on different agar media, both selective and unselective. Further tests with currently circulating clinical, food and environmental isolates would be necessary before implementing it as a potentially stand-alone standard method for routine use., Competing Interests: Declaration of Competing Interest O.J.L, F.G., S.L., M.A., M.P., S.Z., M.B., J.S., H.F., D.D., Y.F., Z.N., J.S., L.O., A.C.V., U.S.J., H.M.H., A.L., S.A., S.P., L.S., A.W., S.R., R.M.H., J.M. and A.B.P. declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. M.C., N.M. and M.K. are Bruker's employees., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: To date, whole genome sequencing has been performed mainly for isolates of Chlamydia trachomatis, C. pneumoniae, C. psittaci and C. abortus, but only a few isolates of C. pecorum have been entirely sequenced and this makes it difficult to understand its diversity and population structure. In this study the genome of two C. pecorum strains isolated from the lung of an Alpine chamois affected with pneumonia (isolate PV7855) and the brain of a water buffalo affected with meningoencephalomyelitis (isolate PV6959), were completely sequenced with MiSeq system (Illumina) and analyzed in their most polymorphic regions., Results: The genome length and GC content of the two isolates were found to be consistent with other C. pecorum isolates and the gene content of polymorphic membrane proteins and plasticity zone was found to be very similar. Some differences were observed in the phospholipase genes for both isolates and in the number of genes in the plasticity zone, such as the presence of some hypothetical proteins in PV6959, not present in any other genomes analyzed in this study. Interestingly, PV6959 possesses an extra pmp and has an incomplete tryptophan biosynthesis operon. Plasmids were detected in both isolates., Conclusions: Genome sequencing of the two C. pecorum strains did not reveal differences in length and GC content despite the origin from different animal species with different clinical disease. In the plasticity zone, the differences in the genes pattern might be related to the onset of specific symptoms or infection of specific hosts. The absence of a tryptophan biosynthesis pathway in PV6959 may suggest a strict relationship between C. pecorum and its host., (© 2022. The Author(s).)

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge for their capability to better adapt to the human host aimed and enhance human-to-human transmission. Mutations in spike largely contributed to adaptation. Viral persistence is a prerequisite for intra-host virus evolution, and this likely occurred in immunocompromised patients who allow intra-host long-term viral replication. The underlying mechanism leading to the emergence of variants during viral persistence in the immunocompromised host is still unknown. Here, we show the existence of an ensemble of minor mutants in the early biological samples obtained from an immunocompromised patient and their dynamic interplay with the master mutant during a persistent and productive long-term infection. In particular, after 222 days of active viral replication, the original master mutant, named MB61 0 , was replaced by a minor quasispecies (MB61 222 ) expressing two critical mutations in spike, namely Q493K and N501T. Isolation of the two viruses allowed us to show that MB61 222 entry into target cells occurred mainly by the fusion at the plasma membrane (PM), whereas endocytosis characterized the entry mechanism used by MB61 0 . Interestingly, coinfection of two human cell lines of different origin with the SARS-CoV-2 isolates highlighted the early and dramatic predominance of MB61 222 over MB61 0 replication. This finding may be explained by a faster replicative activity of MB61 222 as compared to MB61 0 as well as by the capability of MB61 222 to induce peculiar viral RNA-sensing mechanisms leading to an increased production of interferons (IFNs) and, in particular, of IFN-induced transmembrane protein 1 (IFITM1) and IFITM2. Indeed, it has been recently shown that IFITM2 is able to restrict SARS-CoV-2 entry occurring by endocytosis. In this regard, MB61 222 may escape the antiviral activity of IFITMs by using the PM fusion pathway for entry into the target cell, whereas MB61 0 cannot escape this host antiviral response during MB61 222 coinfection, since it has endocytosis as the main pathway of entry. Altogether, our data support the evidence of quasispecies fighting for host dominance by taking benefit from the cell machinery to restrict the productive infection of competitors in the viral ensemble. This finding may explain, at least in part, the extraordinary rapid worldwide turnover of VOCs that use the PM fusion pathway to enter into target cells over the original pandemic strain., (© The Author(s) 2022. Published by Oxford University Press.)

We report the development of a supramolecular structure endowed with photosensitizing properties and targeting capability for antimicrobial photodynamic inactivation. Our synthetic strategy uses the tetrameric bacterial protein streptavidin, labeled with the photosensitizer eosin, as the main building block. Biotinylated immunoglobulin G (IgG) from human serum, known to associate with Staphylococcus aureus protein A, was bound to the complex streptavidin-eosin. Fluorescence correlation spectroscopy and fluorescence microscopy demonstrate binding of the complex to S. aureus . Efficient photoinactivation is observed for S. aureus suspensions treated with IgG-streptavidin-eosin at concentrations higher than 0.5 μM and exposed to green light. The proposed strategy offers a flexible platform for targeting a variety of molecules and microbial species.

This case report describes for the first-time cases of severe gastroenteritis in water buffalo calves due to a new serovar of Salmonella enterica . The study was carried out on fecal matrix collected from live water buffalo calves that showed profuse diarrhea, severe dehydration and fever, exhibiting a systemic course. Culture and molecular investigations identified the pathogens isolated from intestinal contents as two Salmonella serovars, Salmonella enterica enterica O:35 and a new serovar of Salmonella enterica . The isolates showed multi-drug resistance. Timely diagnosis associated with a targeted antimicrobial treatment were found to be sufficient for the survival and recovery of the infected animals. Herd vaccines prepared from isolated pathogens were used to prevent further deaths of the calves.

This study aims to describe trends of mcr-positive Enterobacterales in humans based on laboratory surveillance with a defined catchment population. The data source is the Micro-RER surveillance system, established in Emilia-Romagna region (Italy), to monitor the trend of mcr resistance. Enterobacterales isolates from human clinical samples with minimum inhibitory concentration (MIC) ≥ 2 mg/L for colistin were sent to the study reference laboratory for the detection of mcr genes. Isolates prospectively collected in the period 2018-2020 were considered for the assessment of population rates and trends; further analyses were carried out for the evaluation of clonality and horizontal mcr gene transfer. Previous isolates from local laboratory collection were also described. In the period 2018-2020, 1164 isolates were sent to the reference laboratory, and 51 (4.4%) were confirmed as mcr-positive: 50 mcr-1 (42 Escherichia coli, 6 Klebsiella pneumoniae, 2 Salmonella enterica) and 1 mcr-4 (Enterobacter cloacae). The number of mcr-positive isolates dropped from 24 in the first half of 2018 to 3 in the whole of 2020 (trend p value < 0.001). Genomic analyses showed the predominant role of the horizontal transfer of mcr genes through plasmids or dissemination of transposable elements compared to clonal dissemination of mcr-positive microorganisms. The study results demonstrate a substantial decrease in the circulation of mcr-1 plasmid genes in Emilia-Romagna Region., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Typhoidal and para-typhoidal Salmonella are major causes of bacteraemia in resource-limited countries. Diagnostic alternatives to laborious and resource-demanding serotyping are essential. Fourier transform infrared spectroscopy (FTIRS) is a rapidly developing and simple bacterial typing technology. In this study, we assessed the discriminatory power of the FTIRS-based IR Biotyper (Bruker Daltonik GmbH, Bremen, Germany), for the rapid and reliable identification of biochemically confirmed typhoid and paratyphoid fever-associated Salmonella isolates. In total, 359 isolates, comprising 30 S . Typhi, 23 S . Paratyphi A, 23 S . Paratyphi B, and 7 S . Paratyphi C, respectively and other phylogenetically closely related Salmonella serovars belonging to the serogroups O:2, O:4, O:7 and O:9 were tested. The strains were derived from clinical, environmental and food samples collected at different European sites. Applying artificial neural networks, specific automated classifiers were built to discriminate typhoidal serovars from non-typhoidal serovars within each of the four serogroups. The accuracy of the classifiers was 99.9%, 87.0%, 99.5% and 99.0% for Salmonella Typhi, Salmonella Paratyphi A, B and Salmonella Paratyphi C, respectively. The IR Biotyper is a promising tool for fast and reliable detection of typhoidal Salmonella . Hence, IR biotyping may serve as a suitable alternative to conventional approaches for surveillance and diagnostic purposes.

BackgroundWhole genome sequencing (WGS) is increasingly used for pathogen identification and surveillance.AimWe evaluated costs and benefits of routine WGS through case studies at eight reference laboratories in Europe and the Americas which conduct pathogen surveillance for avian influenza (two laboratories), human influenza (one laboratory) and food-borne pathogens (five laboratories).MethodsThe evaluation focused on the institutional perspective, i.e. the 'investment case' for implementing WGS compared with conventional methods, based on costs and benefits during a defined reference period, mostly covering at least part of 2017. A break-even analysis estimated the number of cases of illness (for the example of Salmonella surveillance) that would need to be avoided through WGS in order to 'break even' on costs.ResultsOn a per-sample basis, WGS was between 1.2 and 4.3 times more expensive than routine conventional methods. However, WGS brought major benefits for pathogen identification and surveillance, substantially changing laboratory workflows, analytical processes and outbreaks detection and control. Between 0.2% and 1.1% (on average 0.7%) of reported salmonellosis cases would need to be prevented to break even with respect to the additional costs of WGS.ConclusionsEven at cost levels documented here, WGS provides a level of additional information that more than balances the additional costs if used effectively. The substantial cost differences for WGS between reference laboratories were due to economies of scale, degree of automation, sequencing technology used and institutional discounts for equipment and consumables, as well as the extent to which sequencers are used at full capacity.

Background: the co-production of carbapenemases and mcr -genes represents a worrisome event in the treatment of Enterobacteriaceae infections. The aim of the study was to characterize the genomic features of two clinical Enterobacter cloacae complex (ECC) isolates, co-producing VIM and MCR enzymes, in Italy., Methods: species identification and antibiotic susceptibility profiling were performed using MALDI-TOF and broth microdilution methods, respectively. Transferability of the bla VIM- and mcr - type genes was verified through conjugation experiment. Extracted DNA was sequenced using long reads sequencing technology on the Sequel I platform (PacBio)., Results: the first isolate showed clinical resistance against ertapenem yet was colistin susceptible (EUCAST 2020 breakpoints). The mcr-9.2 gene was harbored on a conjugative IncHI2 plasmid, while the bla VIM-1 determinant was harbored on a conjugative IncN plasmid. The second isolate, resistant to both carbapenems and colistin, harbored: mcr-9 gene and its two component regulatory genes for increased expression on the chromosome, mcr-4.3 on non-conjugative (yet co-transferable) ColE plasmid, and bla VIM-1 on a non-conjugative IncA plasmid., Conclusions: to our knowledge, this is the first report of co-production of VIM and MCR in ECC isolates in Italy.

Salmonella enterica variants exhibit diverse host adaptation, outcome of infection, and associated risk to food safety. Analysis of the distribution of Salmonella enterica serovar Derby (S. Derby) subtypes in human and swine identified isolates with a distinct PFGE profile that were significantly under-represented in human infections, consistent with further host adaptation to swine. Here we show that isolates with this PFGE profile form a distinct phylogenetic sub-clade within S. Derby and exhibit a profound reduction in invasion of human epithelial cells, and a relatively small reduction in swine epithelial cells. A single missense mutation in hilD, that encodes the master-regulator of the Salmonella Pathogenicity Island 1 (SPI-1), was present in the adapted lineage. The missense mutation resulted in a loss of function of HilD that accounted for reduced invasion in human epithelial cells. The relatively small impact of the mutation on interaction with swine cells was consistent with an alternative mechanism of invasion in this pathogen-host combination.

Paratuberculosis, a chronic disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), in ten scimitar-horned oryxes (SHOs) hosted in an Italian zoological park and originating from a Slovakian flock, was documented by pathology, molecular, cultural, and serological testing. The infection origin in this threatened species was also investigated by genomic analyses. Following the death of six of the 10 SHOs, serial investigations of dead and alive animals were performed. Necropsy, carried out on five out of six animals, identified intestinal thickening and mesenteric lymphadenomegaly in one of the animals. Histopathology (5/6) revealed lepromatous (2/5) and tuberculoid (2/5) intestinal forms or lack of lesions (1/5). Ziehl-Neelsen and immunohistochemistry stains identified two multibacillary, two paucibacillary forms, and one negative case. MAP was identified by quantitative PCR (qPCR) in tissue samples in five out of five SHOs and was microbiologically isolated from two of the three animals whose fresh tissue samples were available. Fecal samples were collected in four of the six dead animals: all four resulted positive to qPCR and in MAP was isolated in three. ELISA identified MAP-specific antibodies in three of the five dead animals whose serum was available. qPCR identified MAP in the freshly deposited feces of two out of the four alive animals. From the feces of these two animals, MAP was microbiologically isolated in one case. All isolates were classified as MAP type C and profiled as INMV2 and MVS27 by molecular analysis. Genomic analysis of a field isolate revealed clusterization with a European clade but was more similar to Italian than East European isolates. Our findings underline that paratuberculosis should always be considered in zoological parks in which endangered species are hosted. Infection can be subclinical, and multiple combined testing techniques may be necessary.

The population structure of human isolates of Listeria monocytogenes in Emilia-Romagna, Italy, from 2012 to 2018 was investigated with the aim of evaluating the presence of genomic clusters indicative of possible outbreaks, the proportion of cluster-associated vs. sporadic isolates and different methods and metrics of genomic analysis for use in routine surveillance. In the 2012-2018 period the notification rate of confirmed invasive cases in Emilia-Romagna was 0.91 per 100,000 population per year, more than twice the average rate of EU countries. Out of the total 283 cases, 268 (about 95%) isolates were typed through whole genome sequencing (WGS) for cluster detection with methods based on core-genome multi-locus sequence typing and single nucleotide polymorphisms. Between 66 and 72% of listeriosis cases belonged to genomic clusters which included up to 27 cases and lasted up to 5 years. This proportion of cluster-associated cases is higher than previously estimated in other European studies. Rarefaction analysis, performed by reducing both the number of consecutive years of surveillance considered and the proportion of isolates included in the analysis, suggested that the observed high proportion of cluster-associated cases can be ascribed to the long surveillance duration (7 years) and the high notification and typing rates of this study. Our findings show that a long temporal perspective and high surveillance intensity, intended as both exhaustiveness of the system to report cases and high WGS-typing rate, are critical for sensitive detection of possible outbreaks within a WGS-based surveillance of listeriosis. Furthermore, the power and complexity of WGS interpretation emerged from the integration of genomic and epidemiological information in the investigation of few past outbreaks within the study, indicating that the use of multiple approaches, including the analysis of the accessory genome, is needed to accurately elucidate the population dynamics of Listeria monocytogenes ., (Copyright © 2020 Scaltriti, Bolzoni, Vocale, Morganti, Menozzi, Re and Pongolini.)

In this report, the draft genome sequence of Listeria monocytogenes serovar 1/2a strain IZSAM&#95;Lm&#95;14-16064, isolated in Italy from a cooked ham, is announced. The genome is similar to that of a clinical strain isolated in 2014., (Copyright © 2020 Di Lollo et al.)

We report here the whole-genome sequence of a Chlamydia avium isolate recovered from a feral pigeon in 1999 in Italy. Only one complete genome of a C. avium strain has been published so far. Future comparative analyses could provide valuable insights on the genomic evolution of the pathogen., (Copyright © 2020 Floriano et al.)

Klebsiella pneumoniae (Kp) is one of the most important nosocomial pathogens worldwide, able to cause multiorgan infections and hospital outbreaks. One of the most widely disseminated lineage of Kp is the clonal group 258 (CG258), which includes the highly resistant "high-risk" sequence types ST258 and ST11. Genomic investigations revealed that very large recombination events have occurred during the emergence of Kp lineages. A striking example is provided by ST258, which has undergone a recombination event that replaced over 1 Mb of the genome with DNA from an unrelated Kp donor. Although several examples of this phenomenon have been documented in Kp and other bacterial species, the significance of these very large recombination events for the emergence of either hypervirulent or resistant clones remains unclear. Here, we present an analysis of 834 Kp genomes that provides data on the frequency of these very large recombination events (defined as those involving >100 kb), their distribution within the genome, and the dynamics of gene flow within the Kp population. We note that very large recombination events occur frequently, and in multiple lineages, and that the majority of recombinational exchanges are clustered within two overlapping genomic regions, which have been involved by recombination events with different frequencies. Our results also indicate that certain lineages are more likely to act as donors to CG258. Furthermore, comparison of gene content in CG258 and non-CG258 strains agrees with this pattern, suggesting that the success of a large recombination depends on gene composition in the exchanged genomic portion., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Acinetobacter baumannii is a known opportunistic pathogen, dangerous for public health mostly due to its ability to rapidly acquire antibiotic-resistance traits. Its genome was described as characterized by remarkable plasticity, with a high frequency of homologous recombinations and proliferation of Insertion Sequences (IS). The SMAL pulsotype is an A. baumannii strain currently isolated only in Italy, characterized by a low incidence and a high persistence over the years. In this present work, we have conducted a comparative genomic analysis on this clone. The genome of 15 SMAL isolates was obtained and characterized in comparison with 24 other assemblies of evolutionary related isolates. The phylogeny highlighted the presence of a monophyletic clade (named ST78A), which includes the SMAL isolates. ST78A isolates have a low rate of homologous recombination and low gene content variability when compared to two related clades (ST78B and ST49) and to the most common A. baumannii variants worldwide (International Clones I and II). Remarkably, genomes in the ST78A clade present a high number of IS, including classes mostly absent in the other related genomes. Among these IS, one copy of IS66 was found to interrupt the gene comEC/rec2 , involved in the acquisition of exogenous DNA. The genomic characterization of SMAL isolates shed light on the surprisingly low genomic plasticity and the high IS proliferation present in this strain. The interruption of the gene comEC / rec2 by an IS in the SMAL genomes brought us to formulate an evolutionary hypothesis according to which the proliferation of IS is slowing the acquisition of exogenous DNA, thus limiting genome plasticity. Such genomic architecture could explain the epidemiological behavior of high persistence and low incidence of the clone and provides an interesting framework to compare ST78 with the highly epidemic International Clones, characterized by high genomic plasticity., (Copyright © 2019 Gaiarsa, Bitar, Comandatore, Corbella, Piazza, Scaltriti, Villa, Postiglione, Marone, Nucleo, Pongolini, Migliavacca and Sassera.)

The study assessed Salmonella carriage in wild boars (Sus scrofa) and compared their isolates with those recovered from the domestic swine population of the same area of northern Italy (Emilia-Romagna), characterized by intensive pig farming and rather high density of wild boars. A total of 189 wild boars hunted during twelve months (2017-2018) were tested for Salmonella in mesenteric lymph nodes (MLN) and faecal samples. Antimicrobial resistance of recovered strains was tested against 14 antimicrobials. Salmonella was detected in 33/189 wild boars (17.5%), specifically from 30/189 MLN (15.9%) and 6/189 faecal samples (3.2%). Three animals were positive in both samples. Thirteen Salmonella serovars were identified, i.e. Typhimurium (the most common), Bovismorbificans, Coeln, Derby, Enteritidis, Gaminara, Hessarek, Houtenae IV, Kottbus, Napoli, Stanleyville, Thompson and Veneziana. Salmonella carriage was higher in warm than in cold months (P = 0.0013). Pregnancy status was never associated with Salmonella carriage, with significant difference in the recovery of the pathogen between non-pregnant and pregnant females (P = 0.003). Only one resistance pattern to streptomycin and tetracycline was found in 15 isolates (41.7%) belonging to Typhimurium (14/14; 100%) and Kottbus (1/3; 33.3%) serovars. Overlap with isolates from farmed pigs was limited at serotype level (Typhimurium, Derby, Enteritis, Bovismorbificans, Kottbus) and absent at PFGE level, and also antimicrobial resistance patterns were substantially different. This evidence indicates a substantial segregation of the two animal populations with regard to infectious contacts, possibly suggesting that biosecurity measures in place at industrial farm level limit the exchange of Salmonella.

Salmonella enterica serovar Napoli (serovar Napoli) is an emerging cause of human salmonellosis in Northern Italy. No specific reservoirs of serovar Napoli have been identified in Italy, so far. However, the environment, especially surface waters, has been hypothesized as an important source of infection based on the observation that genotypically different clusters of serovar Napoli are detected in different geographical macro-areas. To further support the hypothesis of a spatially-restricted pattern of serovar Napoli diffusion, a spatial segregation of serovar Napoli lineages should be observed also at smaller geographical scale. However, classical genotyping techniques used for Salmonella, such as pulsed-field gel electrophoresis (PFGE), did not possess enough discriminatory power to highlight spatial clustering of serovar Napoli within the macro-areas. To this purpose, we performed phylogeographical analyses based on genome-wide single nucleotide polymorphisms to test whether spatio-temporal evolution patterns of serovar Napoli in Northern Italy could be recognized with high geographical resolution, i.e. at local level. Specifically, we analyzed the local spread of the main PFGE clonal group, responsible for more than 60% of human infections in the study area, that did not show any geographical differentiation by PFGE within Northern Italy, i.e. the macro-area considered in the study. Both discrete and continuous phylogeography highlighted the existence of two main geographically-restricted clades: a Southern clade corresponding to the Po Valley and a Northern clade corresponding to the Pre-Alps area. Furthermore, the phylogeographical analyses suggested that the most probable site of origin of the clone was in an area of the Po Valley at the confluence of the Po and Ticino rivers, one of the most important Italian wetlands. These findings provide further support to the hypothesis that environmental transmission may play an important role in the ecology of serovar Napoli., Competing Interests: The authors have declared that no competing interests exist.

During 2014-2015, 300 pig carcasses before chilling and 85 food contact surfaces (FCSs) at cutting lines were tested for Salmonella in three slaughterhouses (namely A, B, and C) of northern Italy. In slaughterhouses A and B, four carcass sites of 100 cm 2 each (from both the exterior and interior side) were swabbed with a single sponge. In abattoir C, four 100 cm 2 sites of the exterior and the interior sides were swabbed with two independent sponges. The population average prevalence of Salmonella-positive carcasses (which takes into account the structure of the study design, with multiple samples collected in a single day) in slaughterhouses A and B was 12.3%, while in slaughterhouse C it was 11.2%. Presence of Salmonella on exterior and interior sides of carcasses showed a low level of concordance (only 3/12 of the contaminated carcasses were positive on both sides). No significant difference was found for FCSs contamination in the three slaughterhouses, with a population average prevalence of Salmonella-positive FCSs of 19.9%. In addition, we found that the clustering due to the day of sampling account for more than 36% and 60% of the overall prevalence variation on carcasses and FCSs, respectively. Eight serovars were identified, with Salmonella Derby as the most common type. The counting of Salmonella on carcasses showed large variability. It was low (<0.0075 most probable number [MPN]/cm 2 ) in 46.6% of the carcasses and as high as 2.7 MPN/cm 2 in 4.7%. Specifically, we found that counts on carcasses fit with "heavy tailed" distributions (lognormal and Weibull with a small shape parameter), suggesting not negligible probability of episodes of high Salmonella contamination. The mean values of contamination obtained with the two distributions ranged from 0.235 to 0.435 MPN/cm 2 .

Objectives: KPC-producing Klebsiella pneumoniae (KPC-Kp) represent a serious problem worldwide. Herein, we describe the evolution of ceftazidime/avibactam resistance by sequencing longitudinal clinical isolates from a patient with KPC-Kp bloodstream infection undergoing ceftazidime/avibactam treatment., Methods: WGS was performed on one ceftazidime/avibactam-susceptible KPC-Kp (BOT-CA-S) and two phenotypically different ceftazidime/avibactam-resistant KPC-Kp with low (BOT-CA-R) and high (BOT-EMO) carbapenem MICs. The population diversity was assessed by the frequency of allele mutations and population analysis profiles (PAPs)., Results: Phylogenetic analysis demonstrated clonal relatedness of the KPC-Kp isolates, all belonging to the clone ST1519. The D179Y mutation in blaKPC-3 was detected in both of the ceftazidime/avibactam-resistant KPC-Kp, whereas it was absent in the ceftazidime/avibactam-susceptible isolate. The mutation emerged independently in the two ceftazidime/avibactam-resistant isolates and was associated with a significant reduction in carbapenem MICs in BOT-CA-R, but not in BOT-EMO. WGS analysis revealed that the frequency of the D179Y mutation was 96.32% and 51.05% in BOT-CA-R and BOT-EMO, respectively. PAP results demonstrated that carbapenem resistance in BOT-EMO was due to the coexistence of mixed subpopulations harbouring WT and mutated blaKPC-3. A bacterial subpopulation with high ceftazidime/avibactam resistance for BOT-EMO KPC-Kp showed low carbapenem MICs, whereas a subpopulation with high meropenem resistance had a low MIC of ceftazidime/avibactam., Conclusions: Our analysis indicates that mixed subpopulations of ceftazidime/avibactam-resistant KPC-Kp emerge after ceftazidime/avibactam treatment. The evolution of different subpopulations that are highly resistant to ceftazidime/avibactam likely contributes to treatment failure, thereby highlighting the need for combination treatment strategies to limit selection of ceftazidime/avibactam-resistant KPC-Kp subpopulations.

Background and aimEpidemiology of human non-typhoid salmonellosis is characterised by recurrent emergence of new clones of the pathogen over time. Some clonal lines of Salmonella have shaped epidemiology of the disease at global level, as happened for serotype Enteritidis or, more recently, for Salmonella 4,[5],12:i:-, a monophasic variant of serotype Typhimurium. The same clonal behaviour is recognisable at sub-serotype level where single outbreaks or more generalised epidemics are attributable to defined clones. The aim of this study was to understand the dynamics of a clone of Salmonella 4,[5],12:i:- over a 3-year period (2012-15) in a province of Northern Italy where the clone caused a large outbreak in 2013. Furthermore, the role of candidate outbreak sources was investigated and the accuracy of multilocus variable-number tandem repeat analysis (MLVA) was evaluated. Methods: we retrospectively investigated the outbreak through whole genome sequencing (WGS) and further monitored the outbreak clone for 2 years after its conclusion. Results: The study showed the transient nature of the clone in the population, possibly as a consequence of its occasional expansion in a food-processing facility. We demonstrated that important weaknesses characterise conventional typing methods applied to clonal pathogens such as Salmonella 4,[5],12:i:-, namely lack of accuracy for MLVA and inadequate resolution power for PFGE to be reliably used for clone tracking. Conclusions : The study provided evidence for the remarkable prevention potential of whole genome sequencing used as a routine tool in systems that integrate human, food and animal surveillance.

Background: Different phleboviruses are important pathogens for humans; most of these viruses are transmitted by sand flies. An increasing number of new phleboviruses have been reported over the past decade, especially in Mediterranean countries, mainly via their detection in sand flies., Results: At least five different phleboviruses co-circulated in sand flies that were collected in three sites in Emilia-Romagna (Italy) in the summer of 2013. The well-known Toscana virus (TOSV) was isolated; three new, closely related phleboviruses differing in their M segments and tentatively named Ponticelli I, Ponticelli II and Ponticelli III virus, respectively, were isolated; a fifth putative phlebovirus, related to the sand fly fever Naples phlebovirus species, was also detected. The co-circulation, in a restricted area, of three viruses characterized by different M segments, likely resulted from reassortment events. According to the phylogenetic analysis of complete genome sequences, the TOSV belongs to clade A, together with other Italian isolates, while the Ponticelli viruses fall within the Salehabad phlebovirus species., Conclusions: Results highlight an unexpected diversity of phleboviruses that co-circulate in the same area, suggesting that interactions likely occur amongst them, that can present challenges for their correct identification. The co-circulation of different phleboviruses appears to be common, and the bionomics of sand fly populations seem to play a relevant role. Such a complex situation emphasizes the need for detailed investigations of the biology of these viruses to better characterize their pathogenic potential for mammals, including humans.

An expert opinion elicitation, based on a modified Delphi technique, was organized to collect the opinion of 16 Italian veterinarians with the aim of conducting a hazard and a welfare promoter characterization for defining and weighing a list of management and housing factors potentially associated with negative or positive welfare outcomes in dairy cows kept in loose housing systems. In addition, the 16 experts judged a set of animal-based measures in order to rate them by appropriateness and by the level of animal pain and suffering due to the welfare consequences they measure. Veterinary experts were asked to score 52 hazards, 47 welfare promoters and 18 animal-based measures. Management and housing hazards, that were determined to be associated with a very high impact on the welfare of dairy cows, were mainly referred to lactating cows (absence of bedding material, presence of inadequate or slippery floor in walking areas, wrong design of the lying area), but also the use of harmful tools for animal handling and the lack of scheduled foot inspection, trimming and foot bathing reached very high impact scores. Management and housing welfare promoters dealing with optimal cow comfort around resting obtained the highest ratings, together with the presence of experienced and trained stockpersons, the implementation of an attentive animal grouping strategy and the control of temperature, humidity and ventilation. Considering animal-based measures, the highest ranking of importance was given to observation of lameness, records of mortality of adult cows and calves, observation of integument alterations and body condition scoring. This study has been the starting point for the development of the first Italian national protocol for the assessment of the welfare of dairy cows farmed in loose housing systems., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study we report the detection of the recently described mcr-4 gene in two human isolates of Salmonella enterica serovar Typhimurium. The strains were isolated from faecal samples of two Italian patients with gastroenteritis, collected in 2016. The identified mcr-4 genes (variant mcr-4.2 ) differed from the mcr-4 gene originally described in a Salmonella strain of swine origin from Italy. Salmonella species could represent a hidden reservoir for mcr genes.

Here we report the complete nucleotide sequence of a 49.257-bp IncL/M conjugative plasmid (pRAY) carrying the blaOXA-48 gene collected from a Klebsiella pneumoniae clinical strain isolated in Italy. The genetic environment of pRAY plasmid revealed that the blaOXA-48 gene was located within a Tn1999.2 transposon. The pRAY plasmid differed from blaOXA-48-harboring IncL/M plasmids by genetic context and size. Comparative analysis demonstrated that pRAY plasmid lacked a region of ~15 kb carrying genes encoding proteins involved in pilus assembly and plasmid conjugative apparatus.

Introduction: Listeria monocytogenes (Lm) is a bacterium widely distributed in nature and able to contaminate food processing environments, including those of dairy products. Lm is a primary public health issue, due to the very low infectious dose and the ability to produce severe outcomes, in particular in elderly, newborns, pregnant women and immunocompromised patients., Methods: In the period between April and July 2015, an increased number of cases of listeriosis was observed in the area of Pavia, Northern Italy. An epidemiological investigation identified a cheesemaking small organic farm as the possible origin of the outbreak. In this work we present the results of the retrospective epidemiological study that we performed using molecular biology and genomic epidemiology methods. The strains sampled from patients and those from the target farm's cheese were analyzed using PFGE and whole genome sequencing (WGS) based methods. The performed WGS based analyses included: a) in-silico MLST typing; b) SNPs calling and genetic distance evaluation; c) determination of the resistance and virulence genes profiles; d) SNPs based phylogenetic reconstruction., Results: Three of the patient strains and all the cheese strains resulted to belong to the same phylogenetic cluster, in Sequence Type 29. A further accurate SNPs analysis revealed that two of the three patient strains and all the cheese strains were highly similar (0.8 SNPs of average distance) and exhibited a higer distance from the third patient isolate (9.4 SNPs of average distance)., Discussion: Despite the global agreement among the results of the PFGE and WGS epidemiological studies, the latter approach agree with epidemiological data in indicating that one the patient strains could have originated from a different source. This result highlights that WGS methods can allow to better.

Usutu virus is an arbovirus closely related to West Nile virus (genus Flavivirus), which circulates between mosquitoes and wild birds. This virus has been increasingly reported in Europe, raising concerns for its possible pathogenic potential for wild birds and humans. This study reports the whole genome sequences of 15 strains of USUV, isolated between 2010 and 2014 from mosquitoes and wild birds in the course of West Nile virus surveillance in the Emilia-Romagna and Lombardy regions of Northern Italy. Both whole and 656 partial genome sequences, obtained from isolated viruses and field samples (mosquitoes and wild birds), were analyzed to describe the temporal and geographical spread of USUV in the surveyed area. The detected sequences belonged to two groups, with one circulating primarily in the northwestern part of the area and the other in the southeastern part. This pattern is likely the result of different routes of introduction from the North (over the Alps) and from the East, respectively. The phylogenetic analysis of obtained sequences and other European sequences demonstrated that the majority of European strains belonged to one main clade, while less common strains, mainly from Western Europe, fell in other two clades. This analysis strongly suggested an autochthonous evolution process of strains of the main clade from a common ancestor with an estimated time of arrival in Europe at the beginning of the 1990s. In addition to causing mass mortality in wild birds, Usutu virus can infect humans and can sporadically cause disease. These factors and the endemization of the Usutu virus in a large area of Europe, sustained by the obtained data, strongly support the need to adequately survey Usutu virus in areas in which its circulation is detected., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Infectious diseases in livestock can be transmitted through fomites: objects able to convey infectious agents. Between-farm spread of infections through fomites is mostly due to indirect contacts generated by on-farm visits of personnel that can carry pathogens on their clothes, equipment, or vehicles. However, data on farm visitors are often difficult to obtain because of the heterogeneity of their nature and privacy issues. Thus, models simulating disease spread between farms usually rely on strong assumptions about the contribution of indirect contacts on infection spread. By using data on veterinarian on-farm visits in a dairy farm system, we built a simple simulation model to assess the role of indirect contacts on epidemic dynamics compared to cattle movements (i.e. direct contacts). We showed that including in the simulation model only specific subsets of the information available on indirect contacts could lead to outputs widely different from those obtained with the full-information model. Then, we provided a simple preferential attachment algorithm based on the probability to observe consecutive on-farm visits from the same operator that allows overcoming the information gaps. Our results suggest the importance of detailed data and a deeper understanding of visit dynamics for the prevention and control of livestock diseases.

Animals' exchanges are considered the most effective route of between-farm infectious disease transmission. However, despite being often overlooked, the infection spread due to contaminated equipment, vehicles, or personnel proved to be important for several livestock epidemics. This study investigated the role of indirect contacts in a potential infection spread in the dairy farm network of the Province of Parma (Northern Italy). We built between-farm contact networks using data on cattle exchange (direct contacts), and on-farm visits by veterinarians (indirect contacts). We compared the features of the contact structures by using measures on static and temporal networks. We assessed the disease spreading potential of the direct and indirect network structures in the farm system by using data on the infection state of farms by paratuberculosis. Direct and indirect networks showed non-trivial differences with respect to connectivity, contact distribution, and super-spreaders identification. Furthermore, our analyses on paratuberculosis data suggested that the contributions of direct and indirect contacts on diseases spread are apparent at different spatial scales. Our results highlighted the potential role of indirect contacts in between-farm disease spread and underlined the need for a deeper understanding of these contacts to develop better strategies for prevention of livestock epidemics., Competing Interests: The authors have declared that no competing interests exist.

Consumption of milk and dairy products is considered one of the main routes of human exposure to Mycobacterium avium subsp. paratuberculosis (MAP). Quantitative data on MAP load in raw cows' milk are essential starting point for exposure assessment. Our study provides this information on a regional scale, estimating the load of MAP in bulk tank milk (BTM) produced in Emilia-Romagna region (Italy). The survey was carried out on 2934 BTM samples (88.6% of the farms herein present) using two different target sequences for qPCR (f57 and IS900). Data about the performances of both qPCRs are also reported, highlighting the superior sensitivity of IS900-qPCR. Seven hundred and eighty-nine samples tested MAP-positive (apparent prevalence 26.9%) by IS900 qPCR. However, only 90 of these samples were quantifiable by qPCR. The quantifiable samples contained a median load of 32.4 MAP cells mL(-1) (and maximum load of 1424 MAP cells mL(-1) ). This study has shown that a small proportion (3.1%) of BTM samples from Emilia-Romagna region contained MAP in excess of the limit of detection (1.5 × 10(1) MAP cells mL(-1) ), indicating low potential exposure for consumers if the milk subsequently undergoes pasteurization or if it is destined to typical hard cheese production., (© 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)