Association between ability to form biofilm and virulence factors of poultry extra-intestinal Campylobacter jejuni and Campylobacter coli.

Campylobacter species are known to be able to produce biofilm, which represents an ideal protective environment for the maintenance of such fragile bacteria. Since the genetic mechanisms promoting biofilm formation are still poorly understood, in this study we assessed the ability of C. jejuni (n = 7) and C. coli (n = 3) strains isolated from diseased poultry, and previously characterized by whole genome sequencing, to form biofilm. The in vitro analyses were carried out by using a microtiter based protocol including biofilm culturing and fixation, staining with crystal violet, and measurement of the optical density (OD570). The ability to form biofilm was categorized into four classes (no, weak, moderate, and strong producers). Potential correlations between OD570 and the presence/absence of virulence determinants were examined. The C. jejuni were classified as no (n = 3), weak (n = 2), and moderate (n = 2) biofilm producers; however, all possessed genes involved in chemotaxis, adhesion, and invasion to the host cells. No genes present exclusively in biofilm producers or in non-biofilm producers were identified. All C. coli were classified as weak producers and showed a similar set of virulence genes between each other. A trend of increased mean OD570 was observed in the presence of flaA and maf7 genes. No association between biofilm production classes and the explanatory variables considered was observed. The results of this study suggest that further investigations are needed to better identify and characterize the genetic determinants involved in extra-intestinal Campylobacter biofilm formation.

Genome Sequence of Campylobacter Strain 19-13652, Isolated from Breeding Pheasants.

We report the whole-genome sequence of a Campylobacter strain that was isolated from breeding pheasants presenting "bulgy eyes" in Italy. Traditional molecular typing methods did not return any reliable result. Whole-genome sequencing and sequence comparison with known genomes did not meet the criteria for assignment to an existing species.

Prevalence, Molecular Characterization and Antimicrobial Susceptibility of Clostridioides difficile Isolated from Pig Carcasses and Pork Products in Central Italy.

In the last decade, the incidence and severity of Clostridioides difficile infections (CDIs) in humans have been increasing and community-associated infections have been described. For these reasons, the interest in C. difficile in food and in food animals has increased, suggesting other possible sources of C. difficile acquisition. This study evaluated the presence of C. difficile on pig carcasses at the slaughterhouse and in pork products in Central Italy. The contamination rate on pig carcasses was 4/179 (2.3%). Regarding food samples, a total of 216 pork products were tested (74 raw meat preparations and 142 ready-to-eat food samples made by cured raw meat). The real-time PCR screening was positive for 1/74 raw meat preparation (1.35%) and for 1/142 ready-to-eat food samples (0.7%) C. difficile was isolated only from the raw meat preparation (pork sausage). All the isolated strains were toxigenic and susceptible to all the tested antibiotics. Strains isolated from carcass samples displayed A+B+CDTa+CDTb+ profile, were toxinotype IV and belonged to the same ribotype arbitrary named TV93, while the one isolated from food samples displayed A+B+CDTa-CDTb- profile and it was not possible to determine ribotype and toxinotype, because it was lost after freeze storage. It was concluded that the prevalence of C. difficile in the pork supply chain is very low.

Extensive Genome Exploration of Clostridium botulinum Group III Field Strains.

In animals, botulism is commonly sustained by botulinum neurotoxin C, D or their mosaic variants, which are produced by anaerobic bacteria included in Clostridium botulinum group III. In this study, a WGS has been applied to a large collection of C. botulinum group III field strains in order to expand the knowledge on these BoNT-producing Clostridia and to evaluate the potentiality of this method for epidemiological investigations. Sixty field strains were submitted to WGS, and the results were analyzed with respect to epidemiological information and compared to published sequences. The strains were isolated from biological or environmental samples collected in animal botulism outbreaks which occurred in Italy from 2007 to 2016. The new sequenced strains belonged to subspecific groups, some of which were already defined, while others were newly characterized, peculiar to Italian strains and contained genomic features not yet observed. This included, in particular, two new flicC types (VI and VII) and new plasmids which widen the known plasmidome of the species. The extensive genome exploration shown in this study improves the C. botulinum and related species classification scheme, enriching it with new strains of rare genotypes and permitting the highest grade of discrimination among strains for forensic and epidemiological applications.

Genomic analysis of extra-intestinal Campylobacter jejuni and Campylobacter coli isolated from commercial chickens.

Campylobacter jejuni and Campylobacter coli have commonly been considered harmless commensal inhabitants of the chicken gut; however, these Campylobacter spp. are known to be able to multiply in the gut and invade other tissues, negatively affecting host health and performance. In this study, fourteen Campylobacter spp. were isolated from chickens showing foci of necrosis on the liver surface resembling lesions observed in cases of avian vibrionic hepatitis/spotty liver disease. The whole genome sequences of the fourteen isolates were analysed and their virulomes compared to those of Campylobacter reference sequences, aiming to investigate the possible association between virulence genes and the observed pathological lesions. Nine C. jejuni and five C. coli were studied. These Campylobacter shared twelve virulence factors with other isolates originated from chicken livers and hosted a higher number of virulence-associated genes in comparison to the reference genomes, including genes encoding for factors involved in adherence to and invasion of the intestinal epithelial cells. Our findings seem to point out that these twelve common virulence-associated genes, together with the presence of a high number of virulence factors involved in adherence, invasion and motility, might be responsible for the extra-intestinal spread of our isolates and the colonization of parenchymatous tissues, possibly causing the pathological lesions observed.

Clostridioides difficile in Calves in Central Italy: Prevalence, Molecular Typing, Antimicrobial Susceptibility and Association with Antibiotic Administration.

The emergence of Clostridioides difficile as the main agent of antibiotic-associated diarrhoea has raised concerns about its potential zoonotic role in different animal species. The use of antimicrobials is a major risk factor for C. difficile infection. Here, we provide data on C. difficile infection in dairy and beef calves in Umbria, a region in central Italy. This cross-sectional study focuses on prevalence, risk factors, ribotypes, toxinotypes and antimicrobial resistance profiles of circulating ribotypes. A prevalence of 19.8% (CI95%, 12-27.6%) positive farms was estimated, and the prescription of penicillins on the farms was associated with C. difficile detection (OR = 5.58). Eleven different ribotypes were found, including the ST11 sublineages RT-126 and -078, which are also commonly reported in humans. Thirteen isolates out of 17 showed resistance to at least one of clindamycin, moxifloxacin, linezolid and vancomycin. Among them, multiple-drug resistance was observed in two isolates, belonging to RT-126. Furthermore, RT-126 isolates were positive for tetracycline resistance determinants, confirming that tetracycline resistance is widespread among ST11 isolates from cattle. The administration of penicillins increased the risk of C. difficile in calves: this, together with the recovery of multi-resistant strains, strongly suggests the need for minimising antibiotic misuse on cattle farms.

Detection of Active BoNT/C and D by EndoPep-MS Using MALDI Biotyper Instrument and Comparison with the Mouse Test Bioassay.

Botulinum neurotoxins (BoNTs) are among the most poisonous known biological substances, and therefore the availability of reliable, easy-to use tools for BoNT detection are important goals for food safety and human and animal health. The reference method for toxin detection and identification is the mouse bioassay (MBA). An EndoPep-MS method for BoNT differentiation has been developed based on mass spectrometry. We have validated and implemented the EndoPep-MS method on a Bruker MALDI Biotyper for the detection of BoNT/C and D serotypes. The method was extensively validated using experimentally and naturally contaminated samples comparing the results with those obtained with the MBA. Overall, the limit of detection (LoD) for both C and D toxins were less than or equal to two mouse lethal dose 50 (mLD50) per 500 µL for all tested matrices with the exception of feces spiked with BoNT/C which showed signals not-related to specific peptide fragments. Diagnostic sensitivity, specificity and positive predictive value were 100% (95% CI: 87.66-100%), 96.08% (95% CI: 86.54-99.52%), and 93.33% (95% CI: 78.25-98.20%), respectively, and accuracy was 97.47% (95% CI: 91.15-99.69%). In conclusion, the tests carried out showed that the EndoPep-MS method, initially developed using more powerful mass spectrometers, can be applied to the Bruker MALDI Biotyper instrument with excellent results including for detection of the proteolytic activity of BoNT/C, BoNT/D, BoNT/CD, and BoNT/DC toxins.

A Longitudinal Case Study on Dissemination of ST398 Methicillin-Resistant Staphylococcus aureus Within a Dairy Cow Herd.

The study was conducted to describe the dynamics of ST398 methicillin-resistant Staphylococcus aureus (MRSA) on a dairy herd in northeastern Italy. MRSA was first identified in this herd of 120 cows in 2016, after which the herd was sampled once every 3 months for 1 year (April 2016-May 2017). Samples collected included nasal swabs and milk samples from cows and nasal swabs from farmworkers. In addition, pen fencing and teat milk liners were swabbed and air samples from cow pens and the milking parlor were collected. All samples were tested for MRSA using a selective medium; positive isolates were confirmed by mecA PCR. A representative set of MRSA isolates was genotyped using spa typing and multilocus sequence typing. Overall, 34 (mean 23%, range 16-30%) milking cows were found harboring MRSA in the mammary gland and only 6 recovered from infection or colonization. The mean incidence rate was 14% (range 8-20%), mean cure rate was 23% (range 13-43%), and estimated basic reproduction number (R0) was 1.08. The average of positive quarters found was 35.1% and most of the positive quarters (82.4%) developed subclinical mastitis. The mean duration of MRSA colonization in quarters during the study was 247 days, but quarters affected by subclinical mastitis harbored MRSA for a longer time than healthy ones (285 days vs. 131 days). After the second sampling, the farmer segregated MRSA-positive cows from the uninfected cows and milked them last. Despite segregation, 25 newly infected or colonized cows were detected. MRSA isolates from cows, environment, and two farmworkers belonged to the same sequence type (ST398) and spa type (t034). This study highlights the ability of ST398 MRSA to cause a persistent infection of the mammary gland and to survive in the farm environment.

Survey, characterization and antimicrobial susceptibility of Clostridium difficile from marine bivalve shellfish of North Adriatic Sea.

Clostridium difficile is a major cause of infectious diarrhea associated to healthcare settings. Community-acquired infections are increasingly reported in the last decade and exposure other than to symptomatic patients rather to contaminated foods or animals is feasible. Occurrence of C. difficile in shellfish raises concern because spores can survive the cooking temperatures given that shellfish is often consumed poorly cooked or raw. Aim of our study was to investigate whether shellfish represents a reservoir of C. difficile human PCR-ribotypes (RTs). 702 shellfish samples of farmed and wild bivalve mollusc species were collected over the 2015-2017 period in North Adriatic Italian Sea to investigate contamination with C. difficile and characterize the isolates in terms of genotypic variability and antimicrobial resistance profile. C. difficile was detected in 16.9% (CI: 14.1%-19.8%) samples: 11.6% mussels and 23.2% clams. Compared to mussels, clams were significantly associated with detection of C. difficile (OR = 2.4, P < 0.01). Overall 113 C. difficile isolates were genotyped and 75 (66.4%) were toxigenic. Fifty-three different RTs were identified. 40.7% C. difficile isolates were among the RTs most commonly involved in human infection in Europe. The profile of antimicrobial susceptibility was determined by E-test; microbiological resistance was frequent against clindamycin (17%), erythromycin (23%), rifampicin (8.8%) and moxifloxacin (10.6%). All isolates were susceptible to metronidazole and one showed MIC > ECOFF for vancomycin. C. difficile strains showed high variety in RTs, most of them already detected in other animals or known as highly virulent and epidemic in humans. These results prompt towards investigating on specific risk mitigation measures against C. difficile and are preliminary for any source attribution and risk assessment study.

Contamination of hospital food with Clostridium difficile in Central Italy.

This study investigates the contamination of foods with Clostridium difficile in 3 hospitals of Central Italy. We used real-time PCR for tpi gene to analyse 350 food samples, including 296 cooked meals and 54 foods to be eaten raw. The molecular screening test was positive for 3 samples, but toxigenic C. difficile was isolated only in two of them. The prevalence in cooked food was 0,3% (1/296), while in uncooked processed foods was 1,9% (1/54). Data support the potential risk of food as a source of toxigenic C. difficile for hospitalized patients, but further investigations are needed.

Virulence gene profiles of rabbit enteropathogenic Escherichia coli strains isolated in Northern Italy.

The virulence gene profile of 26 rabbit enteropathogenic Escherichia coli strains, isolated from 17 colibacillosis outbreaks located in two regions of Northern Italy, was determined using an Echerichia coli virulence DNA microarray. All strains were classified according to their determined biotype, sero- and phylo-group. The distribution of virulence genes encoding for the Locus of enterocyte effacement (LEE), LEE type III secretion system (T3SS), non-LEE T3SS translocated proteins and adherence factors was also determined. All strains but one belonged to phylogroups A and B1. A prevalent association between the O103 serogroup with the rhamnose-negative phenotype (biotype 12 or 14) was found. The most prevalent LEE profile found in tested strains was ler/cesT/espA-1/espB-3/tir-1/eae(beta)/espD-2/escN/eprJ. All strains possessed either the adhesive factor rabbit-2 (afr/2) or the plasmid Rabbit adherence locus (ral) gene and 24 of them an additional individual or combined set of colonization factors efa1/lifA, lpfA and paa genes. Finally, the combined or single presence of a set of LEE and/or non-LEE effector proteins encoding genes, namely espG, cif, map and nle family genes, attested to the genetic potential of investigated strains to induce pathologic lesions to the host. The application of microarray-based technologies in assessing the genetic profile of rabbit E. coli is a reliable, cost-effective candidate for large scale investigations in monitoring programs aimed to survey the circulation of pathogenic strains within rabbit production units, their zoonotic genetic potential and to select E. coli strains eligible for vaccinal prophylaxis in fattening rabbit production.

Detection of Clostridium tetani Neurotoxins Inhibited In Vivo by Botulinum Antitoxin B: Potential for Misleading Mouse Test Results in Food Controls.

The presence of botulinum neurotoxin-producing Clostridia (BPC) in food sources is a public health concern. In favorable environmental conditions, BPC can produce botulinum neurotoxins (BoNTs) outside or inside the vertebrate host, leading to intoxications or toxico-infectious forms of botulism, respectively. BPC in food are almost invariably detected either by PCR protocols targeted at the known neurotoxin-encoding genes, or by the mouse test to assay for the presence of BoNTs in the supernatants of enrichment broths inoculated with the tested food sample. The sample is considered positive for BPC when the supernatant contains toxic substances that are lethal to mice, heat-labile and neutralized in vivo by appropriate polyclonal antibodies raised against purified BoNTs of different serotypes. Here, we report the detection in a food sample of a Clostridium tetani strain that produces tetanus neurotoxins (TeNTs) with the above-mentioned characteristics: lethal for mice, heat-labile and neutralized by botulinum antitoxin type B. Notably, neutralization occurred with two different commercially available type B antitoxins, but not with type A, C, D, E and F antitoxins. Although TeNT and BoNT fold very similarly, evidence that antitoxin B antiserum can neutralize the neurotoxic effect of TeNT in vivo has not been documented before. The presence of C. tetani strains in food can produce misleading results in BPC detection using the mouse test.

Application of MALDI-TOF MS for the subtyping of Arcobacter butzleri strains and comparison with their MLST and PFGE types.

For the first time, this study evaluated the use of MALDI-TOF as a typing tool for Arcobacter butzleri. A total of 104 A. butzleri strains isolated from different sources in an artisanal dairy plant in Italy were identified and typed using MALDI-TOF and compared with their multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) profiles found in previous studies. MALDI-TOF correctly identified all the isolates to species level. No clearly delineated clusters appeared on dendrograms based on either the complete spectra or the significant peaks, but nine clusters were defined using the cophenetic correlation. Interestingly, MALDI-TOF proved able to discriminate A. butzleri strains below species level, confirming its potential use for epidemiological surveys. As expected, the comparative analysis with PFGE and MLST showed that the discriminatory index was lower for MALDI-TOF but roughly comparable to sequence types and pulsotypes. MALDI-TOF appears to be a relatively low cost answer to the urgent need for more rapid, less expensive typing tools suitable for source attribution studies, readily allowing multiple typing methods to be combined. This study provides insights into MALDI-TOF as potential epidemiological tool. Its application in healthcare surveillance systems awaits further exploration to encourage interaction and convergence studies between primary care in humans and animal and food veterinary authorities as part of the One Health concept.

Longitudinal study on antimicrobial consumption and resistance in rabbit farming.

Reliable indicators of antimicrobial consumption (AMC) measured with harmonised data and supported by indicators for antimicrobial resistance (AMR) at herd level are necessary to target antimicrobial misuse in food-producing animals. AMC data in 2010-2015 in 32 Italian industrial rabbit holdings weighted with semester production and standardised with animal daily doses (ADDs) were collected. Herd-level AMR against eight antimicrobials was assessed in Escherichia coli, Enterococcus faecalis and Enterococcus hirae collected in 2014-2015. Escherichia coli were assessed for mcr-1 and mcr-2 genes. To produce 1 kg of live rabbit, a mean of 71.8 ADDs was used. Overall AMC reduced over time (P < 0.05) owing to lowering consumption of tetracyclines (P < 0.05) and colistin (P < 0.01), but consumption of quinolones (P < 0.05), bacitracin (P < 0.01) and sulfonamides (P = 0.017) increased. All except one indicator E. coli were wild-type for cefotaxime, whereas 97% displayed reduced susceptibility to tetracyclines, 89% to trimethoprim, 63% to enrofloxacin, 24% to chloramphenicol and 21% to colistin. mcr-1 was detected in 50/320 E. coli isolates from 15/32 holdings; mcr-2 was not detected in 58 isolates with colistin MIC ≥ 2 mg/L. All 305 enterococci were wild-type for ampicillin, ciprofloxacin and vancomycin and displayed reduced tetracycline susceptibility. The mean antimicrobial resistance index (ARI) was 0.5 for E. coli and 0.3 for enterococci. ARI was significantly correlated with AMC at herd level for enterococci (P = 0.008) but not E. coli where high ARI levels were found in a few holdings with low AMC.

Identification and characterization of Clostridium botulinum group III field strains by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Animal botulism is primarily due to botulinum neurotoxin (BoNT) types C, D or their chimeric variants C/D or D/C, produced by Clostridium botulinum group III, which appears to include the genetically indistinguishable Clostridium haemolyticum and Clostridium novyi. In the present study, we used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI TOF MS) to identify and characterize 81 BoNT-producing Clostridia isolated in 47 episodes of animal botulism. The instrument's default database, containing no entries for Clostridium botulinum, permitted reliable identification of 26 strains at the genus level. Although supplementation of the database with reference strains enhanced the instrument's ability to identify the neurotoxic strains at the genus level, resolution was not sufficient to recognize field strains at species level. Characterization by MALDI TOF confirmed the well-documented phenotypic and genetic differences between Clostridium botulinum strains of serotypes normally implicated in human botulism (A, B, E, F) and other Clostridium species able to produce BoNTs type C and D. The chimeric and non-chimeric field strains grouped separately. In particular, very low similarity was found between two non-chimeric type C field strains isolated in the same outbreak and the other field strains. This difference was comparable with the differences among the various Clostridia species included in the study. Characterization by MALDI TOF confirmed that BoNT-producing Clostridia isolated from animals are closely related and indistinguishable at the species level from Clostridium haemolyticum and Clostridium novyi reference strains. On the contrary, there seem to be substantial differences among chimeric and some non-chimeric type C strains.

Molecular characterization of Clostridium perfringens strains isolated from diseased turkeys in Italy.

One hundred and six Clostridium perfringens field strains, isolated from diseased turkeys in Italy between 2006 and 2015, were toxinotyped by polymerase chain reaction. Strains were derived from intestines (87), livers (17) and subcutaneous tissues (2). In addition to the four major toxins, strains were also screened for NetB toxin, enterotoxin and beta2 toxin encoding genes. The intestinal gross lesions of turkeys with enteric disorders were statistically studied with respect to the presence of C. perfringens beta2 toxin encoding gene and coccidia in the gut. All the isolates belonged to the toxinotype A and were netB negative. Enterotoxin (cpe) and beta2 toxin (cpb2) encoding genes were detected in two (2.63%) and 76 (71.69%) strains, respectively. Toxinotype results agree with the few published reports concerning the genetic characterization of C. perfringens of turkey origin. On the contrary, the presence of netB and cpb2 genes differs from the results of a previous study where these genes were detected respectively in 6.6% and in 0.5% of the tested strains. Necrotic enteritis in turkeys was not statistically correlated either to the presence of cpb2 gene, or to the synergistic effect operated by coccidia, even though a high percentage of birds with these protozoa in the gut showed necrotic enteritis lesions (64.29%).

Molecular characterization and antimicrobial susceptibility of Clostridium difficile isolated from rabbits raised for meat production.

Clostridium difficile is an important cause of enteric disease in humans and animals. Recent studies demonstrated a genetic overlap between C. difficile isolated from animals and humans suggesting animals as possible reservoir for human pathogenic strains. This study was a preliminary investigation on the occurrence of C. difficile in rabbits raised in industrial holdings for food production and aimed to characterise isolates and estimate their antimicrobial susceptibility. C. difficile isolates were characterized by toxin profiles, toxinotyping and PCR-ribotyping. The MICs of six antibiotics were determined using E-test. Between 2007 and 2013, 285 industrial holdings (representing 40% of the national census) submitted rabbits to our laboratory for diagnostic purposes, among these holdings, groups of three to five post-weaned rabbits were sampled once by convenience. 1279 samples of caecal content were collected. The overall isolation rate of C. difficile from the enteric specimen was 3% (38/1279), with no difference among animals affected or not by enteric disorders. Among isolates 66% (25/38) were toxigenic. Sixteen different PCR-ribotypes (RTs) were identified. Among the toxigenic strains RT-014/020, RT-078 and RT-012 were found in at least three rabbit holdings. According to the ECOFF threshold, 82% (31/38) C. difficile isolates displayed a reduced susceptibility to at least one and 18% (7/38) to three tested antimicrobials. Rabbits are colonized by heterogeneous C. difficile ribotypes many of which are commonly isolated in humans. One third of isolates displayed a reduced susceptibility to MTZ, the first choice antimicrobial for human CDI treatment. According to our findings rabbits are a potential source of C. difficile for humans.

Evidence for a natural humoral response in dairy cattle affected by persistent botulism sustained by non-chimeric type C strains.

Bovine botulism is a sporadic acute disease that usually causes catastrophic losses in the herds. The unusual clinical evolution of a persistent mild outbreak in a dairy herd, prompted us to characterize the neurotoxin gene profile of the strain involved and to evaluate whether seroconversion had occurred. Diagnosis was based on mild classical symptoms and was supported by PCR and bacteriological findings, which revealed the involvement of a non-mosaic type C strain. An in-house ELISA was developed to detect antibodies to botulinum neurotoxin type C and its performance was evaluated in a vaccination study. Fifty days after the index case, fecal and serum samples were collected from the 14 animals of the herd and screened for Clostridium botulinum and anti-botulinum neurotoxin antibodies type C, respectively. The in-house developed ELISA was also used to test 100 sera samples randomly collected from 20 herds. Strong ELISA reactions were observed in 3 convalescent and 5 asymptomatic animals involved in the studied outbreak. The ELISA-positive cows all tested positive for non-mosaic C. botulinum type C in the feces and the same strain was also detected in the alfalfa hay, suspected to be the carrier source. Ten out of the 100 randomly collected sera tested positive for anti-botulinum neurotoxin type C antibodies: 7 had borderline values and 3 from the same herd showed titers three times higher than the cut-off. We concluded that type C botulism in cattle may occur with variable severity and that prolonged exposure to sublethal doses of botulinum neurotoxin C may occur, resulting in detectable antibodies.

Isolation of coagulase-positive staphylococci from bitches' colostrum and milk and genetic typing of methicillin-resistant Staphylococcus pseudintermedius strains.

BACKGROUND: Among the coagulase-positive, potentially pathogenic staphylococci, Staphylococcus pseudintermedius has been frequently isolated from bitches' milk. This organism colonizes the mammary gland or causes infection, while S. aureus has been only occasionally reported. The objective of this study was to investigate the occurrence and persistence of coagulase-positive staphylococci in the colostrum and milk of postpartum bitches, either treated or untreated with antimicrobials, and to assess the incidence, antibiotic resistance profile and genetic type of the methicillin-resistant strains. On postpartum D1, D7 and D15, drops of secretion were collected from the mammary glands of 27 postpartum bitches, nine of which were treated with antimicrobials. Coagulase-positive staphylococci were identified, antimicrobial susceptibility and the presence of mecA were tested and the genetic profile of methicillin-resistant strains was assessed. RESULTS: Staphylococcus pseudintermedius was the only coagulase-positive staphylococcus isolated, and its presence was detected in 21 out of 27 bitches and in 66 out of 145 swabs. In a single bitch, it caused puerperal mastitis. In untreated bitches, the frequency of isolation was lower in colostrum than in milk. All of the isolates except one were resistant to at least three antimicrobial classes, while 14 out of 66 S. pseudintermedius strains were methicillin-resistant mecA positive (MRSP) and were isolated from eight bitches housed in the same breeding kennel. A significant association was found between antimicrobial treatment and the presence of MRSP. Six of the 12 typed isolates belonged to spa-type t02 carrying SCCmec II/III, and another six were non-typeable with spa carrying SCCmec IV. The t02-SCCmec II/III isolates were sequence type (ST) 71; four NT-SCCmec IV isolates were ST258 and two were ST369. PFGE showed that isolates from the same dog had identical band patterns, while isolates from different dogs had unique band patterns. MRSP strains showed multidrug resistance profiles. CONCLUSIONS: Our results confirm that S. pseudintermedius is the most frequently isolated coagulase-positive staphylococcus from bitches' milk. The isolation of several different strains of MRSP with different genetic characteristics in the same kennel and the fact that two of the strains belonged to a sequence type (ST) described for the first time are noteworthy findings.

Prevalence and risk factors associated with Clostridium difficile shedding in veal calves in Italy.

The aim of this study is to describe the prevalence and risk factors of Clostridium difficile shedding in six farms belonging to two companies in Northern Italy. Four hundred and twenty veal calves, randomly selected and individually identified, were sampled three times: at 0-16, 90-120, and 150 days after introduction. C. difficile was isolated at least once from 87 out of the 420 calves (20.7%). The prevalence of shedding was 20.24% at the first sampling and dropped to 0.72% at the second sampling. None of the samples obtained at 150 days tested positive. Sampling of cecal contents and carcass swabs at slaughter was stratified according to the herd of origin of the animals. C. difficile was never isolated at slaughter, excluding a prevalence higher than 3.5% on the basis of previous investigations. Therefore, in this work, the veal calf could not be confirmed as a potential source of C. difficile for the consumer. Eight different ribotypes (RT) have been described, but the vast majority of the isolates (87.8%) belonged to three ribotypes only: RT-078, RT-012 and RT-126, which are also among the most common of the ribotypes detected in humans in Europe. Most isolates, and all the RT-078 isolates, harbored genes coding for toxins A and B, the binary toxin, and showed a deletion in the gene encoding toxin C, suggesting that the veal calf was a reservoir for epidemic hyper-virulent strains. A correlation between age and shedding was found: the odds ratio (OR) ranged from 2.79 for 36-45 days of age to 4.57 for 13-28 days of age. The presence of diarrhea at first sampling was significantly associated with the recovery of C. difficile in feces (OR 3.26). A correlation was found between the administration of antimicrobials and shedding: an increased risk was shown when the number of antimicrobials used was higher than 4 (OR 4.02) or 5-6 (OR 5.83) or when polymyxin E or beta-lactams were administered.