The vaginal microbiota of healthy female cats.

The vaginal microbiota of the queen (i.e., female cat) has never been described using culture independent methods. The objectives of the present research were to describe the vaginal microbiota of healthy domestic shorthair queens using both 16S rRNA sequencing and culture, and to assess the effects of age, living environment, and reproductive season on its composition. Thirty queens undergoing elective ovariectomy were included in the study. The vaginal samples were collected just before surgery, from animals under general anaesthesia. Two consecutive mini-swabs were introduced in the queens' vaginal tract. A preliminary study with 10 healthy queens aimed to negate sampling order's effect. Two consecutive samples for sequencing (5 queens, 10 swabs) and culture (5 queens, 10 swabs) were collected, confirming a match (100 % in culture, Bray-Curtis P = 0.96 in sequencing). The experiment included 20 queens that were prospectively grouped based on age (prepubertal N = 10, adult N = 10), living environment (indoor N = 10, outdoor N = 10), and time of the year, whether during the reproductive season (N = 10) or during seasonal anoestrous (N = 10). Bacteria were identified through metataxonomic analysis, amplifying the V1-V2 regions of 16S rRNA gene, and through standard culture followed by MALDI-TOF MS. The feline vaginal microbiota is dominated by Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteria. Escherichia-Shigella, Streptococcus, and Pasteurella were the most abundant genera. Although culture underestimated bacterial richness and diversity compared to sequencing, Escherichia and Streptococcus were the most isolated bacteria. No bacterial growth was observed in 15 % of samples (N = 3/20), whereas growth of one or two bacterial species was observed in 64.7 % (N = 11/17) and 35.3 % (N = 6/17) of cases, respectively. No differences in terms of alpha (Kruskal-Wallis rank sum test P = 0.65) and beta diversity (Bray-Curtis, Unweighted and Weighted UniFrac analyses P > 0.5) were observed. Although a difference in alpha diversity based on phylogenetic tree (P = 0.02) was detected between indoor and outdoor queens. In conclusion, mixed and monoculture of Escherichia coli, Streptococcus canis, Staphylococcus felis, and Enterococcus spp. are normal findings within the cat vagina. Age and reproductive season do not influence the feline vaginal microbiota, whereas further research is needed to elucidate the role of the living environment.

Challenging the Hypothesis of in Utero Microbiota Acquisition in Healthy Canine and Feline Pregnancies at Term: Preliminary Data.

At present, there are no data on the presence of bacteria in healthy canine and feline pregnancies at term. Here, we investigated the uterine microbiome in bitches (n = 5) and queens (n = 3) undergoing elective cesarean section in two facilities. Samples included swabs from the endometrium, amniotic fluid, and meconium, and environmental swabs of the surgical tray as controls. Culture and 16S rRNA gene sequencing were used to investigate the presence of bacteria. Culture was positive for 34.3% of samples (uterus n = 3, amniotic fluid n = 2, meconium n = 4, controls n = 0), mostly with low growth of common contaminant bacteria. With sequencing techniques, the bacterial abundance was significantly lower than in environmental controls (p < 0.05). Sequencing results showed a species-specific pattern, and significant differences between canine and feline bacterial populations were found at order, family, and genus level. No differences were found in alpha and beta diversities between feto-maternal tissues and controls (p > 0.05). Dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria in different proportions based on tissue and species. Culture and sequencing results suggest that the bacterial biomass is very low in healthy canine and feline pregnancies at term, that bacteria likely originate from contamination from the dam's skin, and that the presence of viable bacteria could not be confirmed most of the time.

Salmonella Enteritidis Fatal Septicemia with Meningoencephalitis in a Tiger (Panthera tigris) Cub.

A 15-day-old, female, captive Panthera tigris cub was hospitalized after developing severe hyperthermia, depression, and lack of appetite. The clinical condition rapidly worsened, and the tiger cub died in 72 h after the onset of neurological symptoms, septic shock, and multiple organ dysfunction syndrome. The postmortem main gross findings consisted of a severe and diffuse bilateral fibrino-suppurative meningoencephalitis and ventriculitis, mild fibrinous and sero-hemorrhagic polyserositis and cystitis, severe pulmonary edema, and hemorrhages. Microscopically, the meninges, ependyma, and choroid plexuses were diffusely expanded by abundant infiltration of neutrophils and macrophages, with multifocal fibrinous exudation. Histiocytic interstitial pneumonia, fibrinous and neutrophilic polyserositis, and pyelocystitis were also observed. Vascular thrombosis with multifocal vasculitis and vascular necrosis were frequently observed. Aerobic and anaerobic cultures performed on the brain, lungs, intestine, kidneys, and in pericardial effusion reported the presence of Salmonella enterica subsp. enterica serovar Enteritidis. Environmental and nutritional contamination were identified as putative sources of infections. To the best of the authors' knowledge, this is the first report of Salmonella Enteritidis septicemia with meningoencephalitis in a tiger cub, which highlights the need to further investigate the cause of acute perinatal death to reduce the risk of infectious disease outbreaks.

Case report: Infratentorial Embryonal Tumor with Abundant Neuropil and True Rosettes (ETANTR) in an 8-month-old Maine Coon.

An 8-month-old female Main Coon with a history of recurrent behavioral changes and anorexia was presented with sternal recumbency and depression. Within 5 days, the cat progressively worsened with symptoms of stupor and coma and was euthanized. At post-mortem examination, a solid, grayish infratentorial mass located in the midline rostrally to the cerebellum, was observed. Histologically, highly cellular clusters of small-to-medium undifferentiated cells were intermingled with paucicellular areas with fibrillary eosinophilic (neuropil-like) appearance. Numerous multilayered (ependymoblastic) true rosettes were present. The mitotic activity was frequent (up to 15 mitoses/HPF), involving both undifferentiated cells and rosettes. By immunohistochemistry (IHC), tumor cells were diffusely positive for vimentin, variably for synaptophysin, S-100, and NSE, and focally for NeuN; they were negative for GFAP and CK AE1/AE3. The histological and IHC aspects were consistent with an Embryonal Tumor with Abundant Neuropil and True Rosettes (ETANTR). Embryonal neoplasms of the central nervous system (CNS) are characterized by primitive undifferentiated cells, able to develop toward neuronal, glial, ependymal, and mesenchymal lines. Although extremely rare, juvenile embryonal tumors should be considered in the differentials of CNS disorders in young cats.

Anthroponotic-Based Transfer of Staphylococcus to Dog: A Case Study.

Although usually harmless, Staphylococcus spp. can cause nosocomial and community-onset skin and soft tissue infections in both humans and animals; thus, it is considered a significant burden for healthcare systems worldwide. Companion animals have been identified as potential reservoirs of pathogenic Staphylococcus with specific reference to Methicillin Resistant Staphylococcus aureus (MRSA). In this study, we investigated the circulation and the genetic relationships of a collection of Staphylococcus spp. isolates in a family composed of four adults (a mother, father, grandmother, and grandfather), one child, and a dog, which were sampled over three years. The routes of transmission among humans and between humans and the dog werelyzed. The results displayed the circulation of many Staphylococcus lineages, belonging to different species and sequence types (ST) and being related to both human and pet origins. However, among the observed host-switch events, one of them clearly underpinnthroponotic route from a human to a dog. This suggests that companion animals can potentially have a role as a carrier of Staphylococcus, thus posing a serious concern about MRSA spreading within human and animal microbial communities.

Molecular Investigation of Recent Canine Parvovirus-2 (CPV-2) in Italy Revealed Distinct Clustering.

Canine parvovirus Type 2 (CPV-2) is a worldwide distributed virus considered the major cause of viral gastroenteritis in dogs. Studies on Italian CPV-2 are restricted to viruses circulating until 2017. Only one study provided more updated information on CPV-2 but was limited to the Sicily region. No information regarding the circulation and genetic characteristics of CPV-2 in Northeast Italy has been made available since 2015. The present study investigated the genetic characteristics of CPV-2 circulating in the dog population of Northeast Italy between 2013 and 2019. The VP2 gene of 67 CPV-2 was sequenced, and phylogenetic analysis was performed to identify patterns of distribution. Phylogenetic and molecular analysis highlighted unique characteristics of Northeast Italian CPV-2 and interestingly depicted typical genetic clustering of the Italian CPV-2 strains, showing the existence of distinct CPV-2 genetic groups. Such analysis provided insights into the origin of some Italian CPV-2 genetic clusters, revealing potential introductions from East European countries and the spread of CPV-2 from South/Central to North Italy. This is the first report that describes the genetic characteristics of recent Italian CPV-2. Tracking the genetic characteristics of CPV-2 nationally and globally may have impact on understanding the evolution and distribution of CPV-2, in particular in light of the current humanitarian emergency involving Ukraine, with the massive and uncontrolled movement of people and pet animals.

The evolution of dam-litter microbial flora from birth to 60 days of age.

BACKGROUND: Early bacterial colonization in puppies is still a poorly understood phenomenon. Although the topic is of considerable interest, a big gap in knowledge still exists on the understanding of timing and features of neonatal gut colonization. Thence, the purpose of this study was to evaluate the relationship between dam and litter microbial flora, in vaginally delivered puppies, from birth to two months of age. Bacteria were identified using MALDI-TOF, an accurate and sensitive method, and cluster analysis of data provided a new insight on the investigated topic. METHODS: Six dam-litter units of two medium size breeds were enrolled in the study. Vaginal and colostrum/milk samples were collected from dams after delivery and 48h post-partum, while rectal samples were taken from dams and puppies after delivery and at day 2, 30 and 60 (T2, T30 and T60, respectively) post-partum. Bacterial isolation and identification were performed following standard techniques, then the data were analyzed using a new approach based on bacterial genus population composition obtained using a wide MALDI-TOF screening and cluster analysis. RESULTS: Forty-eight bacteriological samples were collected from the dams and 145 from their 42 puppies. Colostrum/milk samples (n = 12) showed a bacterial growth mainly limited to few colonies. Staphylococci, Enterococci, E. coli, Proteus spp. were most frequently isolated. All vaginal swabs (n = 12) resulted in bacteria isolation (medium to high growth). Streptococci, Enterococci, E. coli were the most frequently detected. E. coli, Proteus mirabilis, Enterococcus spp., Streptococcus spp. were often obtained from dams' and puppies' rectal swabs. Clostridia, not isolated in any other sampling site, were rarely found (n = 3) in meconium while they were more frequently isolated at later times (T2: n = 30; T30: n = 17; T60: n = 27). Analysis of the bacterial genus pattern over time showed a statistically significant reduction (P < 0.01) in the heterogeneity of microbial composition in all time points if compared to birth for each dam-litter unit. These results were confirmed with cluster analysis and two-dimensional scaling. CONCLUSION: This novel data analysis suggests a fundamental role of the individual dam in seeding and shaping the microbiome of the litter. Thus, modulating the dam's microbiota may positively impact the puppy microbiota and benefit their health.

A case report of a rapid development of fetal anasarca in a canine pregnancy at term.

A 5-year-old healthy pluriparous pregnant Flat-coated Retriever bitch was monitored by ultrasound on post-ovulation days 30 and 57: no deviation from normality picture were observed. On day 60, one of the three most caudal fetuses showed ultrasonographic signs of fetal anasarca: subcutaneous edema and anechoic fluid in thoracic and abdominal cavities. There was an increased volume of extra-fetal fluids. On day 64 a Cesarean section was performed and one of the seven pups that were delivered, a female, showed generalized subcutaneous edema and died soon after birth. She weighed 660 g, compared to a mean of 472 g for the other 6 normal fetuses. A total of 295, 40 and 27.5 mL of liquid were collected from subcutaneous tissue, abdominal and thoracic cavity, respectively. Liver showed sub-glissonian necrotic areas. Molecular analyses with PCR method for canine Herpesvirus, Parvovirus, Adenovirus, Leptospira interrogans, Chlamydia spp., Neospora caninum and Toxoplasma gondii from pools of organs (spleen, kidney and brain) and pleural effusion tested negative. This is the first reported case of fetal anasarca with a rapid onset diagnosed on day 60 post-ovulation just three days after observing a normal ultrasonographic pattern in Flat-coated Retriever. Ultrasonographic diagnosis of fetal anasarca is of primary importance when assisting parturition, due to its inherent risk of dystocia. Ultrasonographic monitoring in the immediate prepartum period may be useful in all breeds as it may help to detect ultrasonographic alterations occurring right before term such as anasarca.

Does Bacteria Colonization of Canine Newborns Start in the Uterus?

The assumption that requires the uterus to be a sterile environment to sustain a successful pregnancy has been recently challenged in humans, and is still under debate. The aim of this study was to assess whether bacteria can be isolated from the pregnant uterus and from amniotic fluid and meconium of healthy canine fetuses at term, delivered through cesarean section. Fifteen dams of different breed, age and parity, undergoing either elective (n = 10) or emergency (n = 5) cesarean section after a healthy pregnancy, were included in the study. Swabs for bacterial culture were collected from the uterus, and from amniotic fluid and meconium. Bacteria were isolated from all the sampled sites and materials, irrespective of cesarean type. In most cases, different bacteria were isolated from the different sites. Acinetobacter spp., coagulase-negative Staphylococci and Bacillus spp. were frequently found while Pseudomonas aeruginosa, Micrococcus spp., Moraxella spp., Macrococcus spp., Glutamicibacter spp., Stenotrophomonas spp. and Psychrobacter spp. were only occasionally identified. Our data show that uterus and fetuses may not be sterile in healthy term canine pregnancies.

Effect of sterilization on the canine vaginal microbiota: a pilot study.

BACKGROUND: Surgical sterilization is the most effective method of contraception for dogs. It also prevents pyometra and reduces the risk of mammary tumour development. However, this procedure also has negative effects, such as urinary incontinence. Steroid hormone deprivation following gonadectomy could also affect canine vaginal mucosa conditions and the microbial community colonizing the vaginal tract. This hypothesis was tested by comparing the vaginal cytology and microbial community of two groups of bitches, including 11 in anoestrus and 10 sterilized bitches (post-pubertal sterilization in the last 4 years). Bacteria were identified through metataxonomic analysis, amplifying the V3-V4 regions of 16S rRNA gene, and culturing methods. RESULTS: Vaginal mucosa cytology was suggestive of dystrophic conditions in sterilized bitches, whereas a typical anoestrus pattern with parabasal and intermediate cells was appreciable in anoestrous animals. Metataxonomic analysis revealed large inter-individual variability. Salmonella, Mycoplasma and Staphylococcus were present in moderate quantities in almost all the samples in both groups. Mollicutes (class level) and Tenericutes (phylum level) were commonly present in moderate quantities in anoestrus samples, whereas these microbes were present at high levels in a single sample from the sterilized group. Based on culturing, a higher number of different species were isolated from the anoestrous bitches, and Mycoplasma canis was exclusively identified in an anoestrous bitch. Staphylococcus spp. was the most frequently isolated genus in both groups, followed by Streptococcus spp., and, among gram-negative bacteria, Escherichia spp. and Haemophilus spp. A comparison of the numbers of the most frequently isolated genera of bacteria from vaginal cultures of bitches revealed that Pasteurella and Proteus were the most frequently identified in sterilized animals based on metataxonomic analysis (p-value = 0.0497 and 0.0382, respectively), whereas Streptococcus was significantly and most frequently isolated from anoestrous bitches using culture methods (p value = 0.0436). CONCLUSIONS: In this preliminary investigation, no global patterns of the vaginal bacteria community were noted that characterized the condition of the bitches; however, cytology suggested local modifications. Sterilization after puberty caused minimal alterations in the vaginal microbial community of bitches within 4 years after surgery.

Molecular detection of Pneumocystis in the lungs of cats.

The genus Pneumocystis comprises potential pathogens that reside normally in the lungs of a wide range of mammals. Although they generally behave as transient or permanent commensals, they can occasionally cause life-threatening pneumonia (Pneumocystis pneumonia; PCP) in immunosuppressed individuals. Several decades ago, the presence of Pneumocystis morphotypes (trophic forms and cysts) was described in the lungs of normal cats and cats with experimentally induced symptomatic PCP (after immunosuppression by corticosteroids); yet to date spontaneous or drug-induced PCP has not been described in the clinical feline literature, despite immunosuppression of cats by long-standing retrovirus infections or after kidney transplantation. In this study, we describe the presence of Pneumocystis DNA in the lungs of normal cats (that died of various unrelated causes; n = 84) using polymerase chain reactions (PCRs) targeting the mitochondrial small and large subunit ribosomal RNA gene (mtSSU rRNA and mtLSU rRNA). The presence of Pneumocystis DNA was confirmed by sequencing in 24/84 (29%) cats, with evidence of two different sequence types (or lineages). Phylogenetically, lineage1 (L1; 19 cats) and lineage 2 (L2; 5 cats) formed separate clades, clustering with Pneumocystis from domestic pigs (L1) and carnivores (L2), respectively. Results of the present study support the notion that cats can be colonized or subclinically infected by Pneumocystis, without histological evidence of damage to the pulmonary parenchyma referable to pneumocystosis. Pneumocystis seems most likely an innocuous pathogen of cats' lungs, but its possible role in the exacerbation of chronic pulmonary disorders or viral/bacterial coinfections should be considered further in a clinical setting.

Death Associated to Methicillin Resistant Staphylococcus aureus ST8 Infection in Two Dolphins Maintained Under Human Care, Italy.

The present study describes the isolation of Methicillin-resistant Staphylococcus aureus (MRSA) from respiratory tract of 2 dolphins of different origin, a stranded juvenile Risso's dolphin (Grampus griseus) and a captive born common bottlenose dolphin (Tursiops truncatus) calf, which died in the same institution at 1-month distance from the other. A complete microbiological and genetic investigation confirmed the presence of MRSA clone-complex 8, sequence type (ST) 8, spa-type t008 in both individuals. This strain differs from the one previously reported in walruses and dolphins and has never been described in dolphins before, but it is randomly isolated from Italian human patients. Vertical transmission of the infection may also occurs in other species and considering the description and location of the pathological lesions, this seems to be the most likely route of transmission implied in the young bottlenose dolphin. Staphylococcus aureus is known as an opportunistic agent, usually secondary to other pathogens, but its multiple antibiotic resistance and its zoonotic implications suggest a thorough and strict application of animal management hygiene protocols.

Occurrence and characterization of methicillin-resistant Staphylococcus pseudintermedius in successive parturitions of bitches and their puppies in two kennels in Italy.

BACKGROUND: Multi-drug methicillin-resistant Staphylococcus pseudintermedius (MRSP) detection is rapidly increasing in microbial specimens from pets across Europe. MRSP has also been isolated from bitches and newborns in dog breeding kennels. This study assessed whether MRSP lineage differs between breeding kennels and is maintained over time. Post-partum bitches (at day 3 vaginal and day 3, 9 and 35 milk samples) and their litters (at day 3, 9 and 35 oral and abdominal skin samples) from two Italian breeding kennels (A and B) were sampled and MRSP was subsequently characterized via whole-genome sequencing and antibiotic susceptibility testing. The study was carried out from October 2014 to March 2016 and included successive parturitions from the same animals. RESULTS: The analysis revealed different situations in both investigated kennels. In kennel A, circulating strains were from 7-locus sequence types ST688, ST258 and closely related isolates of ST71, which included most isolates. In kennel B, only a new isolate, ST772, was detected. In addition, most isolates from both kennels had multi-resistant antibiotic profiles. MRSP was only isolated from litters of MRSP-positive bitches, thus suggesting that bitch-litter transmission is likely. CONCLUSIONS: Our data show that MRSP circulation can differ in different settings, that several clonal lineages can circulate together, and that vertical transmission appears common. MRSP colonization did not affect the health conditions of the bitches or of their litters.

Real-time PCR assay for screening Pneumocystis in free-living wild squirrels and river rats in Italy.

We used a real-time PCR (rtPCR) targeting a 150-bp amplicon of the mitochondrial small subunit of ribosomal RNA (mtSSU rRNA) to screen for Pneumocystis DNA in lungs of wild squirrels ( Callosciurus finlaysonii, n = 85) and river rats ( Myocastor coypus, n = 43) in Italy. The rtPCR revealed Pneumocystis DNA in 20 of 85 (24%) squirrels and in 35 of 43 (81%) river rats, and was more sensitive than a nested PCR that targets a portion of the mtSSU rRNA and the mitochondrial large subunit of rRNA (mtLSU rRNA). Phylogenetic analysis based on mtSSU rRNA and mtLSU rRNA sequences showed distinct Pneumocystis sequence types in these rodents. The rtPCR assay should be reliable for screening large populations for this potential pathogen, thereby allowing cost-effective monitoring of the disease in wild animals.

In vitro assessment of quality of citrate-phosphate-dextrose-adenine-1 preserved feline blood collected by a commercial closed system.

BACKGROUND: Optimal procedure for storage of feline blood is needed. Open-collection systems have been employed in feline medicine, thus limiting the possibility for storage. OBJECTIVES: To evaluate indicators of quality of feline blood stored for 35 days at +4°C in a closed-collection system specifically designed for cats. ANIMALS: Eight healthy adult European domestic shorthair cats with a weight of 5-6.8 kg. METHODS: This is a case series study. A bacteriological test, CBC, blood smear, pH, osmotic fragility, 2,3-diphosphoglycerate (2,3-DPG), and adenosine triphosphate (ATP) measurement were performed weekly on whole blood (WB) units from day 1 to day 35 after donation. The hemolysis index, lactate and potassium concentrations, prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen were measured on plasma aliquots. RESULTS: One out of eight blood units (BUs) had bacterial growth (Serratia marcescens) at day 35. No significant differences were found regarding CBC, morphology, pH, and osmotic fragility. Despite high inter-individual variability and low starting levels, significant decreases in the mean concentrations of 2,3-DPG (T0 1.99 mmol/g Hb, SD 0.52, T35 1.25 mmol/g Hb, SD 1.43; P = .003) and ATP (T0 1.45 mmol/g Hb, SD 0.71, T35 0.62 mmol/g Hb, SD 0.51; P < .001) were detected during the study, as opposed to an increase in hemolysis (T0 0.11 mmol/L, SD 0.07, T35 0.84 mmol/L, SD 0.19; P < .001), lactate (T0 3.30 mmol/L, SD 0.86, T35 13.36 mmol/L, SD 2.90; P < .001), and potassium (T0 3.10 mmol/L, SD 0.21, T35 4.12 mmol/L, SD 0.35; P < .001) concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: The commercial BU kit is appropriate for blood collection and conservation of WB in cats. The maintenance of WB quality indicators during storage is essential for future improvements of feline transfusion medicine.

Characterization of Livestock-Associated Methicillin-Resistant Staphylococcus aureus CC398 and mecC-positive CC130 from Zoo Animals in the United Kingdom.

Little is known about the characteristics and diseases associated with methicillin-resistant Staphylococcus aureus (MRSA) in nondomestic animals. Four presumptive MRSA isolates, obtained from clinical (n = 3) and surveillance specimens (n = 1) from dwarf (Helogale parvula) and yellow mongooses (Cynictis penicillata) from a United Kingdom zoo, were analyzed by PCR for detection of mecA and mecC-mediated methicillin resistance, and virulence genes. Isolates were genotyped by multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) and spa sequence typing. Three isolates, obtained from the dwarf mongooses, carried mecA, tetK, and fexA resistance and virulence genes (icaA, icaD, and sec) and were typed to SCCmec IVa, spa type t899, and clonal complex (CC) 398. The fourth MRSA isolate, obtained from the femoral bone marrow of a yellow mongoose showing postmortem findings consistent with septicemia, carried mecC and was oxacillin/cefoxitin susceptible, when tested at 37°C but showed a characteristic MRSA susceptibility profile at 25°C ± 2°C. Furthermore, this isolate exhibited a different genetic background (SCCmecXI/t843/CC130) and had biofilm-associated genes (bap, icaA, and icaD) and tetK tetracycline resistance genes. This work describes the first isolation of livestock-associated MRSA CC398 from two zoo mongoose species where it was associated with both clinical disease and colonization, and the first isolation of mecC MRSA from a zoo species in the United Kingdom. Both reports highlight the potential for zoo species to act as reservoirs for these zoonotic agents.

Environmental surveillance identifies multiple introductions of MRSA CC398 in an Equine Veterinary Hospital in the UK, 2011-2016.

Bacterial environmental and surgical site infection (SSI) surveillance was implemented from 2011-2016 in a UK Equine Referral Veterinary Hospital and identified 81 methicillin-resistant Staphylococcus aureus (MRSA) isolates. A cluster of MRSA SSIs occurred in early 2016 with the isolates confirmed as ST398 by multilocus sequence typing (MLST), which prompted retrospective analysis of all MRSA isolates obtained from the environment (n = 62), SSIs (n = 13) and hand plates (n = 6) in the past five years. Sixty five of these isolates were typed to CC398 and a selection of these (n = 38) were further characterised for resistance and virulence genes, SCCmec and spa typing. Overall, MRSA was identified in 62/540 (11.5%) of environmental samples, 6/81 of the hand-plates (7.4%) and 13/208 of the SSIs (6.3%). spa t011 was the most frequent (24/38) and Based Upon Repeat Pattern (BURP) analysis identified spa t011 as one of the two group founders of the main spa CC identified across the five years (spa CC011/3423). However, 3 singletons (t073, t786, t064) were also identified suggesting separate introductions into the hospital environment. This long-term MRSA surveillance study revealed multiple introductions of MRSA CC398 in a UK Equine Hospital, identifying an emerging zoonotic pathogen so far only sporadically recorded in the UK.

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.

Anthrax phylogenetic structure in Northern Italy.

BACKGROUND: Anthrax has almost disappeared from mainland Europe, except for the Mediterranean region where cases are still reported. In Central and South Italy, anthrax is enzootic, but in the North there are currently no high risk areas, with only sporadic cases having been registered in the last few decades. Regional genetic and molecular characterizations of anthrax in these regions are still lacking. To investigate the potential molecular diversity of Bacillus anthracis in Northern Italy, canonical Single nucleotide polymorphism (canSNP) and Multilocus variable number tandem repeat analysis (MLVA) genotyping was performed against all isolates from animal outbreaks registered in the last twenty years in the region. FINDINGS: Six B. anthracis strains were analyzed. The canSNP analysis indicates the presence of three sublineages/subgroups each of which belong to one of the 12 worldwide CanSNP genotypes: B.Br.CNEVA (3 isolates), A.Br.005/006 (1 isolates) and A.008/009 (2 isolate). The latter is the dominant canSNP genotype in Italy. The 15-loci MLVA analysis revealed five different genotypes among the isolates. CONCLUSIONS: The major B branch and the A.Br.005/006 were recovered in the Northeast region. The genetic structure of anthrax discovered in this area differs from the rest of the country, suggesting the presence of a separate and independent B. anthracis molecular evolution niche. Although the isolates analyzed in this study are limited in quantity and representation, these results indicate that B. anthracis genetic diversity changes around the Alps.

Isolation of methicillin-resistant Staphylococcus pseudintermedius from breeding dogs.

The overuse of antimicrobials can select resistant bacteria strains; staphylococci have the ability to become resistant to all beta-lactam antimicrobials and are a significant concern in human medicine and a growing issue for veterinary medicine. Because antimicrobials are sometimes incorrectly used in breeding kennels, the objective of the work was to assess the occurrence of methicillin-resistant coagulase-positive staphylococci in breeding dogs. The research was carried out in 13 kennels that were allotted to three categories according to the intensity of antimicrobial use. Vaginal and milk swabs were taken from 87 healthy bitches around parturition and also from multiple organs of 27 of their pups that died within the first 2 weeks. Standard bacteriological examinations were carried out and coagulase-positive staphylococci were identified. All the coagulase-positive staphylococci resulted to be Staphylococcus pseudintermedius. Susceptibility to oxacillin and the presence of the mecA gene were tested. Nine out of 89 strains (six isolated from the bitches' milk and three from dead puppies, all belonging to kennels characterized by an excessive use of antimicrobials) were multidrug-resistant, methicillin-resistant and mecA positive. Our results confirm that excessive use of antimicrobials entails the risk of selecting resistant staphylococci strains. Our data also indicate that the bacterial flora of healthy dogs belonging to specific populations may act as a reservoir of resistance genes.