Importance of broth-enrichment culture in equine endometritis diagnosis.

The objective of this study was to investigate the diagnostic accuracy of the standard microbiological protocol to assure the evaluation of bacterial endometritis in the equine clinical practice. Four hundred fifty-two equine uterine swabs were seeded on different types of agar plates and then in a broth-enrichment (Brain Heart Infusion Broth) before plating by using the same media the day after. The prevalence of positivity was 33.7% following direct plating and 66.3% following use of added enrichment-broth phase before seeding on solid media. Furthermore, the prevalence of isolated bacteria included E. coli (29.7%) and Streptococcus equi subsp. zooepidemicus (15.2%), both frequently associated with equine endometritis. Our results indicate that the addition enrichment-broth culture significantly increases the rate of positivity for the detection of bacteria in equine uterine swabs compared to the direct plating of samples alone. Thus, this diagnostic technique may be recommended to increase the sensitivity of bacteriological analysis in mares with endometritis.

Malassezia pachydermatis up-regulates AhR related CYP1A1 gene and epidermal barrier markers in human keratinocytes.

Cytochrome P450 CYP1A1 and CYP1B1 enzymes are regulated by the aryl hydrocarbon receptor (AhR), a transcription factor activated by a variety of ligands among which Malassezia metabolites. In this study, we analyzed the modulation of CYP1A1, CYP1B1, and AhR in human keratinocytes infected with different strains of Malassezia pachydermatis, as well as the upregulation of some genes involved in the epidermal homeostasis. We demonstrated that all the strains induced AhR activation and its nuclear translocation in HaCaT cells infected for 24 h, compared to untreated cells. The expression of CYP1A1 and CYP1B1, prototypical markers of the AhR signaling pathway, were upregulated with the level of CYP1A1 mRNA approximately 100-fold greater than that for CYP1B1. Filaggrin, involucrin, and TGaseI, proteins involved in epidermal differentiation, were all modulated by Malassezia pachydermatis strains, with the strongest induction observed for filaggrin. By contrast, quinone oxidoreductase 1 (NQO1), which is part of the antioxidant defense system involved in detoxification, was not modulated in our experimental model. In conclusions, our findings suggest that Malassezia pachydermatis infection of human keratinocytes induces activation of the AhR, and increases the expression of its responsive genes and markers of epidermal differentiation, paving the way for occurrence/exacerbation of pathological skin conditions.

Ultrastructural alterations of pancreatic beta cells in human diabetes mellitus.

BACKGROUND: Both types of diabetes are characterized by beta-cell failure and death, leading to insulin insufficiency. Very limited information is currently available about the ultrastructural alterations of beta cells in human diabetes. Our aim was to provide a comprehensive ultrastructural analysis of human pancreatic islets in type 1 (T1D) and type 2 (T2D) diabetic patients. METHODS: We performed a morphometric electron microscopy evaluation of beta cells obtained from the pancreas of 8 nondiabetic (ND), 5 T1D, and 8 T2D organ donors. RESULTS: A lower amount of beta cells was found in both T1D and T2D than in ND islets, whereas alpha cells were increased only in T2D. An increased number of bi-hormonal cells (showing both insulin and glucagon granules in their cytoplasm) were found in T1D. Insulin granules were less represented in T2D than in ND beta cells, whereas no significant changes were found in T1D. Volume density of the endoplasmic reticulum was increased in T2D and unchanged in T1D; mitochondria number and volume were significantly higher in T2D than in ND beta cells, whereas no significant differences were found in T1D. In both T1D and T2D, more beta cells showed signs of apoptosis than in ND. CONCLUSIONS: Our results show that in each type of diabetes, beta cells exhibit specific ultrastructural alterations, whose better understanding might improve therapeutic strategies.

2,3,7,8-tetrachlorodibenzo-p-dioxin and the viral infection.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a widespread highly toxic environmental contaminant, suppresses immune response and leads to an increased susceptibility to infectious agents. In particular, several studies have provided evidence that TCDD decreases resistance to numerous viruses. Indeed, in vivo and in vitro investigations showed that the presence of TCDD is able to interfere with the replication of both human and animal viruses, such as influenza A viruses, coxsackie virus B3, immunodeficiency virus type-1 (HIV-1), cytomegalovirus (CMV), herpes simplex II, and bovine herpesvirus 1. Moreover, TCDD could induce an exacerbation of latent infection produced by HIV-1, CMV or Epstein-Barr virus. In this review, we first describe the general effects of TCDD exposure on mammalian cells, then we focus on its influence on the viral infections. Overall, the available data support the concept that TCDD exposure may act as an additional risk factor in promoting of viral diseases.

Correlation between genetic variability and virulence factors in clinical strains of Malassezia pachydermatis of animal origin.

Malassezia pachydermatis is a yeast belonging to the microbiota of the skin and mucous membranes of dog and cat, but it can also act as pathogen, causing dermatitis. The aim of this work was to evaluate the genetic variability of M. pachydermatis strains isolated from symptomatic dogs and cats and determine a correlation between genotype and phenotype. For this purpose eleven strains of M. pachydermatis were molecularly classified by nested-polymerase chain reaction (nested-PCR) based on ITS-1 and ITS-2 regions, specific for fungal rRNA genes. Furthermore, random amplification of polymorphic DNA (RAPD) was applied for genetic typing of M. pachydermatis isolates identifying four different genotypes. Strains belonging to genotype 1 produced the highest amount of biofilm and phospholipase activity. The inflammatory response induced by M. pachydermatis strains in immortalized human keratinocytes (HaCat cells) was significantly different when we compared the results obtained from each strain. In particular, HaCat cells infected with the strains belonging to genotypes 1 and 2 triggered the highest levels of increase in TLR-2, IL-1ß, IL-6, IL-8, COX-2 and MMP-9 expression. By contrast, cells infected with the strains of genotype 3 and those of genotype 4 did not significantly induce TLR-2 and cytokines. The results obtained might suggest a possible association between genotype and virulence factors expressed by M. pachydermatis strains. This highlights the need for a more accurate identification of the yeast to improve the therapeutic approach and to monitor the onset of human infections caused by this emergent zoonotic pathogen.

Mast cells infiltrate pancreatic islets in human type 1 diabetes.

AIMS/HYPOTHESIS: Beta cell destruction in human type 1 diabetes occurs through the interplay of genetic and environmental factors, and is mediated by immune cell infiltration of pancreatic islets. In this study, we explored the role of mast cells as an additional agent in the pathogenesis of type 1 diabetes insulitis. METHODS: Pancreatic tissue from donors without diabetes and with type 1 and 2 diabetes was studied using different microscopy techniques to identify islet-infiltrating cells. The direct effects of histamine exposure on isolated human islets and INS-1E cells were assessed using cell-survival studies and molecular mechanisms. RESULTS: A larger number of mast cells were found to infiltrate pancreatic islets in samples from donors with type 1 diabetes, compared with those from donors without diabetes or with type 2 diabetes. Evidence of mast cell degranulation was observed, and the extent of the infiltration correlated with beta cell damage. Histamine, an amine that is found at high levels in mast cells, directly contributed to beta cell death in isolated human islets and INS-1E cells via a caspase-independent pathway. CONCLUSIONS/INTERPRETATION: These findings suggest that mast cells might be responsible, at least in part, for immune-mediated beta cell alterations in human type 1 diabetes. If this is the case, inhibition of mast cell activation and degranulation might act to protect beta cells in individuals with type 1 diabetes.

Are we overestimating the loss of beta cells in type 2 diabetes?

AIMS/HYPOTHESIS: Previous work has demonstrated that beta cell amount (whether measured as beta cell mass, beta cell volume or insulin-positive area) is decreased in type 2 diabetes; however, recent findings suggest that mechanisms other than death may contribute to beta cell failure in this disease. To better characterise beta cell mass and function in type 2 diabetes, we performed morphological, ultra-structural and functional studies using histological samples and isolated islets. METHODS: Pancreases from ten non-diabetic (ND) and ten matched type 2 diabetic organ donors were studied by insulin, glucagon and chromogranin A immunocytochemistry and electron microscopy (EM). Glucose-stimulated insulin secretion was assessed using isolated islets and studies were performed using independent ND islet preparations after 24 h exposure to 22.2 mmol/l glucose. RESULTS: Immunocytochemistry showed that the fractional islet insulin-positive area was lower in type 2 diabetic islets (54.9 ± 6.3% vs 72.1 ± 8.7%, p < 0.01), whereas glucagon (23.3 ± 5.4% vs 20.2 ± 5.3%) and chromogranin A (86.4 ± 6.1% vs 89.0 ± 5.5%) staining was similar between the two groups. EM showed that the proportion of beta cells in type 2 diabetic islets was only marginally decreased; marked beta cell degranulation was found in diabetic beta cells; these findings were all reproduced after exposing isolated ND islets to high glucose. Glucose-stimulated insulin secretion was 40­50% lower from type 2 diabetic islets (p < 0.01), which again was mimicked by culturing non-diabetic islets in high glucose. CONCLUSIONS/INTERPRETATION: These results suggest that, at least in subgroups of type 2 diabetic patients, the loss of beta cells as assessed so far might be overestimated, possibly due to changes in beta cell phenotype other than death, also contributing to beta cell failure in type 2 diabetes.

Methicillin-resistant Staphylococcus pseudintermedius: epidemiological changes, antibiotic resistance, and alternative therapeutic strategies.

Staphylococcus pseudintermedius is a major opportunistic bacterial pathogen that belongs to the skin and mucosal microbiota of the dog. Since its global emergence around 2006, multidrug - methicillin-resistant S. pseudintermedius (MRSP) clones have become endemic worldwide. MRSP strains pose a significant threat to animal health and make antimicrobial therapy difficult due to their typical multidrug resistance phenotypes. The difficulty to treat MRSP infections using the current antimicrobials licensed for veterinary use has intensified research efforts to develop new treatment strategies and alternative anti-infective approaches to conventional antimicrobial therapy. The present narrative review outlines the latest changes in the epidemiology of MRSP with focus on the geographical distribution variability and antimicrobial resistance profiles in the main MRSP lineages. It also provides an overview of the effectiveness of currently available antimicrobials and the status of anti-infective alternatives to conventional antimicrobials.Recent studies have reported notable changes in the population structure of MRSP, with the emergence of new epidemic lineages, such as ST258, ST123, ST496, and ST551 in European countries and ST45, ST181, ST258, ST496 in non-European countries, which partly or totally replaced those that were initially prevalent, such as ST71 in Europe and ST68 in the US. Due to methicillin resistance often associated with the resistance to a broader number of antimicrobials, treating canine MRSP skin infection is challenging. Several alternative or supplementary treatment options to conventional antibiotics, especially for topical treatment, such as a novel water-soluble hydroxypyridinone-containing iron-chelating 9 kDa polymer (DIBI), antimicrobial peptides (AMPs), nanoparticles, and bacteriophages seem to be particularly interesting from a clinical perspective.

Multi-locus sequence typing and in vitro antimicrobial resistance of equine Streptococcus equi subspecies zooepidemicus strains.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the most important pathogens frequently associated with the main causes of equine infertility. In this study, we surveyed 22 strains of S. zooepidemicus collected during 2021 from cervico-uterine swabs of mares with endometritis. The genetic variability of the isolated strains was studied by multi-locus sequence typing (MLST) from whole-genome sequencing (WGS) data. The average length of reconstructed genomes was 2,088,286 bp (95% CI: 2,061,569 bp-2,114,967 bp), which was expected for S. zooepidemicus genomes. The assembled genomes were assigned to sequence types (STs) using the S. zooepidemicus scheme targeting seven loci (arcC, nrdE, proS, spi, tdk, tpi, yqiL) available in PubMLST database. MLST revealed a wide variability of STs with two (9.1%) novel STs identified in this study, precisely ST521 with two isolates and ST522 with one isolate. Furthermore, 4/22 (18.2%) isolates were assigned to ST92, 3/22 (13.6%) to ST205, 2/22 (9.1%) to ST475, and one strain (4.5%) for each of the following STs: ST10, ST30, ST39, ST49, ST101, ST132, ST147, ST314, ST369, ST467. Isolates were also tested for antimicrobial resistance using Kirby-Bauer disk diffusion method. Resistance to amoxicillin-clavulanate, ampicillin, amikacin, gentamicin, streptomycin, enrofloxacin, sulfamethoxazole-trimethoprim, tetracycline, oxytetracycline represented the most common resistance profile (13/22, 59.1%). No correlation between specific ST and antimicrobial resistance profile was found. Our study provides a comprehensive insight into the epidemiology, ST diversity and antimicrobial resistance profile of S. zooepidemicus strains, isolated in Italy, causing subfertility problems in mares.

Staphylococcus pseudintermedius and Pseudomonas aeruginosa Lubbock Chronic Wound Biofilm (LCWB): a suitable dual-species model for in vitro studies.

Antimicrobial treatment of methicillin-resistant Staphylococcus pseudintermedius associated with canine wounds represents an important challenge. The aim of this study was to create a canine wound infection model, Lubbock Chronic Wound Biofilm (LCWB), with a focus on S. pseudintermedius, drawing inspiration from the established human model involving Staphylococcus aureus. Methicillin-resistant S. pseudintermedius 115 (MRSP) and Pseudomonas aeruginosa 700 strains, isolated from dog wounds, were used to set up the LCWB at 24, 48 and 72 h. The LCWBs were evaluated in terms of volume, weight, and microbial CFU/mg. The microbial spatial distribution in the LCWBs was assessed by SEM and CLSM imaging. The best incubation time for the LCWB production in terms of volume (3.38 cm3 ± 0.13), weight (0.86 gr ± 0.02) and CFU/mg (up to 7.05 × 106 CFU/mg ± 2.89 × 105) was 48 h. The SEM and CLSM images showed a major viable microbial colonization at 48 h with non-mixed bacteria with a prevalence of MRSP on the surface and P. aeruginosa 700 in the depth of the wound. The obtained findings demonstrate the capability of S. pseudintermedius to grow together P. aeruginosa in the LCWB model, representing the suitable model to reproduce the animal chronic wound in vitro.

Hygiene Assessment of Buffalo Milking Parlours in Campania Region, Italy: A Preliminary Study by Using ATP Luminometry and Bacteriological Investigation.

Careful cleaning of a milking parlour and its equipment is fundamental to guarantee good raw milk quality and prevent the dissemination of bacteria and improve animal welfare. This study aimed to investigate, using an ATP-bioluminescence assay and bacteriological analysis, the bacterial contamination of milking parlours on milking parlour surfaces of buffalo farms in the Campania Region, evaluating the seasonal dynamics during the year 2022. Eight farms were selected by the Italian ClassyFarm system, which assesses the level of animal welfare and biosecurity according to risk analysis. Before sampling, all dairy farm owners filled out a questionnaire on milking management, animal hygiene, and health. The questionnaires evidenced similar cleaning procedures but an absence of a standardised cleaning protocol among the different farms. ATP bioluminescence results evidenced similar levels of contamination in all the selected buffalo farms, and the season comparison showed no significant differences. A variation in the percentages of bacterial isolates during the different seasons was observed, with a higher prevalence of Enterobacteriaceae (38%) in summer. A small number of samples exhibited an absence of bacterial growth. Identifying bacteria is crucial for understanding the microorganisms present in the milking parlour, yet employing ATP luminometry could offer broad and accurate applications in buffalo milking parlours. In conclusion, the use of ATP bioluminescence for evaluating the hygiene of a buffalo milking parlour could represent a further important advancement in dairy farming technology.

Cannabis sativa L. essential oil: chemical characterisation and antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius.

Cannabis sativa L. essential oil has attracted the interest of the scientific community thanks to its numerous biological activities. Several studies have evaluated EOs as alternative therapeutic approaches to limit the use of antibiotics; the present study aimed to evaluate the in vitro inhibitory and bactericidal activity of the essential oils obtained from the leaves and inflorescences of two hemp genotypes against twenty-one multidrug-resistant, methicillin-resistant Staphylococcus pseudintermedius strains isolated from canine clinical samples. Both EOs were mainly represented by sesquiterpene hydrocarbons, with a prevalence of ß-caryophyllene and α-humulene. However, different relative amounts of phytocannabinoids were also detected. Microbiological results evidenced better outcomes for the EO characterised by the highest content of phytocannabinoids, which in turn showed no differences among the tested strains. Nevertheless, both the EOs showed better inhibitory and bactericidal activities than their main constituent, ß-caryophyllene, tested individually, highlighting the presence of synergistic effects among the EO compounds.

Use of cytobrush for bacteriological and cytological diagnosis of endometritis in mares.

Background and Aim: A combined microbial and cytological examination of uterine samples is the main diagnostic method for endometritis in mares. This study aimed to describe a procedure for using the same uterine cytobrush (CB) for both bacteriological and cytological evaluation. Material and Methods: The procedure consists of rolling the CB onto a sterilized glass slide immediately after collection and before the transfer into a sterile saline solution. In Experiment 1, a comparison between bacteriological results of the cotton swab (CS) and CB or pellet was made in 10 mares; in Experiment 2, bacteriological and cytological results were compared between different processing methods of CB in 28 mares; in other 6 mares, a CB was processed for cytology only, to investigate the reasons for the low cellularity of the pellet. Results: The agreement between culture results from the CB and CS was evaluated, and a comparison between the cytological data obtained by different processing methods of CB was performed. The perfect agreement between the CB and CS microbiological results was found. The described procedure enables useful diagnostic smears for cytology. Moreover, the seeding of both the tip of CB and the saline solution used for the transport produced accurate bacteriological results. Conclusion: The protocol described in this study for the use of CB for both cytological and bacteriological analysis could be used for the diagnosis of endometritis. To maximize diagnostic sample quality, cytology slides must be prepared with meticulous care in the field to preserve cellular integrity and minimize artifacts.

Evidence and antibiotic resistance profiles of clinical Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) and non-ACB complex members in companion animals: A 2020-2022 retrospective study.

To evaluate the frequency of Acinetobacter spp., belonging to both Acinetobacter calcoaceticus-baumannii (ACB) and non-ACB complex, and their antibiotic resistance profiles in veterinary medicine, a three-year (2020-2022) retrospective study was carried out on sick companion animals. Epidemiological data from different clinical canine, feline, and equine samples, were acquired. For each strain, MALDI-TOF MS identification and susceptibility to a panel of 11 antibiotics, by Kirby-Bauer and E-test methods, were performed. Out of 628 bacteriological examinations, 2.5% resulted positive for strains belonging to Acinetobacter genus. Frequencies of 2.3%, 1.9%, and 3% were obtained from both in-visiting and hospitalized dogs, cats, and horses, respectively. Members of ACB-complex accounted for 50% of isolates. Since all strains resulted susceptible to aminoglycosides and polymyxins, no pandrug-resistant (PDR) species were recorded. While 12.5% A. baumannii resulted extensively-drug resistant (XDR), a higher percentage of multidrug-resistant strains was recorded among non-ACB strains (35.5%) than ACB strains (25%). Susceptibility was observed in the same percentage in both groups (62.5%). All ACB strains confirmed their intrinsic resistances. Non-ACB species showed lower resistances against antipseudomonal penicillins plus beta-lactamase inhibitors (P=0.1306), III generation cephalosporins (P=0.0547), and tetracyclines (P=0.0209) than ACB species. Carbapenem-resistance was observed for XDR A. baumannii (12.5%) and, in particular for MDR non-ACB complex members (25%). To our knowledge, A. lactucae represents the first description in two sick dogs in Italy. Furthermore, our results emphasize the role of non-ACB-complex species as important zoonotic pathogens, which could be reservoirs of clinically relevant resistance profiles.

The biological variation of serum 1,25-dihydroxyvitamin D and parathyroid hormone, and plasma fibroblast growth factor 23 in healthy individuals.

BACKGROUND AND AIMS: Measuring 1,25-dihydroxyvitamin D (1,25(OH)2D), parathyroid hormone 1-84 (PTH 1-84) and intact FGF23 (iFGF23) is crucial for diagnosing a variety of diseases affecting bone and mineral homeostasis. Biological variability (BV) data are important for defining analytical quality specifications (APS), the usefulness of reference intervals, and the significance of variations in serial measurements in the same subject. The aim of this study was to pioneer the provision of BV estimates for 1,25(OH)2D and to improve existing BV estimates for iFGF23 and PTH 1-84. MATERIALS AND METHODS: Serum and plasma-EDTA samples of sixteen healthy subjects have been collected for seven weeks and measured in duplicate by chemiluminescent immunoassay on the DiaSorin Liaison platform. After variance verification, within-subject (CVI) and between-subject (CVG) BV estimates were assessed by either standard ANOVA, or CV-ANOVA. The APSs were calculated according to the EFLM-BV-model. RESULTS: We found the following CVI estimates with 95% confidence intervals:1,25(OH)2D, 22.2% (18.9-26.4); iFGF23, 16.1% (13.5-19.5); and PTH 1-84, 17.9% (14.8-21.8). The CVG were: 1,25(OH)2D, 21.2% (14.2-35.1); iFGF23, 21.1% (14.5-35.8); and PTH 1-84, 31.1% (22.1-50.8). CONCLUSIONS: We report for the first time BV estimates for 1,25(OH)2D and enhance existing data about iFGF23-BV and PTH 1-84-BV through cutting-edge immunometric methods.

Bovine herpesvirus type 4 infection modulates autophagy in a permissive cell line.

Bovine herpesvirus type 4 (BoHV-4), like other herpesviruses, induces a series of alterations in the host cell that modify the intracellular environment in favor of viral replication, survival and spread. This research examined the impact of BoHV-4 infection on autophagy in BoHV-4 infected Madin Darby bovine kidney (MDBK) cells. Protein extracts of BoHV-4 infected and control MDBK cells were subjected to Western blot. The concentrations of the autophagy and apoptosis-related proteins Beclin 1, p21, PI3 kinase, Akt1/2, mTOR, phospho mTOR, p62 and the light chain three (LC3) were normalized to the actin level and expressed as the densitometric ratio. Western blot analysis of virus-infected cells revealed that autophagic degradation pathway was induced in the late phase of BoHV-4 infection. After 48 h post-infection the protein LC3II, which is essential for autophagy was found to be markedly increased, while infection of MDBK cells with BoHV-4 resulted in a depletion of p62 levels. Becline 1, PI3 kinase, Akt1/2 and p21 expression increased between 24 and 48 h post-infection. Surprisingly, mTOR and its phosphorylated form, which are negative regulators of autophagy, also increased after 24 h post-infection. In conclusion, our findings suggest that BoHV-4 has developed mechanisms for modulation of autophagy that are probably part of a strategy designed to enhance viral replication and to evade the immune system. Additional studies on the relationship between autophagy and BoHV-4 replication and survival, in both lytic and latent replication phases, are needed to understand the role of autophagy in BoHV-4 pathogenesis.

Editorial on the Research Topic of the Special Issue "Current Status of Acinetobacter Infections".

Emerging bacterial infections will continue to be an important issue for public health, mostly because of the constant changes on our earth [...].

Antimicrobial Resistant Staphylococcus Species Colonization in Dogs, Their Owners, and Veterinary Staff of the Veterinary Teaching Hospital of Naples, Italy.

This study aimed to identify Staphylococcus species isolated from nasal swabs of both healthy and diseased dogs, and those of human origin, obtained from nasal swabs of both owners and veterinary staff. Firstly, pet owners were requested to complete a questionnaire relating to the care and relationship with their pets, whose results mainly showed a statistically significant higher frequency of hand washing in diseased dogs' owners than in healthy dogs' owners. Canine nasal swabs were obtained from 43 diseased dogs and 28 healthy dogs, while human nasal swabs were collected from the respective dogs' owners (71 samples) and veterinary staff (34 samples). The isolation and identification of Staphylococcus spp. were followed by disk diffusion method to define the antimicrobial resistance profiles against 18 different molecules. Staphylococcus pseudintermedius was the most frequent isolated strain in both diseased (33.3%) and healthy (46.1%) dogs. Staphylococcus epidermidis was the most frequent isolated bacterium in diseased dogs' owners (66.6%), while in nasal samples of healthy dogs' owners, the same frequency of isolation (38.4%) was observed for both Staphylococcus epidermidis and Staphylococcus aureus. All the isolated strains showed good susceptibility levels to the tested antimicrobials; however, the carriage of oxacillin-resistant strains was significantly higher in diseased dogs than in healthy ones (71% and 7.7%, respectively). Only in three cases the presence of the same bacterial species with similar antimicrobial resistance profiles in dogs and their owners was detected, suggesting the potential bacterial transmission. In conclusion, this study suggests potential transmission risk of staphylococci from dogs to humans or vice versa, and highlights that the clinical relevance of Staphylococcus pseudintermedius transmission from dog to human should not be underestimated, as well as the role of Staphylococcus aureus from human to dog transmission.

Staphylococcus microti Strains Isolated from an Italian Mediterranean Buffalo Herd.

S. microti is a new species among non-aureus staphylococci (NAS) frequently found in bovine milk samples and associated with subclinical mastitis (SCM). The aim of this study was to analyze the presence of S. microti in 200 composite milk samples and 104 milking parlor surface swabs collected at a buffalo farm in Southern Italy to define its presence in milk and a milking parlor environment. The samples were inoculated onto different agar plates, and the isolates were identified by MALDI-TOF MS. The strains identified as S. microti (54/304 samples, 17.8%) were collected, and their purified genomic DNA was subjected to PCR amplification and whole 16S rRNA gene sequencing. Furthermore, their phenotypic resistance profiles were evaluated by a disk diffusion method, and the genotypic characterization of the tetracycline resistance was performed for the tetM and tetK genes by multiplex PCR. Four and forty-seven S. microti isolates from milk samples of lactating animals with subclinical mastitis (SCM) and intramammary infection (IMI), respectively, and three isolates from milking parlor surfaces were recovered. The genomic DNA was purified from the bacterial isolates, and the amplification and sequencing of the 16S gene further supported the proteomic identification as S. microti. No clinical mastitis was detected in the herd during the study period. The antimicrobial susceptibility testing revealed a worrisome 100% resistance to tetracyclines, genotypically mediated by the tetM gene for all strains. This study highlights that S. microti may be commonly isolated from dairy buffalo milk and milking parlor equipment. Its association with SCM or IMI remains to be established.

On effect of poultry manure treatment with Effective Microorganisms with or without zeolite.

The decomposition process of poultry manure is generally mediated by microorganisms, whose degradation activity has beneficial effects on soil fertility but, on the other hand, leads to the generation of malodour gas. Indeed, a relevant problem of poultry farms is represented by the release of bad smells, which are mainly a consequence of decomposition process of chicken feces, chicken bedding, plumes, dropped feed, and dust. Furthermore, the unpleasant odour, associated with poultry manure degradation, not only limits its use in agriculture but also negatively affects the housing communities located near the farms. This study aimed at evaluating the effects in vitro of different doses of Effective Microorganisms (EM), mainly consisting of live communities of lactic acid bacteria, photosynthetic bacteria, and yeasts, on poultry manure alone or with zeolite, a porous mineral with absorbent and ion-exchange properties, belonging to the family of aluminosilicates. The obtained results demonstrated that these treatments were able to reduce the poultry manure malodours, associated mainly with a decrease in the ammonia (NH3) levels with respect to controls. The pH tended to increase, the nitrogen to go down, and the phosphorus to go up. Thus, all the effects described above were evident, testifying to a slower degradation of proteins, both with EM alone or in combination with zeolite. The presence of a pool of pesticides (65 components) was evaluated, and no variation was observed in the different experimental conditions versus control, as well as for REEs and metals. In conclusion, these preliminary results demonstrated that the use of EM with or without the addition of zeolite is a valid tool to eliminate the bad smell of manure and to make it a useful product as a fertilizer.