Long-term outcomes of the Italian Mycobacterium avium subspecies paratuberculosis control programme for dairy cattle.

BACKGROUND: The considerable epidemiological and economic implications of paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), have placed importance on control efforts aimed at preventing MAP transmission. In this context, Italy issued national guidelines for the control and status certification of MAP in dairy cattle in 2013. METHODS: We assessed the long-term outcomes of the Italian MAP control programme for 14 dairy farms located in northern Italy by retrospectively reviewing the results of yearly serological tests, presence of clinical cases, MAP faecal shedding in serologically positive animals, farm management and health ranking as indicators of herd health between 2014 and 2021. RESULTS: A significantly higher number of serologically positive animals were observed between 2014 and 2016 than between 2017 and 2021, as well as an improving trend in the paratuberculosis health ranking for nine of the 14 farms. No clinical cases were reported. MAP shedding was detected in 9.4% of serologically positive animals. Discarding colostrum and prioritised culling of seropositive animals assisted by adoption of standardised serological testing were presumed to have a key role in MAP control, despite the reluctance of some farmers to address hygienic issues and improve the separation of calves from adult animals. LIMITATIONS: The small number of farms included in this study and the fact that these were not randomly selected may limit the generalisability of the findings. CONCLUSIONS: The Italian paratuberculosis control plan has provided measures to limit the uncontrolled spread of MAP infection within and between herds by promoting animal trading between farms certified as negative or low risk.

Label-Free Quantitative Analysis of Pig Liver Proteome after Hepatitis E Virus Infection.

Hepatitis E represents an emerging zoonotic disease caused by the Hepatitis E virus (HEV), for which the main route of transmission is foodborne. In particular, infection in humans has been associated with the consumption of contaminated undercooked meat of pig origin. The aim of this study was to apply comparative proteomics to determine if porcine liver protein profiles could be used to distinguish between pigs seropositive and seronegative for HEV. Preliminarily, an ELISA was used to evaluate the presence of anti-HEV antibodies in the blood serum of 136 animals sent to slaughter. Among the analyzed samples, a seroprevalence of 72.8% was estimated, and it was also possible to identify 10 animals, 5 positive and 5 negative, coming from the same farm. This condition created the basis for the quantitative proteomics comparison between homogeneous animals, in which only the contact with HEV should represent the discriminating factor. The analysis of the proteome in all samples of liver exudate led to the identification of 554 proteins differentially expressed between the two experimental groups, with 293 proteins having greater abundance in positive samples and 261 more represented in negative exudates. The pathway enrichment analysis allowed us to highlight the effect of the interaction between HEV and the host biological system in inducing the potential enrichment of 69 pathways. Among these, carbon metabolism stands out with the involvement of 41 proteins, which were subjected to interactomic analysis. This approach allowed us to focus our attention on three enzymes involved in glycolysis: glucose-6-phosphate isomerase (GPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and fructose-bisphosphate aldolase A (ALDOA). It therefore appears that infection with HEV induced a strengthening of the process, which involves the breakdown of glucose to obtain energy and carbon residues useful for the virus's survival. In conclusion, the label-free LC-MS/MS approach showed effectiveness in highlighting the main differences induced on the porcine liver proteome by the interaction with HEV, providing crucial information in identifying a viral signature on the host metabolism.

Detection of zoonotic enteropathogens in captive large felids in Italy.

AIMS: Within the One Health paradigm, infectious disease surveillance have been developed for domestic and wild animals, leaving the role of captive non-domestic populations, especially felids in zoos and circuses, less explored. This study addresses the proximity of these captive animals to urban areas, necessitating focused monitoring for potential zoonotic enteropathogens. The present work aimed to investigate the presence of such zoonotic enteropathogens in faecal samples from captive large felid populations. METHODS AND RESULTS: A total of 108 faecal samples were collected in three circuses, five zoos and one rescue centre across Italy. Salmonella spp. isolation, serotyping and antimicrobial susceptibility testing were conducted on all samples. Additionally, 60 samples were also examined for gastrointestinal parasites using standard coprological techniques. Giardia spp. detection employed direct immunofluorescent staining and specific PCR, while Toxoplasma gondii was detected using PCR targeting B1 gene. A total of 51 Salmonella enterica subsp. enterica were isolated, with predominant serovariants including Infantis (43.1%), Coeln (11.8%) and Newport (11.8%). The captive felids likely act as asymptomatic carriers of foodborne Salmonella, with notable resistance ampicillin and trimethoprim-sulfamethoxazole, no resistance to enrofloxacin was noted. Microscopic analysis revealed Toxascaris leonina eggs in 11 faecal samples (18.3%) and Giardia duodenalis cysts in one animal (1.7%). CONCLUSIONS: Captive animals in public settings may act as sources of Salmonella infection and enteroparasitosis for both occupational and general exposure. The study emphasizes the role of captive animals in antimicrobial resistance dynamics, highlighting the need for routine pathogen screening in the management practices of zoological structures.

Comparison between clinical evaluations and laboratory findings and the impact of biofilm on antimicrobial susceptibility in vitro in canine otitis externa.

BACKGROUND: In canine otitis externa (OE), biofilm-producing bacteria are frequently present but biofilm may be underdiagnosed clinically. HYPOTHESIS/OBJECTIVES: The study aimed to investigate an association between clinical and cytological findings with bacteriological data from dogs with OE, to establish, through Environmental Scanning Electron Microscope (ESEM) examination, whether the presence of biofilm in vivo can be predicted and to evaluate the impact of biofilm on antimicrobial susceptibility tests. MATERIALS AND METHODS: Fifty-six dogs showing clinical signs of OE were enrolled. One cotton swab each was collected for ESEM, bacterial culture and susceptibility testing and for cytology. Staphylococcus pseudintermedius (n = 42, 48.8%) and Pseudomonas aeruginosa (n = 26, 30.2%) were tested for their ability to form biofilm. Minimum Inhibitory Concentrations (MIC), Minimal Biofilm Inhibitory Concentrations (MBIC) and Minimal Biofilm Eradication Concentrations (MBEC) towards enrofloxacin, gentamicin, polymyxin B and rifampicin were determined. RESULTS: Pseudomonas aeruginosa was positively associated with the biofilm clinical evaluation (p < 0.01) and neutrophils (p < 0.05), nuclear streaks (p < 0.01) and rods bacteria (p < 0.01) on cytology. S. pseudintermedius was associated with a low presence of neutrophils. There was a statistical correlation between clinical and cytological biofilm presence (p ≤ 0.01), but none with the biofilm production assay nor ESEM biofilm detection. No differences were found comparing the results of MIC and MBIC. MBEC results showed higher values than MIC and MBIC for all antimicrobials tested (p ≤ 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: Biofilm presence in OE was often underdiagnosed. Even if there is no specific clinical or cytological pattern related to biofilm, its presence should always be suspected.

Inhibition of Staphylococcus pseudintermedius Efflux Pumps by Using Staphylococcus aureus NorA Efflux Pump Inhibitors.

One promising approach in treating antibiotic-resistant bacteria is to "break" resistances connected with antibacterial efflux by co-administering efflux pump inhibitors (EPIs) with antibiotics. Here, ten compounds, previously optimized to restore the susceptibility to ciprofloxacin (CIP) of norA-overexpressing Staphylococcus aureus, were evaluated for their ability to inhibit norA-mediated efflux in Staphylococcus pseudintermedius and synergize with CIP, ethidium bromide (EtBr), gentamycin (GEN), and chlorhexidine digluconate (CHX). We focused efforts on S. pseudintermedius as a pathogenic bacterium of concern within veterinary and human medicine. By combining data from checkerboard assays and EtBr efflux inhibition experiments, the hits 2-arylquinoline 1, dihydropyridine 6, and 2-phenyl-4-carboxy-quinoline 8 were considered the best EPIs for S. pseudintermedius. Overall, most of the compounds, except for 2-arylquinoline compound 2, were able to fully restore the susceptibility of S. pseudintermedius to CIP and synergize with GEN as well, while the synergistic effect with CHX was less significant and often did not show a dose-dependent effect. These are valuable data for medicinal chemistry optimization of EPIs for S. pseudintermedius and lay the foundation for further studies on successful EPIs to treat staphylococcal infections.

Diagnostic characteristics of refractometry cut-off points for the estimation of immunoglobulin G concentration in mare colostrum.

BACKGROUND: Feeding foals with poor quality colostrum predisposes them to failure of passive transfer (FPT). FPT is a major risk factor for neonatal infections. OBJECTIVES: To assess the optimal cut-offs for the optical (OR) and digital (DR) refractometer and determine their accuracy for poor quality colostrum diagnosis. STUDY DESIGN: A diagnostic validation study. METHODS: Eighty-one colostrum samples and sera were collected from broodmares and their neonatal foals, respectively. Colostral and serum IgG concentrations were measured by radial immunodiffusion (RID), DR and OR. Correlation coefficients were calculated. ROC curves were generated to identify optimal cut-offs for the refractometers and their diagnostic characteristics were evaluated. RESULTS: The optimal cut-offs for DR and OR were ≤23.75% and 23.9%, respectively. The sensitivity and specificity of the DR were 93.3% (95% CI: 66.0-99.7) and 87.9% (95% CI: 77.0-94.3) to detect colostral IgG <60 g/L, respectively. The sensitivity and specificity of the OR were 93.3% (95% CI: 66.0-99.7) and 81.8% (95% CI: 70.0-89.9), respectively. DR and OR had negative predictive values of 98.3% (95% CI: 89.7-99.9) and 98.2% (95% CI: 89.0-99.9), respectively, whilst positive predictive values were lower. No maternal variable, including breed, significantly influenced colostral IgG concentrations. Fifteen out of 81 colostrum samples had IgG <60 g/L. FPT and PFPT were diagnosed in 4/81 and 10/81 foals, respectively. Nine out of 14 animals with FPT/PFPT suckled colostrum with IgG <60 g/L. A moderate correlation (rs 0.542; P = .01) was observed between IgG concentrations measured by RID in sera and colostrum. MAIN LIMITATIONS: A smaller number of samples than the size requirement based on a priori estimate of specificity and the low prevalence of poor quality colostrum. CONCLUSIONS: The method has the potential to reliably differentiate between good and poor quality colostrum. Assessing colostrum quality by refractometry may be an indicator of passive transfer of immunity.

INTRODUCTION/CONTEXTE: Nourrir les poulains avec du colostrum de mauvaise qualité prédispose à l'échec du transfert d'immunité passive (FPT). FPT constitue un risque majeur pour les infections néonatales. OBJECTIFS: Évaluer les valeurs limites optimales au réfractomètre optique (RO) et digital (RD) et déterminer leur précision pour le diagnostic du colostrum de pauvre qualité. TYPE D'ÉTUDE: Étude de validation diagnostique. MÉTHODES: Quatre-vingt-un colostrums et sérums ont été recueillis à partir de juments de reproduction et de leur poulains nouveaux-nés respectivement. Les concentrations d'IgG dans le sérum et le colostrum ont été mesurées par immunodiffusion radiale (IDR), RO et RD. Les coefficients de corrélation ont été calculés. Des graphes d'air sous la courbe (ASC) ont été générés afin d'identifier les valeurs limites optimales aux différents réfractomètres et leurs caractéristiques diagnostiques ont été évaluées. RÉSULTATS: Les valeurs limites optimales pour les RD et RO étaient ≤23.75% et 23.9% respectivement. La sensibilité et la spécificité du RD étaient 93.3% (95% IC: 66.0-99.7) et 87.9% (95% IC: 77.0-94.3) pour la détection des IgG colostraux <6000 mg/dl, respectivement. La sensibilité et spécificité du RO étaient de 93.3% (95% IC: 66.0-99.7) et 81.8% (95% IC: 70.0-89.9), respectivement. Les RD et RO avaient une valeur prédictive négative de 98.3% (95% IC: 89.7-99.9) et 98.2% (95% IC: 89.0-99.9) respectivement, alors que les valeurs prédictives positives étaient plus basses. Aucune variable maternelle, incluant la race, n'a influencé significativement les concentrations colostrales en IgG. Quinze des 81 échantillons colostraux avaient une valeur d'IgG <6000 mg/dl. FPT et PFPT ont été diagnostiqué chez 4/81 et 10/81 poulains respectivement. Neuf des 14 animaux avec FPT/PFPT ont reçu du colostrum ayant des valeurs d'IgG <6000 mg/dl. Une corrélation modérée (rs 0.542; p= 0.01) a été observée entre les concentrations d'IgG mesurées par IDR dans le sérum et le colostrum. LIMITES PRINCIPALES: Le nombre d'échantillon est inférieur à celui recommandé basé sur unestimé a priori de la spécificité et considérant la faible prévalence de colostrum de pauvre qualité. CONCLUSIONS: La méthodologie utilisée pourrait différencier de façon fiable les colostrums de pauvre et bonne qualité. L'évaluation de la qualité du colostrum par réfractométrie pourrait représenter un indice du transfert d'immunité passive.

Mucosal exposure to non-tuberculous mycobacteria elicits B cell-mediated immunity against pulmonary tuberculosis.

Bacille Calmette-Guerin (BCG) is the only licensed vaccine against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease. However, BCG has limited efficacy, necessitating the development of better vaccines. Non-tuberculous mycobacteria (NTMs) are opportunistic pathogens present ubiquitously in the environment. TB endemic countries experience higher exposure to NTMs, but previous studies have not elucidated the relationship between NTM exposure and BCG efficacy against TB. Therefore, we develop a mouse model (BCG + NTM) to simulate human BCG immunization regime and continuous NTM exposure. BCG + NTM mice exhibit superior and prolonged protection against pulmonary TB, with increased B cell influx and anti-Mtb antibodies in serum and airways, compared with BCG alone. Notably, spatial transcriptomics and immunohistochemistry reveal that BCG + NTM mice formed B cell aggregates with features of germinal center development, which correlate with reduced Mtb burden. Our studies suggest a direct relationship between NTM exposure and TB protection, with B cells playing a crucial role.

Detection of Mycoplasma columbinasale in Cases of Respiratory Disease in Domestic Pigeons (Columba livia var. domestica).

In 2017, respiratory disease and low mortality were reported in domestic flying pigeons (Columba livia var. domestica) trained as hunting live bait in a breeding farm in Umbria, Italy. Clinically, open beak breathing, dropped wings, and pharyngeal and laryngeal hyperaemia were observed. Three birds were submitted for necropsy. Gross pathological evaluation revealed in all cases diffuses hyperaemia of the tracheal mucosa in association with mild emaciation and multiorgan congestion. Microscopically, diffuse epithelial hyperplasia of the trachea (n = 3) and diffuse lymphocytic infiltration of the lamina propria (n = 3) were observed. No lesions were reported in other organs. Based on reported clinical signs and lesions, Mycoplasma spp. were suspected, and molecular detection was performed on tracheal specimens leading to the identification of Mycoplasma columbinasale. Immunohistochemistry was subsequently performed to localize the microorganism within tissue lesions. Immunohistochemistry confirmed the presence of Mycoplasma species on the tracheal epithelial cells of all birds. Following tylosin administration, complete resolution of the clinical condition and lack of recurrence of clinical signs were reported in the breeding farm. These findings suggest that M. columbinasale could potentially have a role in the respiratory disease and low mortality in domestic pigeons.

Drug efflux transporters in Staphylococcus pseudintermedius: in silico prediction and characterization of resistance.

OBJECTIVES: To perform an in silico prediction of drug efflux pumps (EPs) in Staphylococcus pseudintermedius and investigate their role in conferring resistance to antibiotic and biocidal agents and biofilm formation. METHODS: A S. pseudintermedius efflux mutant was obtained by stimulating an isogenic line (ATCC 49444) with increasing concentrations of an efflux system substrate. Changes in antimicrobial susceptibility and biofilm-forming capability were evaluated in the presence/absence of the EP inhibitors (EPIs) thioridazine and reserpine and the efflux activity was assayed by fluorometry. Homologues of EPs of Staphylococcus aureus and Staphylococcus epidermidis were searched by exploratory GenBank investigations. Gene expression analyses and sequencing were then conducted on selected genes. RESULTS: Susceptibility to chlorhexidine, gentamicin and ciprofloxacin, but not enrofloxacin, was affected by the increased efflux and it was variably restored by the EPIs. The efflux mutant showed much greater biofilm formation that the original strain, which was significantly inhibited by thioridazine and reserpine at MIC/2. A high expression of norA, which was mgrA-independent, was found in the S. pseudintermedius efflux mutant, apparently regulated by an 11 bp deletion in its promoter region, whilst lmrB was transitorily overexpressed. icaA, which encodes the polysaccharide intercellular adhesin forming the extracellular matrix of staphylococcal biofilm, was also up-regulated. CONCLUSIONS: EPs, particularly NorA, are supposed to have complex involvement in multiple stages of resistance development. Overexpression of EPs appears to be correlated with a remarkable increase of S. pseudintermedius biofilm production; however, the regulatory mechanisms remain to be explored.

Paenibacillus amylolyticus osteomyelitis in a Poodle dog: case report and literature review.

Paenibacilli are gram-variable, endospore-forming bacteria that occupy various ecologic niches. These microorganisms have been known to infect humans occasionally at various anatomic sites. However, in humans, as well as in other vertebrate animals, the relationship between disease and isolation of Paenibacillus spp. remains poorly understood. We report here a case of infection in an adult Poodle dog. The animal had nodules in the lungs and multifocal osteolytic expansile bone lesions. From bone, Paenibacillus amylolyticus was recovered by culture and identified by MALDI-TOF mass spectroscopy and 16S rDNA sequencing; pyogranulomatous inflammation was observed in lung and bone specimens. The microorganism was resistant to clindamycin and imipenem. Four-month treatment with amoxicillin-clavulanate resulted in clinical resolution of disease in this dog. Nevertheless, therapy for more prolonged periods should be considered because recurrent infections can occur as a result of the transition of Paenibacillus spores to vegetative cells. Disease caused by a Paenibacillus species has not been reported previously in dogs, to our knowledge.

A new Montanide™ Seppic IMS1313-adjuvanted autogenous vaccine as a useful emergency tool to resolve a Salmonella enterica subsp. enterica serovar abortus equi abortion outbreak in mares.

Background: In Italy, an autogenous registered vaccine, adjuvanted with aluminum hydroxide, can be administrated to contrast Salmonella enterica subsp. enterica serovar abortus equi infection, coupled to a specific antimicrobial treatment. Case Description: Here, we report the case of an abortion outbreak by Salmonella abortus equi in Central Italy where mares were vaccinated but immediately developed a strong local reaction, maybe due to the adjuvant. Promptly, another autogenous vaccine, substituting the aluminum hydroxide with a new generation adjuvant (Montanide™ Seppic IMS1313), was produced and administrated. The new formulated vaccine did not cause any adverse outcome and conferred high protection titers against the infection. To the best of our knowledge, this is the first reported case of immunization by a vaccine adjuvanted with Montanide™ Seppic IMS1313 in horses. Conclusion: This approach may be used as a preventive strategy for further outbreaks in association with the application of recommended biosafety principles.

Inclusion Body Disease and Columbid Alphaherpesvirus 1 Infection in a Eurasian Eagle-Owl (Bubo bubo) of Central Italy.

Hepatosplenitis or inclusion body disease is a fatal disease in owls caused by Columbid alphaherpesvirus 1 (CoHV-1). A few old case reports describe it worldwide. In Italy, knowledge regarding virus circulation and disease development is lacking. Four Eurasian eagle-owls (Bubo bubo), two adults and two juveniles, were submitted for postmortem examination showing aspecific clinical signs a few hours before death. Grossly disseminated petechial hemorrhages on serosal surfaces (n = 4), hepatic and splenic necrosis (n = 3), bilateral and symmetric necrosis of pharyngeal tonsils (n = 2), and diffuse and bilateral dark-red discoloration and firmness in lungs (n = 2) were seen. Tissues were sampled for histology, bacteriology, molecular testing, and transmission electron microscopy (TEM). On histology, disseminated petechial hemorrhages (n = 4) and necrosis of liver (n = 3) and spleen (n = 3) were seen, as well as lympho-histiocytic interstitial pneumonia and meningoencephalitis (n = 2). Intranuclear inclusion bodies (INIBs) were detected in one case. A panherpesviral PCR led to positive results in one case, identified in sequencing as CoHV-1. On TEM, intranuclear and intracytoplasmic virions with herpesviral morphology were seen in the same case. For the other three birds, the lack of PCR positivity, INIBs, and TEM detection could be linked to a possible reduction of the virus to undetectable levels. Death possibly occurred secondarily to bacterial infections, supposedly established during the acute phase of CoHV-1 infection. This paper reports the presence of CoHV-1in Italy and the development of a fatal form of the disease in a Eurasian eagle-owl.

Enfermedad con cuerpos de inclusión e infección por Alfaherpesvirus de las columbiformes 1 en un búho real euroasiático (Bubo bubo) del centro de Italia. La hepatoesplenitis o enfermedad con cuerpos de inclusión es una enfermedad mortal en los búhos causada por el Alfaherpesvirus de las columbiformes 1 (CoHV-1). Algunos informes de casos antiguos lo describen en todo el mundo. En Italia, falta conocimiento sobre la circulación del virus y el desarrollo de enfermedades. Cuatro búhos reales euroasiáticos (Bubo bubo), dos adultos y dos juveniles, fueron sometidos a examen post mortem mostrando signos clínicos específicos unas horas antes de la muerte. Se observaron hemorragias petequiales muy diseminadas en las superficies serosas (n = 4), necrosis hepática y esplénica (n = 2), necrosis bilateral y simétrica de las tonsilas faríngeas (n = 2) y decoloración difusa y bilateral de color rojo oscuro y firmeza en los pulmones (n = 2). Se recolectaron muestras de tejidos para histología, bacteriología, pruebas moleculares y microscopía electrónica de transmisión (TEM). En la histología se observaron hemorragias petequiales diseminadas (n = 4) y necrosis de hígado (n = 3) y bazo (n = 3), así como neumonía intersticial linfohistiocítica y meningoencefalitis (n = 2). En un caso se detectaron cuerpos de inclusión intranucleares (INIB). Un método de PCR panherpesviral arrojó resultados positivos en un caso, identificado en la secuenciación como CoHV-1. Mediante microscopía electrónica de transmisión, se observaron viriones intranucleares e intracitoplasmáticos con morfología herpesviral en el mismo caso. Para las otras tres aves, la falta de positividad de PCR, la ausencia de cuerpos de inclusión intranucleares y de detección por microscopía electrónica de transmisión podría estar relacionada con una posible reducción del virus a niveles no detectables. La muerte posiblemente ocurrió de forma secundaria a infecciones bacterianas, posiblemente establecidas durante la fase aguda de la infección por el CoHV-1. Este artículo reporta la presencia de CoHV-1 en Italia y el desarrollo de una forma mortal de la enfermedad en un búho real euroasiático.

Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test.

The indiscriminate use of first-line drugs contributed to the spread of resistant bacteria, a major concern for both human and veterinary medicine. Methicillin resistance is acquired through the mecA gene, which encodes for the PBP2a protein and lends the resistance to ß-lactams. Verifying the correspondence between gene harboring and protein expression and accelerating methicillin resistance diagnosis is critical to improve the management of antimicrobial administration and to reduce the spread of drug resistances. We tested the applicability of immunofluorescence targeting PBP2a protein to identify a new potential methicillin resistance screening test, ancillary to conventional culture methods. We collected 26 clinical Staphylococcus pseudintermedius (SP) isolates: 25 from canine pyoderma and 1 from dermatitis in a dog owner. SP is one of the most important etiological agents in canine pyoderma and can harbor the mecA gene. We performed PCR for mecA gene detection, broth microdilution (BMD) for phenotypic methicillin resistance, and immunofluorescence targeting PBP2a protein. Compared to the PCR as the gold standard, immunofluorescence showed an apparent prevalence of 34.6% vs. a true prevalence of 53.8%, with 100% specificity, 64.3% sensitivity, and 80.8% diagnostic accuracy. PBP2a expression showed isolate-dependent variability: in some isolates, most of the bacterial cells showed an intense and clearly membranous pattern, while in others only a few of them could be detected. Performing the assay in duplicate improved the diagnostic accuracy. Since the mecA gene is shared among the members of the Staphylococcus genus, the test can be applied to identify methicillin resistance independently from the staphylococcal species, both in human and animal samples. Being a rapid and easy method and providing the unique possibility to study the expression of PBP2a by directly visualizing the morphology, it could represent a new interesting tool for both research and diagnostics. To accelerate methicillin resistance diagnosis, it would be worth further testing of its performance on cytological samples.

Comparative Performances of Vitek-2, Disk Diffusion, and Broth Microdilution for Antimicrobial Susceptibility Testing of Canine Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is the primary cause of canine cutaneous infections and is sporadically isolated as a pathogen from humans. Rapidly emerging antibiotic-resistant strains are creating serious health concerns so that accurate and timely antimicrobial susceptibility testing (AST) is crucial for patient care. Here, the performances of the AST methods Vitek-2, disk diffusion (DD) and broth microdilution (BMD) were compared for the determination of susceptibility of 79 S. pseudintermedius isolates from canine cutaneous infections and one from human pyoderma to oxacillin (OXA), amoxicillin/clavulanate (AMC), cephalothin (CEF), gentamicin (GEN), enrofloxacin (ENR), doxycycline (DOX), clindamycin (CLI), inducible clindamycin resistance (ICR), mupirocin (MUP), and trimethoprim-sulfamethoxazole (SXT). Overall, the agreement of DD and Vitek-2 using the veterinary AST-GP80 card with reference BMD was ≥90%, suggesting reliable AST performances. While DD generated mainly minor errors and one major error for OXA, Vitek-2 produced one very major error for GEN, and it failed in identifying one ICR-positive isolate. Moreover, five bacteria were diagnosed as ICR-positive by Vitek-2, but they showed a noninduction resistance phenotype with manual methods. All S. pseudintermedius isolates were interpreted as susceptible or intermediately susceptible to DOX using CLSI breakpoints for human staphylococci that match the DOX concentration range included in AST-GP80. However, this could lead to inappropriate antimicrobial prescription for S. pseudintermedius infections in companion animals. Considering the clinical and epidemiological importance of S. pseudintermedius, we encourage updating action by the system manufacturer to address AST for this bacterium.

Ethidium bromide exposure unmasks an antibiotic efflux system in Rhodococcus equi.

BACKGROUND: This study introduces a newly created strain (Rhodococcus equiEtBr25) by exposing R. equi ATCC 33701 to ethidium bromide (EtBr), a substrate for MDR transporters. Such an approach allowed us to investigate the resulting phenotype and genetic mechanisms underlying the efflux-mediated resistance in R. equi. METHODS: R. equi ATCC 33701 was stimulated with increasing concentrations of EtBr. The antimicrobial susceptibility of the parental strain and R. equiEtBr25 was investigated in the presence/absence of efflux pump inhibitors (EPIs). EtBr efflux was evaluated by EtBr-agar method and flow cytometry. The presence of efflux pump genes was determined by conventional PCR before to quantify the expression of 30 genes coding for membrane transporters by qPCR. The presence of erm(46) and mutations in 23S rRNA, and gyrA/gyrB was assessed by PCR and DNA sequencing to exclude the occurrence of resistance mechanisms other than efflux. RESULTS: R. equi EtBr25 showed an increased EtBr efflux. Against this strain, the activity of EtBr, azithromycin and ciprofloxacin was more affected than that of rifampicin and azithromycin/rifampicin combinations. Resistances were reversed by combining the antimicrobials with EPIs. Gene expression analysis detected a marked up-regulation of REQ_RS13460 encoding for a Major Facilitator Superfamily (MFS) transporter. G→A transition occurred in the transcriptional repressor tetR/acrR adjacent to REQ_RS13460. CONCLUSIONS: Exposure of R. equi to EtBr unmasked an efflux-mediated defence against azithromycin and ciprofloxacin, which seemingly correlates with the overexpression of a specific MFS transporter. This genotype may mirror an insidious low-level resistance of clinically important isolates that could be countered by EPI-based therapies.

Detection of anti-HEV antibodies and RNA of HEV in pigs from a hyperendemic Italian region with high human seroprevalence.

BACKGROUND: Pigs are considered the main reservoir of genotypes 3 and 4 of hepatitis E virus (HEV), which is the major cause of acute hepatitis of viral origin in humans worldwide. An increasing number of autochthonous HEV infections have been observed in recent years in industrialized countries, most likely as a result of zoonotic transmission through the consumption of raw or undercooked meat products. METHODS: Two hundred and thirty-three blood and liver samples were collected at four different local slaughterhouses from domestic pigs bred in Abruzzo, a region of south-central Italy, where there is the highest human seroprevalence to HEV compared with the rest of Italy. An indirect enzyme-linked immunosorbent assay kit was used for detecting anti-HEV IgG in the sera, while the presence of HEV RNA was investigated by performing a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Between 87.3% and 100% of swine serum samples collected in different slaughterhouses of Abruzzo were positive for anti-HEV antibodies. Conversely, none of the liver samples collected from the same animals were positive for HEV by real-time RT-PCR. CONCLUSIONS: The hypothesis of foodborne zoonotic transmission from local pigs as responsible for the hyperendemic status of Abruzzo cannot be corroborated. However, the high seroprevalence observed in pigs indicates that HEV is highly circulating in these territories. We propose to further investigate the role of wild fauna and trade in carrier pigs, and the maintenance of HEV virulence in the environment and meat supply chain to shed light on the possible sources of human infection and the degree of occupational risk.

Preclinical Models of Nontuberculous Mycobacteria Infection for Early Drug Discovery and Vaccine Research.

Nontuberculous mycobacteria (NTM) represent an increasingly prevalent etiology of soft tissue infections in animals and humans. NTM are widely distributed in the environment and while, for the most part, they behave as saprophytic organisms, in certain situations, they can be pathogenic, so much so that the incidence of NTM infections has surpassed that of Mycobacterium tuberculosis in developed countries. As a result, a growing body of the literature has focused attention on the critical role that drug susceptibility tests and infection models play in the design of appropriate therapeutic strategies against NTM diseases. This paper is an overview of the in vitro and in vivo models of NTM infection employed in the preclinical phase for early drug discovery and vaccine development. It summarizes alternative methods, not fully explored, for the characterization of anti-mycobacterial compounds.

Phenotypic Characterization of Rhodococcus equi Biofilm Grown In Vitro and Inhibiting and Dissolving Activity of Azithromycin/Rifampicin Treatment.

Microbial biofilm has been implicated in a wide range of chronic infections. In spite of the fact that Rhodococcus equi is a recognized cause of chronic disease in animals and humans, few studies have focused on the sessile phenotype of R. equi. The aim of this research was to phenotypically characterize the biofilm development of R. equi and its answerability for hypo-responsiveness to macrolides and rifampicin. Biofilm formation is initiated by bacterial adhesion to the surface. In this work, the ability of R. equi to adhere to the surface of human lung epithelial cells was detected by a fluorometric adhesion test performed on 40 clinical isolates. Subsequently, the capability of R. equi to produce biofilm was investigated by colorimetric, fluorescence and scanning electron microscopy analysis, revealing a general slow growth of rhodococcal biofilm and different sessile phenotypes among field isolates, some also including filamented bacteria. Azithromycin treatment produced a higher long-term inhibition and dissolution of R. equi biofilms than rifampicin, while the two antibiotics combined boosted the anti-biofilm effect in a statistically significant manner, although this was not equally effective for all R. equi isolates. Increasing the MIC concentrations of drugs tenfold alone and in combination did not completely eradicate pre-formed R. equi biofilms, while a rifampicin-resistant isolate produced an exceptionally abundant extracellular matrix. These results have strengthened the hypothesis that biofilm production may occur as an antibiotic tolerance system in R. equi, potentially determining persistence and, eventually, chronic infection.

Temporal efficacy of antimicrobials against aerobic bacteria isolated from equine endometritis: an Italian retrospective analysis (2010-2017).

This study aimed to describe bacteria isolated from the reproductive tract of mares and to identify changes in antimicrobial susceptibility patterns to those antibiotics commonly used for the treatment of equine endometritis. A total of 4122 equine uterine swabs were collected from mares suffering from reproductive tract disorders in the period 2010-2017. Aerobic culture and antimicrobial susceptibility testing using agar disc diffusion were performed on each sample. Aerobic bacteria were isolated from 3171 of 4122 (76.9 per cent) samples. The most frequently isolated microorganisms were Escherichia coli (885/3171, 27.9 per cent) and Streptococcus equi subspecies zooepidemicus (791/3171, 24.9 per cent), confirming previous findings from the literature. Antimicrobial susceptibility patterns of E coli, S equi subspecies zooepidemicus and Klebsiella pneumoniae changed over time. A statistically significant decrease in antimicrobial efficacy of cefquinome against E coli was observed over the years, as well as of ampicillin, cefquinome and penicillin against S equi subspecies zooepidemicus The high frequency of resistant bacteria isolated in the present work proceeds in the same way as indicated by surveillance data on the huge antibiotic use in Italy. As a result, testing and monitoring programmes of antimicrobial efficacy are crucial to consciously using antibiotics and preserving their effectiveness both for veterinary and human medicine.