Isolation and characterization of novel Staphylococcus aureus bacteriophage Hesat from dairy origin.

A novel temperate phage, named Hesat, was isolated by the incubation of a dairy strain of Staphylococcus aureus belonging to spa-type t127 with either bovine or ovine milk. Hesat represents a new species of temperate phage within the Phietavirus genus of the Azeredovirinae subfamily. Its genome has a length of 43,129 bp and a GC content of 35.11% and contains 75 predicted ORFs, some of which linked to virulence. This includes (i) a pathogenicity island (SaPln2), homologous to the type II toxin-antitoxin system PemK/MazF family toxin; (ii) a DUF3113 protein (gp30) that is putatively involved in the derepression of the global repressor Stl; and (iii) a cluster coding for a PVL. Genomic analysis of the host strain indicates Hesat is a resident prophage. Interestingly, its induction was obtained by exposing the bacterium to milk, while the conventional mitomycin C-based approach failed. The host range of phage Hesat appears to be broad, as it was able to lyse 24 out of 30 tested S. aureus isolates. Furthermore, when tested at high titer (108 PFU/ml), Hesat phage was also able to lyse a Staphylococcus muscae isolate, a coagulase-negative staphylococcal strain. KEY POINTS: • A new phage species was isolated from a Staphylococcus aureus bovine strain. • Pathogenicity island and PVL genes are encoded within phage genome. • The phage is active against most of S. aureus strains from both animal and human origins.

Antibacterial activity in egg samples from small-spotted catshark Scyliorhinus canicula and nursehound Scyliorhinus stellaris: A preliminary investigation.

The study aims to identify antibacterial properties in unfertilized eggs of Scyliorhinus canicula and Scyliorhinus stellaris. Despite challenging marine conditions, these eggs retain their integrity for extended periods and remain unaffected by pathogens. The antibacterial activity was measured using minimum inhibitory and minimum bactericidal concentration analysis. The eggs of S. stellaris exhibited a slight inhibitory effect against Staphylococcus aureus and Listeria monocytogenes, whereas both species' eggs showed no activity against gram-negative microorganisms.

Feline Parvovirus Lethal Outbreak in a Group of Adult Cohabiting Domestic Cats.

Feline panleukopenia is a highly contagious and often fatal disease in cats. The virus, known as feline panleukopenia virus (FPV), primarily affects kittens and unvaccinated cats. It is transmitted through contact with infected cats or their bodily fluids, as well as contaminated objects and environments. The diagnosis of FPV infection can be confirmed through a combination of clinical signs, blood tests, and fecal testing. Prevention through vaccination is recommended for all cats. This case report describes an outbreak of feline panleukopenia in a group of unvaccinated domestic cats that resulted in acute mortality. The lesions were evaluated using histopathology, and the specific viral strain was characterized using molecular techniques. The clinical course of the outbreak was peracute, with a hemorrhagic pattern and 100% of lethality. The observed clinical-pathological pattern was unusual; nevertheless, molecular studies did not highlight peculiar genomic features of the parvovirus isolate. The outbreak affected 3 out of 12 cats in a very short time. However, the prompt application of biosecurity measures and vaccination resulted in an effective interruption of virus spread. In conclusion, we could assume that the virus found the ideal conditions to infect and replicate at high titers, resulting in a particularly aggressive outbreak.

Colistin Resistance Genes in Broiler Chickens in Tunisia.

Colistin is a polymyxin antibiotic that has been used in veterinary medicine for decades, as a treatment for enterobacterial digestive infections as well as a prophylactic treatment and growth promoter in livestock animals, leading to the emergence and spread of colistin-resistant Gram-negative bacteria and to a great public health concern, considering that colistin is one of the last-resort antibiotics against multidrug-resistant deadly infections in clinical practice. Previous studies performed on livestock animals in Tunisia using culture-dependent methods highlighted the presence of colistin-resistant Gram-negative bacteria. In the present survey, DNA extracted from cloacal swabs from 195 broiler chickens from six farms in Tunisia was tested via molecular methods for the ten mobilized colistin resistance (mcr) genes known so far. Of the 195 animals tested, 81 (41.5%) were mcr-1 positive. All the farms tested were positive, with a prevalence ranging from 13% to 93%. These results confirm the spread of colistin resistance in livestock animals in Tunisia and suggest that the investigation of antibiotic resistance genes by culture-independent methods could be a useful means of conducting epidemiological studies on the spread of antimicrobial resistance.

Antimicrobial Resistant Coagulase-Negative Staphylococci Carried by House Flies (Musca domestica) Captured in Swine and Poultry Farms.

House flies (Musca domestica) are very diffuse insects attracted by biological materials. They are abundantly present in farm environments and can frequently come in contact with animals, feed, manure, waste, surfaces, and fomites; consequently, these insects could be contaminated, carry, and disperse several microorganisms. The aim of this work was to evaluate the presence of antimicrobial-resistant staphylococci in house flies collected in poultry and swine farms. Thirty-five traps were placed in twenty-two farms; from each trap, 3 different kinds of samples were tested: attractant material present in the traps, the body surface of house flies and the body content of house flies. Staphylococci were detected in 72.72% of farms, 65.71% of traps and 43.81% of samples. Only coagulase-negative staphylococci (CoNS) were isolated, and 49 isolates were subjected to an antimicrobial susceptibility test. Most of the isolates were resistant to amikacin (65.31%), ampicillin (46.94%), rifampicin (44.90%), tetracycline (40.82%) and cefoxitin (40.82%). Minimum Inhibitory concentration assay allowed to confirm 11/49 (22.45%) staphylococci as methicillin-resistant; 4 of them (36.36%) carried the mecA gene. Furthermore, 53.06% of the isolates were classified as multidrug-resistant (MDR). Higher levels of resistance and multidrug resistance were detected in CoNS isolated from flies collected in poultry farms than in swine farms. Therefore, house flies could carry MDR and methicillin-resistant staphylococci, representing a possible source of infection for animals and humans.

House Flies (Musca domestica) from Swine and Poultry Farms Carrying Antimicrobial Resistant Enterobacteriaceae and Salmonella.

The house fly (Musca domestica) is a very common insect, abundantly present in farm settings. These insects are attracted by organic substrates and can easily be contaminated by several pathogenic and nonpathogenic bacteria. The aim of this survey was to evaluate the presence of Salmonella spp. and other Enterobacteriaceae in house flies captured in small-medium size farms, located in Northwest Tuscany, Central Italy, and to evaluate their antimicrobial resistance; furthermore, isolates were tested for extended spectrum ß-lactamase and carbapenems resistance, considering the importance these antimicrobials have in human therapy. A total of 35 traps were placed in seven poultry and 15 swine farms; three different kinds of samples were analyzed from each trap, representing attractant substrate, insect body surface, and insect whole bodies. Enterobacteriaceae were isolated from 86.36% of farms, 82.87% of traps, and 60.95% of samples; high levels of resistance were detected for ampicillin (61.25% of resistant isolates) and tetracycline (42.5% of resistant isolates). One extended spectrum ß-lactamase producer strain was isolated, carrying the blaTEM-1 gene. Salmonella spp. was detected in 36.36% of farms, 25.71% of traps, and 15.24% of samples. Five different serovars were identified: Kentucky, Kisarawe, London, Napoli, and Rubislaw; some isolates were in R phase. Resistance was detected mainly for ampicillin (31.21%) and tetracycline (31.21%). House flies could represent a serious hazard for biosecurity plans at the farm level, carrying and sharing relevant pathogenic and antimicrobial resistant bacteria.

In Vitro Antimicrobial Activity of Thymus vulgaris, Origanum vulgare, Satureja montana and Their Mixture against Clinical Isolates Responsible for Canine Otitis Externa.

Otitis externa is a frequent inflammation among dogs, mainly caused by bacteria and yeasts that are often resistant to conventional drugs. The aim of the present study was to evaluate the in vitro antibacterial and antifungal activities of commercial essential oils (EOs) from Origanum vulgare, Satureja montana, and Thymus vulgaris, as well as a mixture of these three components, against 47 clinical bacterial strains (Staphylococcus sp., Streptococcus sp., Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens) and 5 Malassezia pachydermatis strains, previously cultured from the ears of dogs affected by otitis externa. The tested Gram-positive bacteria were sensible to the analysed EOs with MICs ranging from 1.25% (v/v) to <0.0195% (v/v); Gram-negative isolates, mainly P. aeruginosa, were less sensitive with MICs from >10% (v/v) to 0.039% (v/v). M. pachydermatis isolates were sensitive to all EOs with MICs from 4.25% (v/v) to 2% (v/v). However, the mixture was active against all bacterial (except one P. aeruginosa strain) and fungal tested isolates. The three EOs and their mixture seem to be an interesting alternative for treating canine otitis externa when conventional antimicrobials are not active.

Retrospective Molecular Survey on Bacterial and Protozoan Abortive Agents in Roe Deer (Capreolus capreolus) from Central Italy

Bacterial and protozoan agents can determine abortion and other reproductive disorders in domestic ruminants, but data regarding their occurrence in wild ruminants are scanty worldwide, including in Italy. The aim of this retrospective study was to verify the occurrence of the main bacterial and protozoan abortive agents in 72 spleen samples previously collected from roe deer (Capreolus capreolus) living in mountain areas of Central Italy. All samples were collected and submitted to DNA extraction for other investigations. Molecular analyses were carried out on the DNA samples to detect Brucella spp., Chlamydia abortus, Coxiella burnetii, Salmonella enterica, Listeria monocytogenes, Neospora caninum, and Toxoplasma gondii. Three (4.16%) roe deer resulted PCR positive for C. burnetii and one (1.38%) for T. gondii. These findings suggest that roe deer living in the investigated areas do not act as important reservoirs of the searched agents. However, the tested animals lived in a closed area without contact with domestic animals that are usually involved in the epidemiology of the investigated pathogens. Monitoring of wild ruminants is pivotal to verify changes in the epidemiological scenario from a One Health perspective, too.

Occurrence of Bacterial and Protozoan Pathogens in Red Foxes (Vulpes vulpes) in Central Italy.

Most surveys of pathogens in red foxes (Vulpes vulpes) have focused on particular agents. The aim of this study was to verify, with bacteriological and molecular analyses, the occurrence of the main bacterial and protozoan pathogens that are able to infect canids, in red foxes regularly hunted in Central Italy. Spleen, brain, kidney and fecal samples from red foxes were submitted to bacteriological and/or molecular analyses to detect Salmonella spp., Yersinia spp., Listeria monocytogenes, Brucella spp., Ehrlichia canis, Anaplasma phagocytophilum, Coxiella burnetii, Leptospira spp., Neospora caninum, Hepatozoon canis, Babesia spp. and microsporidia. Two (9.1%) strains of Yersinia enterocolitica biotype 1 and 2 (9.1%) of Yersinia frederiksenii were isolated from 22 fecal samples. Among the 22 spleen samples, seven (31.8%) were PCR-positive for H. canis and 3 (13.6%) for Babesia vulpes. Kidneys from two (2.9%) foxes, among 71 tested, were PCR-positive for L. interrogans. Even though the analyses were carried out on a small number of animals, the results suggested that red foxes from the selected geographic area may act as reservoirs of some investigated pathogens.

Editorial for the Special Issue: Colistin Resistance-The Need for a One Health Approach.

Colistin is an "old" antimicrobial belonging to the class of polymyxins, initially discovered in 1947 [...].

Virulence Genes of Pathogenic Escherichia coli in Wild Red Foxes (Vulpes vulpes).

Different pathotypes of Escherichia coli can cause severe diseases in animals and humans. Wildlife may contribute to the circulation of pathogenic pathotypes, including enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), and enterohemorrhagic E. coli (EHEC). This study analyzed 109 DNA samples previously extracted from fecal specimens collected from red foxes (Vulpes vulpes) to detect E. coli virulence genes eaeA, hlyA, stx1, and stx2, that characterize the EPEC, STEC, and EHEC strains. Thirty-one (28.4%) samples were positive for at least one investigated virulence gene: eaeA gene was detected in 21 (19.2%) samples, hlyA in 10 (9.1%), stx1 in 6 (5.5%), and stx2 in 4 (3.6%). Nine DNA samples resulted positive for two or three virulence genes: five (4.6%) samples were positive for eaeA and hlyA genes, two (1.8%) for eaeA and stx1, one (0.9%) for hlyA and stx1, one (0.9%) for eaeA, hlyA and stx2. Red foxes seem to be involved in the epidemiology of these infections and their role could be relevant because they may be source of pathogenic E. coli for other wild animals, as well as domestic animals and humans.

Antimicrobial-Resistant Enterococcus spp. in Wild Avifauna from Central Italy.

Bacteria of the genus Enterococcus are opportunistic pathogens, part of the normal intestinal microflora of animals, able to acquire and transfer antimicrobial resistance genes. The aim of this study was to evaluate the possible role of wild avifauna as a source of antimicrobial-resistant enterococci. To assess this purpose, 103 Enterococcus spp. strains were isolated from the feces of wild birds of different species; they were tested for antimicrobial resistance against 21 molecules, vancomycin resistance, and high-level aminoglycosides resistance (HLAR). Furthermore, genes responsible for vancomycin, tetracycline, and HLAR were searched. E. faecium was the most frequently detected species (60.20% of isolates), followed by E. faecalis (34.95% of isolates). Overall, 99.02% of the isolated enterococci were classified as multidrug-resistant, with 19.41% extensively drug-resistant, and 2.91% possible pan drug-resistant strains. Most of the isolates were susceptible to amoxicillin/clavulanic acid (77.67%) and ampicillin (75.73%), with only 5.83% of isolates showing an ampicillin MIC ≥ 64 mg/L. HLAR was detected in 35.92% of isolates, mainly associated with the genes ant(6)-Ia and aac(6')-Ie-aph(2″)-Ia. Few strains (4.85%) were resistant to vancomycin, and the genes vanA and vanB were not detected. A percentage of 54.37% of isolates showed resistance to tetracycline; tet(M) was the most frequently detected gene in these strains. Wild birds may contribute to the spreading of antimicrobial-resistant enterococci, which can affect other animals and humans. Constant monitoring is essential to face up to the evolving antimicrobial resistance issue, and monitoring programs should include wild avifauna, too.

Molecular survey on the presence of arthropod-borne bacteria and protozoans in roe deer (Capreolus capreolus) and ticks from Central Italy.

Environmental changes, due to climatic emergency and to anthropogenic activities severely impact on the epidemiology of vector borne diseases, mostly when transmitted by ticks. The data about the distribution of microorganisms responsible for them in roe deer (Capreolus capreolus) population living in Italy are scanty and completely lacking in Tuscany, so a molecular survey was carried out to estimate the prevalence of some zoonotic tick-borne pathogens in roe deer, and ticks removed from them, living in areas of Central Italy with high risk of arthropod exposure. Spleen samples from 72 roe deer were tested by PCR for Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Francisella tularensis and piroplasms. Moreover, 345 ticks were removed from 65 roe deer, morphologically or molecularly identified and grouped into 162 pools that were submitted to PCR for detecting the same pathogens. Forty-six (63.88%) roe deer were positive for at least one investigated pathogen: 43 (59.72%) for A. phagocytophilum, 2 (2.78%) for Babesia capreoli, 1 (1.39%) for B. burgdorferi, and 1 (1.39%) for Babesia sp. No animals were PCR positive for F. tularensis.  All ticks were identified as Ixodes ricinus. Seventy-six (46.91%) tick pools showed DNA of one or more pathogens: 66 (40.74%) were positive for A. phagocytophilum, 22 (13.58%) for B. burgodorferi s.l., 6 (3.70%) for B. venatorum and 3 (1.85%) for B. capreoli. No pools were positive for F. tularensis. Two or three pathogens were detected in 23 (14.19%) pools.

Molecular Detection of Avipoxvirus in Wild Birds in Central Italy.

Avipoxviruses (APVs) are important pathogens of both domestic and wild birds. The associated disease is characterized by skin proliferative lesions in the cutaneous form or by lesions of the first digestive and respiratory tracts in the diphtheritic form. Previous studies investigated these infections in symptomatic wild birds worldwide, including Italy, but data about the circulation of APVs in healthy avian wildlife are not available. The present study tested spleen samples from 300 wild birds without typical lesions to detect Avipoxvirus DNA. Overall, 43.33% of the samples scored positive. Aquatic birds were more frequently infected (55.42%) than other animals (26.40%), and in Anseriformes, high positivity was found (52.87%). The obtained results suggest that wild birds could be asymptomatic carriers of Avipoxviruses, opening new possible epidemiological scenarios.

Low Level of Colistin Resistance and mcr Genes Presence in Salmonella spp.: Evaluation of Isolates Collected between 2000 and 2020 from Animals and Environment.

Salmonellosis is one of the most important zoonoses in Europe and the world. Human infection may evolve in severe clinical diseases, with the need for hospitalization and antimicrobial treatment. Colistin is now considered an important antimicrobial to treat infections from multidrug- resistant Gram-negative bacteria, but the spreading of mobile colistin-resistance (mcr) genes has limited this option. We aimed to evaluate colistin minimum inhibitory concentration and the presence of mcr (mcr-1 to mcr-9) genes in 236 Salmonella isolates previously collected from different animals and the environment between 2000 and 2020. Overall, 17.79% of isolates were resistant to colistin; no differences were observed in relation to years of isolation (2000-2005, 2009-2014, and 2015-2020), Salmonella enterica subspecies (enterica, salamae, diarizonae, and houtenae), origin of samples (domestic animals, wildlife, and environment), or animal category (birds, mammals, and reptiles); only recently isolated strains from houseflies showed the most resistance. Few isolates (5.93%) scored positive for mcr genes, in particular for mcr-1, mcr-2, mcr-4, mcr-6, and mcr-8; furthermore, only 2.54% of isolates were mcr-positive and colistin-resistant. Detected resistance to colistin was equally distributed among all examined Salmonella isolates and not always related to the presence of mcr genes.

In vitro antimicrobial activity of selected essential oils against bacteria and yeasts isolated from the genital tract of mares.

The aim of the present study, the first to test clinical equine isolates, was to evaluate the antimicrobial activity of commercial essential oils (EOs) obtained from Origanum vulgare, Thymus vulgaris, Cinnamomum zeylanicum, Cymbopogon citratus, Aloysia triphylla and Syzygium aromaticum against Streptococcus spp., Staphylococcus spp., Candida albicans and Rhodotorula spp. previously isolated from uteri of mares with reproductive disorders. T. vulgaris showed the best antibacterial activity against all the selected agents, with MICs ranging from 0.07 mg/mL for S. equi subsp. zooepidemicus to 0.29 mg/mL versus Streptococcus sp. and Staphylococcus spp. isolates and versus C. albicans and Rhodotorula spp., with MICs of 0.46 mg/mL and 1.86 mg/mL, respectively. O. vulgare, S. aromaticum, C. zeylanicum EOs showed good antibacterial activity with MICs ranging from 0.07 mg/mL to 0.29 mg/mL, 0.16 mg/mL to 0.32 mg/mL, 0.07 mg/mL to 0.63 mg/mL, respectively; A. triphylla was the most active EO against Rhodotorula spp. (0.43 mg/mL).

Tetracycline Resistance Genes in Wild Birds from a Wildlife Recovery Centre in Central Italy.

Wild animals are less likely to be exposed directly to clinical antimicrobial agents than domestic animals or humans, but they can acquire antimicrobial-resistant bacteria through contact with humans, animals, and the environment. In the present study, 254 dead free-living birds belonging to 23 bird species were examined by PCR for the presence of tetracycline resistance (tet) genes. A fragment of the spleen was collected from each bird carcass. A portion of the intestine was also taken from 73 of the 254 carcasses. Extracted DNA was subjected to PCR amplification targeting the tet(L), tet(M), and tet(X) genes. In total, 114 (45%) of the 254 birds sampled belonging to 17 (74%) of the 23 bird species tested were positive for one or more tet genes. The tet(M) gene showed a higher frequency than the other tested genes, both in the spleen and in the intestine samples. These results confirm the potential role of wild birds as reservoirs, dispersers, or bioindicators of antimicrobial resistance in the environment.

Characterization and Comparison of Enterococcus spp. Isolates from Feces of Healthy Dogs and Urine of Dogs with UTIs.

Enterococcus spp. are opportunistic pathogens of both humans and animals characterized by high resistance to antimicrobials. Dogs could be intestinal carriers or suffer from Enterococcus infections, mainly urinary tract infections (UTIs). This study aimed to analyze and compare Enterococcus spp. isolated from healthy dog stools and sick dog urine. Overall, 51 isolates (29 from stools and 22 from UTI) were characterized at species level and tested for antimicrobial resistance, biofilm production and presence of resistance and virulence genes. E. faecium and E. faecalis resulted as equally distributed in stools samples, while E. faecalis predominated among UTI isolates. HLAR phenotype was detected in 47.1% isolates; 64.7% isolates were resistant to ampicillin (47.1% with a MIC ≥ 64 µg/mL). High levels of resistance were recorded for fluoroquinolones (enrofloxacin 74.5%, ciprofloxacin 66.7%), clindamycin (84.3%), tetracycline (78.4%) and quinupristin-dalfopristin (78.4%). No vancomycin resistant strains were detected. All but one isolate were multidrug-resistant. Most detected resistance genes were tetM (70.5%), pbp4 (52.9%) and aph(3')-IIIa (39.2%). All isolates were able to produce biofilm, but isolates from UTIs and belonging to E. faecalis more frequently resulted in strong biofilm producers. Most detected virulence genes were asa1 (52.9%), gelE (41.2%), cylA (37.3%) and esp (35.3%); all of them resulted as more frequently associated to E. faecalis. No particular differences emerged between isolates from feces and UTI, considering all evaluated aspects. Our results confirm pet dogs as carriers of multidrug-resistant enterococci; stool microflora could be considered as the most probable source of enterococcal UTI and E. faecalis carried by dogs seems to be more virulent than E. faecium, justifying its more frequent involvement in urinary tract infections.

Tear Production, Intraocular Pressure, Ultrasound Biometric Features and Conjunctival Flora Identification in Clinically Normal Eyes of Two Italian Breeds of Chicken (Gallus gallus domesticus).

Given the abundance of chickens in Italy, it is important for veterinarians to know the normal state of chickens' eyes in order to identify any ophthalmic pathological changes. The aim of this study was to determine the normal values of select ocular parameters and to evaluate conjunctival microflora in two Italian chicken breeds. Sixty-six healthy chickens underwent a complete ophthalmic examination, which included a phenol red thread test (PRTT) for the evaluation of tear production and the assessment of intraocular pressure by rebound tonometry. B-mode ultrasound biometric measurements and conjunctival microflora identification were also performed in twenty-seven chickens. Mean PRTT was 23.77 ± 2.99 mm/15 s in the Livorno breed and 19.95 ± 2.81 mm/15 s in the Siciliana breed. Mean intraocular pressure was 14.3 ± 1.17 mmHg in the Livorno breed and 14.06 ± 1.15 mmHg in the Siciliana breed. Reference ranges for morphometric parameters were reported in the two breeds. Twenty-three chickens (85.18%) were bacteriologically positive. Chlamydia spp. antigen was detected in 14.81% of chickens. No positive cultures were obtained for fungi. Normal reference range values for selected ophthalmic parameters were obtained in clinically healthy chickens, which could facilitate accurate diagnosis and better management of ophthalmic diseases in these animals.