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.

In Vitro Antimicrobial Activity of Essential Oils Against Salmonella enterica Serotypes Enteritidis and Typhimurium Strains Isolated from Poultry.

Salmonella enterica serotype Enteritidis and S. enterica serotype Typhimurium are frequently present among poultry and are associated with outbreaks of human salmonellosis. The study investigated the in vitro antimicrobial activity of essential oils (EOs) obtained from Aloysia triphylla, Cinnamomum zeylanicum, Cymbopogon citratus, Litsea cubeba, Mentha piperita, Syzygium aromaticum against S. Enteritidis and S. Thyphimurium strains previously isolated from poultry. A 1:1 mixture of C. zeylanicum and S. aromaticum was also tested. The activity of all compounds was evaluated against the yeast Saccharomyces cerevisiae, commonly used as probiotic. The highest antibacterial activity was observed for C. zeylanicum (minimum inhibitory concentrations (MICs) ranging from 1.26 mg/mL to 0.63 mg/mL), S. aromaticum (MICs from 2.637 mg/mL to 0.164 mg/mL) and the mixture (MICs from 1.289 mg/mL to 0.322 mg/mL). No activity was recorded against S. cerevisiae. The results suggest a possible use of C. zeylanicum and S. aromaticum, alone or in combination, in the farm environment for disinfection and in poultry diet, combined with S. cerevisiae administration, for an integrated approach to avoid Salmonella intestinal colonization.

Antimicrobial Activity of Five Essential Oils against Bacteria and Fungi Responsible for Urinary Tract Infections.

Urinary tract infections are frequently encountered in small animal practice. Escherichia coli and Enterococcus spp. are the most common agents associated to these infections, even though other bacteria and yeasts, such as Candida albicans and Candida famata, may be involved. In view of the increasing problem of the multi-drug resistance, the aim of this study was to investigate the antimicrobial activity of essential oils obtained from star anise (Illicium verum Hook.f.), basil (Ocimum basilicum L.), origanum (Origanum vulgare L.), clary sage (Salvia sclarea L.) and thymus (Thymus vulgaris L.) against multidrug-resistant strains of Escherichia coli, Enterococcus spp., Candida albicans and Candida famata previously isolated from dogs and cats with urinary tract infections. Enterococci were resistant to Illicium verum and Salvia sclarea, such as Candida to Salvia sclarea. Thymus vulgaris and Origanum vulgare essential oils showed the best activity against all the tested pathogens, so they could be proposed for the formulation of external and/or intravesical washes in small animals.

Activity of Salvia dolomitica and Salvia somalensis Essential Oils against Bacteria, Molds and Yeasts.

Essential oils (EOs) from Salvia dolomitica and Salvia somalensis, widely employed in the cosmetic and perfume industry, were analyzed for composition and tested against bacterial and fungal pathogens isolated from clinical and environmental specimens. The analyses were carried out against Staphylococcus aureus, Staphylococcus pseudointermedius, Pseudomonas aeruginosa, Escherichia coli, Streptococcus canis, Streptococcus pyogenes, Klebsiella pneumoniae, Proteus mirabilis, Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Aspergillus niger, Aspergillus flavus, Candida albicans, Candida krusei, Mucor sp. and Trichothecium roseum. Both EOs showed similar percentages of total monoterpenes and sesquiterpene hydrocarbons. The main constituents were 1,8-cineole and ß-caryophyllene in S. dolomitica and bornyl acetate and camphor in S. somalensis. The selected EOs have no relevant antifungal or antibacterial activities if compared to conventional drugs.

Molecular Survey on the Occurrence of Tick-Borne Bacteria in Wild Birds from Central Italy.

Birds are known to be carriers of ticks infected by tick-borne pathogens, including bacteria. However, not many studies have been carried out on avian tissues to detect these agents. The aim of the present survey was to investigate, using PCR, the presence of Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Chlamydia psittaci, Coxiella burnetii, Ehrlichia canis, Francisella tularensis, and Rickettsia spp. in the spleens collected from 300 wild birds of different orders and species from Central Italy. A total of 53 (17.67%) samples were PCR positive for at least one investigated pathogen. One (0.33%) bird was positive for Bartonella spp., five (1.67%) birds were positive for C. burnetii, eleven (3.67%) for B. burgdorferi s.l., and thirty-six (12%) for C. psittaci. No coinfection was detected. All samples were negative for A. phagocytophilum, E. canis, F. tularensis, and Rickettsia spp. The findings showed that wild birds may harbor different zoonotic tick-borne bacteria; therefore, they can contribute to the diffusion of these agents.

Occurrence of Antimicrobial-Resistant Enterococcus spp. in Healthy Chickens Never Exposed to Antimicrobial Agents in Central Italy.

Enterococci are part of the natural flora of the gastrointestinal tract of mammals, including humans, birds and invertebrates. They can cause infection, mainly among hospitalized patients, as well as acquire and transfer antimicrobial resistance genes. The present study allowed the isolation of 98 Enterococcus (73.47% E. faecium, 23.47% E. faecalis, 3.06% E. avium) strains from 120-day-old healthy chickens that had never been treated with antimicrobials. Their antimicrobial resistance was evaluated by the agar disk diffusion method; high-level aminoglycoside (streptomycin and gentamicin) and vancomycin resistance were established using the microbroth dilution method. The highest percentages of resistant isolates were detected with quinupristin-dalfopristin (88.78%), rifampicin (64.29%), tetracyclines (45.92%), and enrofloxacin (41.84%). High percentages of susceptible strains were found with teicoplanin (100%), amoxicillin-clavulanic acid (97.96%), nitrofurantoin (94.90%), ampicillin (92.86%), chloramphenicol (90.82%), and linezolid (88.78%). About 60% of the strains were classified as MDR (multidrug-resistant). Moreover, PCR was carried out to investigate genes encoding for tetracyclines resistance determinants: tet(M), tet(L), tet(O), tet(K), and Int-Tn. Genes were detected in 68 (69.38%) strains: 36 were shown to be resistant with the agar disk diffusion method, while 28 were intermediate, and 2 were susceptible. The present study showed that chickens never treated with antimicrobials potentially harbor enterococci having phenotypic and genotypic characters of antimicrobial resistance.

Preliminary evaluation of probiotic potential of Lactobacillus plantarum strains isolated from Italian food products.

The aim of this study was to investigate some probiotic properties of 42 wild Lactobacillus plantarum strains isolated from different Italian foods of animal origin. The strains were first screened for their antibiotic resistance profile (chloramphenicol, erythromycin, gentamicin, and tetracycline), subsequently they were tested for their in vitro resistance to lysozyme (100 mg L⁻¹), low pH (3.0, 2.5 and 2.0) and bile salts (0.3, 0.5 and 1.0 %). Moreover, agglutination property was studied (adhesion to Saccharomyces cerevisiae cells), as well as the presence of bsh and msa genes. The strains with the best characteristics were subjected to a further trial in order to evaluate their ability to survive to multiple stresses over time (lysozyme, low pH and bile salts) and the effect of these treatments on adhesion to yeast cells. All the strains were susceptible to chloramphenicol, erythromycin and gentamicin, while 6 strains were excluded from further evaluation because of their resistant phenotype against tetracycline. All the strains were able to grow in presence of lysozyme, as well as in MRS broth at pH 3.0. Only 4 strains showed a growth rate lower than 80 % when grown in MRS broth at pH 2.5, while a relevant growth rate decrease was observed after exposure to pH 2.0. Bile salts didn't affect the viability of the L. plantarum cells. Twenty-one strains out of 33 tested strains were able to adhere to S. cerevisiae cells. Presence of both bsh and msa genes was detected in 6 strains. The strains resistant to all the stresses, positive to agglutination with S. cerevisiae and showing bsh and msa genes were selected for further evaluation and subjected to different stress treatments over time. The assessment of growth rates showed that exposure to lysozyme significantly increased low pH resistance in L. plantarum. This increase ranged from 2.35 to 15.57 %. The consequential lysozyme and low pH exposures didn't affect the growth rate values after bile salts treatment, as well as the ability of the strains to adhere to yeast cells wasn't modified by previous treatments (lysozyme, low pH and bile salts). The present work allows to increase knowledge about non starter lactic acid bacteria from Italian food products. The studied L. plantarum strains showed a good potential for their use as probiotic cultures. However, more in vivo tests are necessary to confirm this potentiality.

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.

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.

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.

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.

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 [...].

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.

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.

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.

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.

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.

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).