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BackgroundSnakes are globally considered as pet animals, and millions of ophidians are bred in captivity.Pseudomonas aeruginosais a ubiquitous Gram-negative bacterium that can act as an opportunistic pathogen of man and animals and is frequently present in the oral and cloacal microbiota of healthy ophidians. It can cause severe clinical diseases and often shows antibiotic resistance. The aim of this study was to evaluate the prevalence and antibiotic resistance profiles ofP. aeruginosaisolated from the cloacal microbiota of a large population sample of healthy captive ophidians and to evaluate the statistical associations with farming conditions.MethodsA total of 419 cloacal swabs were collected from snakes belonging to the Boidae (n = 45), Colubridae (n = 48) and Pythonidae (n = 326) families and inoculated onto complete culture media. Food, water and bedding samples were also analyzed. The antimicrobial susceptibility ofP. aeruginosaisolates was evaluated through the Kirby-Bauer agar diffusion test. Statistical analyses were performed with the chi-square test.ResultsThe prevalence ofP. aeruginosawas 59.9%, and 35.5% of these strains were multidrug resistant (MDR). The prevalence of MDRP. aeruginosawas significantly higher in adult samples than in young samples, and widespread resistance to Cephalosporins, Polymyxins and Sulfonamides was observed. Statistically significant differences in the prevalence ofP. aeruginosawere observed depending on the farm size and snake family.Feeding thawed prey was associated with a higherP. aeruginosaand MDRP. aeruginosaprevalence. Moreover, snakes fed home-raised prey had a significantly higher MDRP. aeruginosaprevalence than snakes fed commercially available feed. Less frequent terrarium cleaning was associated with a higher MDRP. aeruginosaprevalence. On the other hand, snake reproductive status was not significantly associated withP. aeruginosaor MDRP. aeruginosaprevalence. All food, water and bedding samples were negative forP. aeruginosapresence.DiscussionThe overallP. aeruginosaprevalence found in this study was lower than that found by other authors, but a high proportion of the isolates were MDR. This study highlighted the presence of constitutive (such as age and taxonomic family) and managerial (farm size, cleaning cycle frequency and food type) factors associated withP. aeruginosaand/or MDRP. aeruginosaprevalence. Good breeding management and proper antibiotic treatment ofP. aeruginosainfections could help reduce the presence ofP. aeruginosaand MDRP. aeruginosain the gut microbiota of snakes and consequently reduce the risk to public health.

Antimicrobial resistance (AMR) in a wide range of infectious agents is a growing public health threat. Birds of prey are considered indicators of the presence of AMR bacteria in their ecosystem because of their predatory behaviour. Only few data are reported in the literature on AMR strains isolated from animals housed in zoos and none about AMR in raptors housed in zoological gardens. This study investigated the antibiotic sensitivity profile of the isolates obtained from the conjunctival and cloacal bacterial flora of 14 healthy birds of prey, 6 Accipitriformes, 3 Falconiformes and 5 Strigiformes, housed in an Italian zoological garden. Staphylococcus spp. was isolated from 50% of the conjunctival swabs, with S. xylosus as the most common species. From cloacal swabs, Escherichia coli was cultured from all animals, while Klebsiella spp. and Proteus spp. were isolated from a smaller number of birds. Worthy of note is the isolation of Escherichia fergusonii and Serratia odorifera, rarely isolated from raptors. Staphylococci were also isolated. All the isolates were multidrug resistant (MDR). To the author's knowledge, this is the first report regarding the presence of MDR strains within raptors housed in a zoological garden. Since resistance genes can be transferred to other pathogenic bacteria, this represents a potential hazard for the emergence of new MDR pathogens. In conclusion, the obtained data could be useful for ex‐situ conservation programmes aimed to preserve the health of the endangered species housed in a zoo.

The bacterium Pseudomonas aeruginosa (PA) and the yeast Candida albicans (CA) are pathogens that cohabit the mucosa of the respiratory tracts of animals and humans. Their virulence is largely determined by chemical communication driven by quorum sensing systems (QS), and the cross perception of their quorum sensing molecules (QSM) can modulate the prevalence of one microorganism over the other. Aiming to investigate whether some of the protein components dissolved in the mucus layering the respiratory mucosa might interfere with virulence and cross-communication of these, and eventually other microorganisms, ligand binding assays were carried out to test the scavenging potential of the bovine and porcine forms of the Lipocalin odorant binding protein (OBP) for several QSMs (farnesol, and acylhomoserine lactones), and for pyocyanin, a toxin produced by PA. In addition, the direct antimicrobial activity of the OBPs was tested by time kill assay (TKA) against CA, PA and other bacteria and yeasts. The positivity of all the ligand binding assays and the antimicrobial activity determined for CA, and for some of the other microorganisms tested, let hypothesize that vertebrate OBPs might behave as humoral components of innate immunity, active against pathogenic bacteria and fungi. In addition, TKAs with mutants of bovine OBP with structural properties different from those of the native form, and with OBP forms tagged with histidines at the amino terminal, provided information about the mechanisms responsible of their antimicrobial activity and suggested possible applications of the OBPs as alternative or co-adjuvants to antibiotic therapeutic treatments.

Naja atra subsp. atra cardiotoxin 1 (CTX-1), produced by Chinese cobra snakes, belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. Using as template mainly the tip and the subsequent β-strand of the first "finger" of this toxin, different sequences of 20 amino acids linear peptides have been designed in order to avoid toxic effects but to maintain or even strengthen the partial antimicrobial activity already seen for the complete toxin. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently 4 other variant sequences of NCP-0 were developed. These synthesized variant sequences have shown microbicidal activity towards a panel of reference and field strains of Gram-positive and Gram-negative bacteria. The sequence named NCP-3, and its variants NCP-3a and NCP-3b, have shown the best antimicrobial activity, together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 μg/ml for most of the tested bacterial strains. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 50-6.3 μg/ml), and against the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Moreover, NCP-3 has shown virucidal activity on Bovine Herpesvirus 1 (BoHV1) belonging to Herpesviridae family. The bactericidal activity is maintained even in a high salt concentration medium (125 and 250 mM NaCl) and phosphate buffer with 20% Mueller Hinton (MH) medium against E. coli, methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, the search for active sequences within proteins presenting an intrinsic microbicidal activity could provide a new way for discovering a large number of novel and promising antimicrobial peptides families.

Background Antimicrobial resistance is a growing threat to public health. Pseudomonas aeruginosa is a relevant pathogen causing human and animal infections, frequently displaying high levels of resistance to commonly used antimicrobials. The increasing difficulty to develop new effective antibiotics have discouraged investment in this area and only a few new antibiotics are currently under development. An approach to overcome antibiotic resistance could be based on antimicrobial peptides since they offer advantages over currently used microbicides. Methods The antimicrobial activity of the synthetic peptide AMP2041 was evaluated against 49 P. aeruginosa clinical strains with high levels of antimicrobial resistance, isolated from humans (n = 19) and animals (n = 30). In vitro activity was evaluated by a microdilution assay for lethal dose 90% (LD90), while the activity over time was performed by time-kill assay with 12.5 µg/ml of AMP2014. Evidences for a direct membrane damage were investigated on P. aeruginosa ATCC 27853 reference strain, on animal isolate PA-VET 38 and on human isolate PA-H 24 by propidium iodide and on P. aeruginosa ATCC 27853 by scanning electron microscopy. Results AMP2041 showed a dose-dependent activity, with a mean (SEM) LD90 of 1.69 and 3.3 µg/ml for animal and human strains, respectively. AMP2041 showed microbicidal activity on P. aeruginosa isolates from a patient with cystic fibrosis (CF) and resistance increased from first infection isolate (LD90 = 0.3 μg/ml) to the mucoid phenotype (LD90 = 10.4 μg/ml). The time-kill assay showed a time-dependent bactericidal effect of AMP2041 and LD90 was reached within 20 min for all the strains. The stain-dead assay showed an increasing of membrane permeabilization and SEM analysis revealed holes, dents and bursts throughout bacterial cell wall after 30 min of incubation with AMP2041. Conclusions The obtained results assessed for the first time the good antimicrobial activity of AMP2041 on P. aeruginosa strains of human origin, including those deriving from a CF patient. We confirmed the excellent antimicrobial activity of AMP2041 on P. aeruginosa strains derived from dog otitis. We also assessed that AMP2041 antimicrobial activity is linked to changes of the P. aeruginosa cell wall morphology and to the increasing of membrane permeability.

The emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria has become a medical and veterinary problem. Antimicrobial peptides (AMPs) show potential to overcome antibiotic resistance and could be used therapeutically. A novel AMP (AMP2041) was developed in silico and its microbiocidal activity against MDR clinical strains isolated from cattle (n = 6), dogs (n = 8), and pigs (n = 20) was evaluated. AMP2041 showed strong antimicrobial activity against all Gram-positive and Gram-negative MDR clinical strains tested. Within 20 min of incubation, there was complete killing of Pseudomonas aeruginosa ATCC 27953 and a 90% reduction of colony count for Escherichia coli ATCC 25922. For Staphylococcus aureus ATCC 25923, a 90% reduction of colony count was observed within 120 min of incubation.

Antimicrobial-peptide-based therapies could represent a reliable alternative to overcome antibiotic resistance, as they offer potential advantages such as rapid microbicidal activity and multiple activities against a broad spectrum of bacterial pathogens. Three synthetic antimicrobial peptides (AMPs), AMP72, AMP126, and also AMP2041, designed by using ad hoc screening software developed in house, were synthesized and tested against nine reference strains. The peptides showed a partial β-sheet structure in 10-mM phosphate buffer. Low cytolytic activity towards both human cell lines (epithelial, endothelial, and fibroblast) and sheep erythrocytes was observed for all peptides. The antimicrobial activity was dose dependent with a minimum bactericidal concentration (MBC) ranging from 0.17 to 10.12 μM (0.4–18.5 µg/ml) for Gram-negative and 0.94 to 20.65 μM (1.72-46.5 µg/ml) for Gram-positive bacteria. Interestingly, in high-salt environment, the antibacterial activity was generally maintained for Gram-negative bacteria. All peptides achieved complete bacterial killing in 20 min or less against Gram-negative bacteria. A linear time-dependent membrane permeabilization was observed for the tested peptides at 12.5 µg/ml. In a medium containing Mg2+ and Ca2+, the peptide combination with EDTA restores the antimicrobial activity particularly for AMP2041. Moreover, in combination with anti-infective agents (quinolones or aminoglycosides) known to bind divalent cation, AMP126 and AMP2041 showed additive activity in comparison with colistin. Our results suggest the following: (i) there is excellent activity against Gram-negative bacteria, (ii) there is low cytolytic activity, (iii) the presence of a chelating agent restores the antimicrobial activity in a medium containing Mg2+ and Ca2+, and (iv) the MBC value of the combination AMPs–conventional antibiotics was lower than the MBC of single agents alone. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

The aims of the study were to identify microbial flora in boar semen under field conditions in northern Italy, to investigate antibiotic resistance and sensitivity of isolated bacteria, and to evaluate elimination of bacteria after storage in two types of extenders added with different antibiotics (amikacin vs gentamicin). A total of 60 boars were collected in 13 pig farms. Bacteriological and mycological investigations were performed immediately on raw semen samples, then at 48 and 120 h of storage on semen diluted randomly in a new short-term modified extender (ME-S) or in a commercial one (CRONOSTM). Bacterial contamination was found in 63% of raw semen samples and different bacterial species were isolated: E.coli, Serratia marcescens, Staphylococcus epidermidis and aureus, Proteus spp., Streptococcus spp. and Pseudomonas aeruginosa. E. coli was the most isolated contaminant (53%); Pseudomonas aeruginosa was found only in one semen sample. The analysis of variance of factors affecting contamination levels was significant for the farm of origin (P

Background Pseudomonas aeruginosa (PA) may cause suppurative otitis externa with severe inflammation and ulceration in dogs. Multidrug resistance is commonly reported for this organism, creating a difficult therapeutic challenge. Objective The aim of this study was to evaluate the in vitro antimicrobial activity of a gel containing 0.5 μg/mL of antimicrobial peptide AMP2041, 0.07% chlorhexidine digluconate (CLX), 0.4% Tris and 0.1% EDTA on 30 clinical isolates of PA from canine otitis externa. Materials and Methods Antimicrobial activity was evaluated through minimal bactericidal concentration (MBC). Standardized bacterial suspensions were incubated with different concentrations of the gel at 37°C for 30 min and plated for colony forming unit (CFU) counts. Time-to-kill kinetics were evaluated with the undiluted product and at MBC for each PA strain at 30 s, 1, 5, 10, 15, 30 min, 24 and 48 h. Results The MBC was 1:64 for two of 30 strains, 1:128 for 15 of 30 strains and 1:256 for 13 of 30 strains. The geometric mean was 1:165, equivalent to a concentration of 0.003 μg/mL AMP2041 + 0.0004% CLX + 0.0024%Tris + 0.0006% EDTA. Time-to-kill assays with the undiluted product showed complete bactericidal effect within 30 s for all isolates, whereas at the MBC this effect was reached within 5 min for 20 of 30 isolates and within 30 min for all isolates. Bactericidal activity was maintained after 48 h for all isolates. Conclusion This gel has shown rapid, complete and long-lasting activity against a panel of 30 PA isolates from cases of canine otitis externa.

Background Lichtheimia corymbifera (previously Absidia corymbifera) is a filamentous zygomycetes belonging to the order Mucorales and to the family Lichtheimiaceae. Members of genus Lichtheimia spp. are cosmopolitan and ubiquitous in nature. Lichtheimia corymbifera is a recognized agent of diseases in man and animals. In cattle it causes abortion and mastitis. Three cases of bovine abortion occurred in a herd located in the Po Valley. Serological examinations were performed on fetal and mother's blood. One of the aborted fetus was referred to our laboratory. The paper describes the isolation and characterization of Lichtheimia corymbifera from a bovine aborted fetus. Methods Serological examinations were performed on fetal and mother's blood. Lesions on fetal tissues and placenta leaded the diagnostic suspect towards a mycotic aetiology. Tissues were then put in culture, and at the same time an histological examination was performed, together with bacteriological and virological tests. The isolate from placenta and fetal tissues was identified and characterized by PCR and RFLP, using the ITS region as a target sequence and AclI restriction site within the amplicon to distinguish Lichtheimia corymbifera among the other fungi. Results Serological, bacteriological and virological tests gave aspecific results. Histological examination evidenced numerous PAS positive hyphae within the necrotic cotiledons and numerous fungal nonseptate hyphae to the GMS stain. Colonies with typical morphological features of fungi grew up on Sabouraud agar from fetal skin and placenta. On the developed colonies the microscopic examination has shown a large number of nonseptate hyphae and sporangia consistent with Mucorales. PCR and RFLP allowed the identification of the isolate as Lichtheimia corymbifera. Conclusion The present report describes the isolation and the molecular characterisation of a fungal isolate from bovine aborted fetus and placenta. The diagnostic protocol allowed to identify and characterise the strain. This is the first isolation in Italy of Lichtheimia corymbifera in a bovine aborted fetus.