The prevalence of Pseudomonas aeruginosa and multidrug resistant Pseudomonas aeruginosa in healthy captive ophidian.

BACKGROUND: Snakes are globally considered as pet animals, and millions of ophidians are bred in captivity. Pseudomonas aeruginosa is 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 of P. aeruginosa isolated from the cloacal microbiota of a large population sample of healthy captive ophidians and to evaluate the statistical associations with farming conditions. METHODS: A 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 of P. aeruginosa isolates was evaluated through the Kirby-Bauer agar diffusion test. Statistical analyses were performed with the chi-square test. RESULTS: The prevalence of P. aeruginosa was 59.9%, and 35.5% of these strains were multidrug resistant (MDR). The prevalence of MDR P. aeruginosa was 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 of P. aeruginosa were observed depending on the farm size and snake family. Feeding thawed prey was associated with a higher P. aeruginosa and MDR P. aeruginosa prevalence. Moreover, snakes fed home-raised prey had a significantly higher MDR P. aeruginosa prevalence than snakes fed commercially available feed. Less frequent terrarium cleaning was associated with a higher MDR P. aeruginosa prevalence. On the other hand, snake reproductive status was not significantly associated with P. aeruginosa or MDR P. aeruginosa prevalence. All food, water and bedding samples were negative for P. aeruginosa presence. DISCUSSION: The overall P. aeruginosa prevalence 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 with P. aeruginosa and/or MDR P. aeruginosa prevalence. Good breeding management and proper antibiotic treatment of P. aeruginosa infections could help reduce the presence of P. aeruginosa and MDR P. aeruginosa in the gut microbiota of snakes and consequently reduce the risk to public health.

Novel Naja atra cardiotoxin 1 (CTX-1) derived antimicrobial peptides with broad spectrum activity.

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 ß-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.

Activity of AMP2041 against human and animal multidrug resistant Pseudomonas aeruginosa clinical isolates.

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.

In vitro antimicrobial activity of a gel containing antimicrobial peptide AMP2041, chlorhexidine digluconate and Tris-EDTA on clinical isolates of Pseudomonas aeruginosa from canine otitis.

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.

Antibiotic resistance in conjunctival and enteric bacterial flora in raptors housed in a zoological garden.

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.