Antibacterial activities of Manuka and Honeydew honey-based membranes against bacteria that cause wound infections in animals.

INTRODUCTION: In this study, membranes composed of honey (Manuka or Honeydew) and pectin were developed, and the ISO 22196 method was used to evaluate their antibacterial activities against multidrug-resistant bacteria (i.e., Staphylococcus pseudointermedius, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa) that cause wound infection in animals. The results demonstrated that both Manuka and Honeydew honey-based membranes had strong antibacterial activities against the strain of methicillin-resistant S. pseudointermedius tested. Specifically, membranes composed of Manuka honey were effective in inhibiting the growth of Gram-negative bacteria within 3 h, whereas those composed of Honeydew honey needed 24 h to neutralise bacterial growth. The antimicrobial activities of both membranes developed in this study suggest that they can be effectively used as wound dressing in veterinary clinical medicine.

Dans le cadre de cette étude, on a fabriqué des membranes à base de miel (miel de Manuka et miel de miellat) et de pectine et on a testé, selon le processus ISO 22196, leur activité antibactérienne sur des germes multirésistants provenant de blessures d'animaux (Staphylococcus pseudointermedius, E. coli, Proteus mirabilis und Pseudomonas aeruginosa). Les résultats montrent que les deux types de membranes ont une forte activité bactéricide sur les souches de Staphylococcus pseudointermedius résistantes à la méthicilline. Les membranes à base de miel de Manuka étaient également actives contre tous les germes gram négatifs ét réduisaient leur nombre en 3 heures, alors qu'un contact de 24 heures était nécessaire pour que les membranes à base de miel de miellat réduisent la croissance bactérienne. L'activité antibactérienne des membranes utilisées dans la présente étude justifie leur emploi dans la médecine vétérinaire clinique.

Evaluation of antimicrobial activity of Italian honey for wound healing application in veterinary medicine.

INTRODUCTION: Honey as a topical treatment for infected wounds dates back to ancient times. However, few studies have been reported concerning the medical properties of Italian honey. In this study, the microbial contamination, the antimicrobial activity and the antibiotic residues of 6 different varieties of Piedmont honeys were evaluated. The antimicrobial activity of honeys was tested by agar well diffusion method and 1 honey for each variety has been selected and tested by broth micro-dilution test to determine Minimum Inhibitory Concentrations (MICs) and evaluated by Minimum Bactericidal Concentrations (MBCs). The honeys with a high level of antibacterial activity were analyzed for the presence of tetracyclines, sulfonamides and macrolide residues. The agar well diffusion method showed the greatest antimicrobial activity for honeydew, chestnut and lime tree honeys. The MICs and MBCs identified the close similarity to the medical manuka honey of honeydew, polyfloral and chestnut honey. The levels of antibiotic residues on these honeys were below the limit of quantification. Based on our results the Italian variety of honeydew showed the best antimicrobial activity and can be considered for the treatment of infected wounds in animals.

INTRODUCTION: L'utilisation du miel pour le traitement des plaies infectées remonte à loin dans l'antiquité. Dans le présent travail, on étudie les contaminations microbiennes, l'activité antimicrobienne et les résidus d'antibiotiques dans 6 sortes de miels différentes provenant du Piémont. L'activité antimicrobienne a été mesurée au moyen d'une méthode de diffusion sur gel d'agar et un échantillon de chaque sorte de miel a été examiné quant à sa concentration minimale inhibitrice (CMI) et sa concentration minimale bactéricide (CMB) au moyen d'un test de micro-dilution. Les échantillons présentant une haute activité antibactérienne ont été analysés quant à la présence de tétracycline, de sulfamidés et de macrolides. Au test de diffusion sur agar, le miel de miellat ainsi que ceux de châtaignier et de tilleul ont démontré la plus grande activité antimicrobienne. Les CIM et CBM permettent de reconnaitre une grande similitude entre les miels de miellat, de nectar et de tilleul avec le miel de Manuka utilisé à des fins thérapeutiques. Les résidus d'antibiotiques de ces échantillons se situaient en dessous des limites de détection. Sur la base de ces constatations, les divers miels de miellat italiens présentent la plus grande activité antimicrobienne et peuvent être utilisés pour le traitement de plaies infectées chez les animaux.

Porous CS based membranes with improved antimicrobial properties for the treatment of infected wound in veterinary applications.

Recently, much attention has been given to the use of innovative solution for the treatment of infected wounds in animals. Current applied treatments are often un-effective leading to infection propagation and animal death. Novel engineered membranes based on chitosan (CS) can be prepared to combine local antimicrobial effect, high flexibility and easy manipulation. In this work, CS crosslinked porous membranes with improved antimicrobial properties were prepared via freeze-drying technique to promote wound healing and to reduce the bacterial proliferation in infected injuries. Silver nanoparticles (AgNPs) and gentamicin sulfate (GS) were incorporated into the CS matrices to impart antibacterial properties on a wild range of strains. CS based porous membranes were tested for their physicochemical, thermal, mechanical as well as swelling and degradation behavior at physiological condition. Additionally, GS release profile was investigated, showing a moderate burst effect in the first days followed by a decreasing release rate which it was maintained for at least 56 days. Moreover, porous membranes loaded with GS or AgNPs showed good bactericidal activity against both of Gram-positive and Gram-negative bacteria. The bacterial strains used in this work were collected in chelonians after carapace injuries to better mimic the environment after trauma.

Occurrence and functionality of cycle inhibiting factor, cytotoxic necrotising factors and cytolethal distending toxins in Escherichia coli isolated from calves and dogs in Italy.

Escherichia coli isolated from animals up to three months of age, with diarrhea (255 calves and 29 dogs (pups)), without diarrhea (21 calves and 11 pups, used as controls), and 58 adult dogs with cystitis were tested to investigate the occurrence and functional expression of cyclomodulins cycle inhibiting factor (CIF), cytotoxic necrotizing factors (CNFs) and cytolethal distending toxins (CDTs). In cyclomodulin-positive isolates the association was assessed with other virulence genotypes and phylogenetic groups. Of 374 E. coli isolates, 80 (21.4%) were positive for at least one cyclomodulin and 14 of the latter (3.7%) showed different combinations of more than one. cif-positive isolates showed a low number of additional virulence factors, and were commonly associated with phylogroup B1, while cnf- and cdt-positive isolates, harboring many extraintestinal virulence factors, belonged to phylogroups B2 and D. Almost all isolates showed an irreversible cytopathic effect (CPE), displaying functionality of cyclomodulins. Five isolates that presented a mutation of cif were CPE-negative.

Prevalence of Campylobacter jejuni, Campylobacter coli and enteric Helicobacter in domestic and free living birds in North-Western Italy.

In order to investigate the prevalence of some thermophilic Campylobacter (C. jejuni and C. coli) and enteric Helicobacter (H. pullorum and H. canadensis) in domestic and wild birds, a total of 278 bird caecal samples were analyzed over a 2 year period in North-Western Italy. Samples were collected from poultry raised in intensive farming at the slaughterhouse (n=102, group A) and in small scale rural farms (n=60, group B) as well as from wild birds (n=116, group C). PCR amplifications were carried out on DNA extracted from caecal samples. Molecular assays targeted the hipO gene for C. jejuni, the asp gene for C. coli and the 16S rRNA gene of H. pullorum/H. canadensis. To differentiate H. pullorum from H. canadensis, PCR products were subjected to an ApaLI digestion assay. Prevalence of thermophilic Campylobacter and enteric Helicobacter was significantly different among groups (p<0.0001). Campylobacter infections were detected in all three bird groups (78.4% group A, 18.3% group B and 38.8% group C, respectively), Helicobacter infections were only detected in poultry, with H. pullorum infecting 68.6% of group A and 21.7% of group B birds. H. canadensis was detected in Guinea fowls (group A) and for the first time in pheasants (group B). Mixed infections by enteric Campylobacter and Helicobacter were shown in 53.9% of group A and in 5.0 % of group B. Our results show that both microorganisms commonly infect poultry, especially intensive farming animals. Only hooded crows among the wild bird group (group C), proved to be highly sensitive to Campylobacter infection.

Phylogenetic background of attaching and effacing Escherichia coli isolates from animals.

Detection and distribution of eae gene in forty-four attaching and effacing Escherichia coli (AEEC) strains of animal origin were investigated. Association of distinct intimin alleles with phylogenetic background were assessed among strains in comparison with different serogroups. Phylogenetic analysis showed that 31 EHEC/eae+ STEC strains belong to groups A, B1 and E, 13 EPEC strains segregated in B1 and B2. Moreover, group A possessed the eae gamma2/theta type, group B1 the eae beta1, eae kappa, eae zeta, and eae epsilon types, group B2 the eae alpha1, eae alpha2 and eae iota types, while the group E possessed the eae gamma1 type. The presence of numerous eae-types show that EPEC and EHEC/eae+ STEC tested have a high genetic homology within each phylogenetic group.

Identification of intimin alleles in pathogenic Escherichia coli by PCR-restriction fragment length polymorphism analysis.

A rapid two-step identification method based on PCR-RFLP analysis of the intimin gene was developed to differentiate specific alleles in pathogenic Escherichia coli. This technique, tested on isolates eae-positive, accurately detects eae and resolves alleles encoding the alpha1, alpha2, beta, gamma1, gamma2/theta, kappa, epsilon, zeta, and iota intimin variants.

Identification of enteric Helicobacter in avian species.

The presence of enteric Helicobacter species was investigated in poultry (n=130) and in pet and ornamental birds (n=50) using a PCR sequencing method which permits the differentiation of many Helicobhacter species derived from animal tissues. All samples were of Italian origin, except for 21 Guinea fowl from a French flock. About 80% of poultry (chickens, laying hens, Guinea fowl) were positive to Helicobacter DNA. H. pullorum was most frequently (62.1%) identified whereas H. pylori and 3 H. sp. hamster B strains were seen in only 3 cases each. Pet and ornamental birds were all negative. H. canadensis was found in all Guinea fowl from a French farm. This is the first report on the occurrence of this bacterium in poultry.

In vitro antimicrobial activity of marbofloxacin and enrofloxacin against bacterial strains isolated from companion animals.

Fluoroquinolones were originally developed for the Gram-negative aerobic spectrum, but the newer generation agents are also highly effective against some Gram-positive pathogens and cause few adverse effects. Owing to these characteristics, fluoroquinolones are often used in first line therapy in small animal practice. However, their widespread use has raised concern over emerging bacterial resistance. In this study we evaluated the in vitro efficacy of two fluoroquinolones, marbofloxacin and enrofloxacin, on field strains isolated from clinical infections between 2002 and 2005. Our data show that most of the isolates are still sensitive to both antimicrobials and marbofloxacin was more effective than enrofloxacin, especially against P. aeruginosa and beta-Streptococci (P < 0.01). beta-Streptococci demonstrated the greatest resistance to the two study drugs.

Isolation of Acinetobacter lwoffii from a lovebird (Agapornis roseicollis) with severe respiratory symptoms.

Although Acinetobacter lwoffii is generally considered an ubiquitous and opportunistic bacterium, this germ has been isolated from the pulmonary and abdominal air sac swabs obtained from a Lovebird (Agapornis roseicollis), which died of a severe respiratory disease. Bacteriological tests (phenotypic and genotypic) led to the identification of A. lwoffii in pure culture. All the other parrots in the breeding centre were treated orally with oxytetracycline for 14 days and 3 months later no bird showed any signs of respiratory symptoms.

Paratuberculosis in red deer (Cervus elaphus hippelaphus) in the western Alps.

During the hunting seasons 1995-96 to 1997-98, 19 red deer from the Upper Susa Valley (Cottian Alps) were examined for paratuberculosis (Johne's disease). Specific DNA amplification on mesenteric lymph nodes detected Mycobacterium avium paratuberculosis in 17 animals. Ten of these red deer were tested for serum antibodies by the AGID and ELISA tests, nine being negative. Three isolates from infected deer were genetically characterized by an arbitrarily primed polymerase chain reaction, and showed similar genetic polymorphism to that of bovine strains isolated in different Italian areas. The study showed that paratuberculosis is present in red deer of the Upper Susa Valley and that serological tests are not an efficient means for monitoring this infection.

Expression and antigenic characterization of recombinant Mycoplasma agalactiae P48 major surface protein.

The gene encoding the P48 major surface lipoprotein of M. agalactiae has been recently characterised. Since its product plays an important role in the immune response of infected animals, in this study we analysed a recombinant P48 expressed in E. coli. Multiple point mutations were introduced by site directed mutagenesis in order to convert four tryptophan TGA codons, which are a typical feature of the mycoplasma genetic code, into the standard TGG. The mutated p48 gene was subcloned into pGex-2T and expressed in fusion with glutathione-S transferase. Following purification steps, P48 was eluted from carrier protein by thrombin digestion and used in Western blot and indirect ELISA using well-characterised sheep sera. Results demonstrate that specific antibodies against P48 are detected 3 weeks after onset of clinical disease and the recombinant P48 is a diagnostically relevant marker of M. agalactiae infection.

Mycobacterium paratuberculosis infection in two free-ranging Alpine ibex.

The authors report two cases of Mycobacterium paratuberculosis infection in free-ranging Alpine ibex (Capra ibex) from two different herds in the Western Alps, Italy. One ibex, found dead in October 1998, was in poor condition. The second animal died due to trauma following capture with a dart gun. The only gross lesions observed were the enlargement of the mesenteric and iliac lymph nodes. Samples from both ibex tested positive to polymerase chain reaction for a primer set specific for the M. paratuberculosis insertion sequence IS900 and one ibex also tested positive to the Zielh-Nielsen stain. Isolation by bacterial culture was not successful. The infected ibex originated from herds in which seroreactors to M. paratuberculosis had been found previously. Seroreactors to M. paratuberculosis were also detected in sympatric cattle.

P48 major surface antigen of Mycoplasma agalactiae is homologous to a malp product of Mycoplasma fermentans and belongs to a selected family of bacterial lipoproteins.

A major surface antigenic lipoprotein of Mycoplasma agalactiae, promptly recognized by the host's immune system, was characterized. The mature product, P48, showed significant similarity and shared conserved amino acid motifs with lipoproteins or predicted lipoproteins from Mycoplasma fermentans, Mycoplasma hyorhinis, relapsing fever Borrelia spp., Bacillus subtilis, and Treponema pallidum.

Effects of the combination antibiotic--EDTA-Tris in the treatment of chronic bovine endometritis caused by antimicrobial-resistant bacteria.

The combined effects of the uterine infusion of EDTA-Tris solution and antibiotics have been evaluated in 75 cases of slight, moderate or severe bovine endometritis which did not respond to local routine antimicrobial therapy. Antibiotic-resistant bacteria were isolated from uterine swabs. The cows were divided into three groups on the basis of the severity of endometritis and treated with 100 ml of sterile EDTA-Tris solution (250 mM EDTA and 50 mM tris, pH 8) and the same antibiotic used in the first unsuccessful treatment (oxytetracycline, enrofloxacin, lincomycin-spectinomycin or amikacin). Control groups consisting of six animals treated with antibiotic alone were used. Clinical evaluations performed 2, 15, 21, 42 and 63 days after treatment revealed good therapeutic results, as 53 cows showed a complete recovery with renormalization of the subsequent oestrus cycle. Artificial insemination was followed by pregnancy in about 90% of treated cows. In control animals the second treatment performed using only the antibiotic gave variable and unsatisfactory results, particularly in animals affected by severe endometritis.