In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections.

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy.

Dynamics of bovine intramammary infections due to coagulase-negative staphylococci on four farms.

The objectives of this study were to compare the impact of different coagulase-negative species (CNS) on udder health measured in terms of individual quarter milk somatic cell count (SCC) and duration of intramammary infection, and to get some insight into most likely routes of infection for different CNS species. This longitudinal observational study was performed on four farms that were sampled at 4-week intervals for a total of 12 visits each. Quarters infected with CNS were followed through time with milk samples being submitted for bacteriological culture and SCC determination. PCR amplification of the internal transcribed spacer region and sequencing of the sodA and rpoB genes were used for species allocation. Pulsed-field gel electrophoresis (PFGE) was performed to assess strain identity. The percentage of quarters affected per farm varied between 6 and 35%, with the most frequently isolated CNS species being Staphylococcus epidermidis, followed by Staph. simulans, Staph. chromogenes and Staph. haemolyticus. It was possible to follow 111 intramammary infections due to CNS through time. Duration of infection had a mean of 188 d and was not significantly different between CNS species. Geometric mean quarter SCC overall was 132 000 cells/ml and was also not significantly different between CNS species. Despite the possibility of a different epidemiology of infection, the impact in terms of udder health seems to be similar for different CNS species.

Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis.

Streptococcus agalactiae (Group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (Group C Streptococcus, GCS) and Streptococcus uberis are relevant mastitis pathogens, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production. The aims of this study were the evaluation of antimicrobial drug resistance patterns, particularly important for streptococcal mastitis control and the identification of strain molecular features. Antimicrobial resistance was assessed by disk diffusion against amoxicillin-clavulanic acid, cefazolin, cefoperazone, pirlimycin-PRL, rifaximin, streptomycin, chloramphenicol, erythromycin-ERY, gentamicin, tetracycline-TET and vancomycin. Genotypic relationships were identified using pulsed-field gel electrophoresis (PFGE), macrolide and/or tetracycline resistance gene profiling, GBS capsular typing, GBS virulence gene profiling and GBS and S. uberis multi locus sequence typing (MLST). The majority of the isolates were susceptible to all drugs except to aminoglycoside, macrolide, lincosamide and tetracycline. Close to half of the TET resistant isolates have tetO and tetK and almost all ERY-PRL resistant isolates have ermB. A high degree of intra-species polymorphism was found for GCS. The GBS belonged to ST-2, -554, -61, -23 lineages and five new molecular serotypes and human GBS insertion sequences in the cpsE gene were found. Also, GBS of serotype V with scpB and lmb seem to be related with GBS isolates of human origin (same ST-2 and similar PFGE). Overall our results suggested that different therapeutic programs may have been implemented in the different farms and that in most cases clones were herd-specific.

Deterministic model to evaluate the impact of lactational treatment of subclinical mastitis due to coagulase-negative staphylococci.

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria from milk samples in several studies worldwide. Despite their relative frequency, specific measures aiming at their control are not well established. One possible measure to include in a control programme is lactational antimicrobial treatment. The decision to perform such treatment, as well as other actions on farm, should be based on the likelihood of financial return. A deterministic model was used to evaluate whether performing an antimicrobial treatment during the lactation for quarters infected with CNS was financially justifiable. Input variables for the impact of CNS on udder health were based on a previous study by the same authors and on available literature on the subject. Prices included in the model were based on 2009/2010 conditions in Portugal. The average result per antimicrobial treated quarter was a net loss of €38·74. Performing a sensitivity analysis to evaluate how systematic variation of the input variables of the model would lead to outcome changes showed that variation in input variables nearly always led to a negative outcome, with the greatest variation in losses observed for variation in the length of treatment and milk withdrawal period (-€46·26 to -€28·49). The situations in which a net benefit was to be expected included the bulk tank somatic cell count decreasing to a level corresponding to a premium payment or to penalties being avoided, and the prevention of transmission of CNS in the milking parlour when the possibility of transmission was at its highest level. For most situations, lactational treatment of CNS subclinical mastitis was not financially justifiable.

Virulence gene pool detected in bovine group C Streptococcus dysgalactiae subsp. dysgalactiae isolates by use of a group A S. pyogenes virulence microarray.

A custom-designed microarray containing 220 virulence genes of Streptococcus pyogenes (group A Streptococcus [GAS]) was used to test group C Streptococcus dysgalactiae subsp. dysgalactiae (GCS) field strains causing bovine mastitis and group C or group G Streptococcus dysgalactiae subsp. equisimilis (GCS/GGS) isolates from human infections, with the latter being used for comparative purposes, for the presence of virulence genes. All bovine and all human isolates carried a fraction of the 220 genes (23% and 39%, respectively). The virulence genes encoding streptolysin S, glyceraldehyde-3-phosphate dehydrogenase, the plasminogen-binding M-like protein PAM, and the collagen-like protein SclB were detected in the majority of both bovine and human isolates (94 to 100%). Virulence factors, usually carried by human beta-hemolytic streptococcal pathogens, such as streptokinase, laminin-binding protein, and the C5a peptidase precursor, were detected in all human isolates but not in bovine isolates. Additionally, GAS bacteriophage-associated virulence genes encoding superantigens, DNase, and/or streptodornase were detected in bovine isolates (72%) but not in the human isolates. Determinants located in non-bacteriophage-related mobile elements, such as the gene encoding R28, were detected in all bovine and human isolates. Several virulence genes, including genes of bacteriophage origin, were shown to be expressed by reverse transcriptase PCR (RT-PCR). Phylogenetic analysis of superantigen gene sequences revealed a high level (>98%) of identity among genes of bovine GCS, of the horse pathogen Streptococcus equi subsp. equi, and of the human pathogen GAS. Our findings indicate that alpha-hemolytic bovine GCS, an important mastitis pathogen and considered to be a nonhuman pathogen, carries important virulence factors responsible for virulence and pathogenesis in humans.

Diagnosis of intramammary infection in samples yielding negative results or minor pathogens in conventional bacterial culturing.

Up to half of quarter milk samples submitted for mastitis diagnosis are culture-negative results or lead to identification of coagulase-negative staphylococci or Corynebacterium bovis in conventional culturing, the so-called minor pathogens. The interpretation and usefulness of these results in terms of udder and animal health management is limited, even though the amount of resources spent is relatively high. This work aimed to test two methods of analysis of milk samples with the goal of increasing detection of intramammary pathogens. In the first study, 783 milk samples were processed in duplicate: before and after freezing at -20°C for 24 h, using standard bacteriological techniques. There was a significant difference between the two methods with samples frozen for 24 h yielding significantly fewer Gram-positive catalase-positive cocci, Gram-negative bacilli, Gram-positive bacilli and significantly more samples leading to no growth, than samples before freezing. The number of samples yielding Gram-positive catalase-negative cocci was not significantly affected by freezing. In the second study, a real-time PCR-based test was performed on milk samples with an individual quarter somatic cell count above 500,000 cells/ml that were either negative (n=51 samples) or that led to the isolation of minor pathogens in culturing: Corynebacterium bovis (n=79 samples) or non-aureus staphylococci (NAS, n=32). A mastitis pathogen, beyond the result obtained with standard bacteriology, was detected on 47% of the no-growth samples, on 35% of the samples from which C. bovis had been isolated and on 25% of the samples from which NAS had been isolated. The most commonly detected major pathogen was Escherichia coli, followed by Streptococcus uberis, Arcanobacterium pyogenes/Peptoniphilus indolicus and Streptococcus dysgalactiae. These results suggest that simply freezing milk samples for 24 h does not increase the detection of intramammary bacteria in milk samples and therefore should not be recommended. However, use of the real-time PCR-based test may be useful in diagnosing intramammary infections when milk samples with high somatic cell counts are culture-negative or when culturing results in the detection of minor pathogens.

Observed reduction in recovery of Corynebacterium spp. from bovine milk samples by use of a teat cannula.

Although Corynebacterium bovis and coagulase-negative staphylococci are frequently the most commonly isolated bacteria from milk samples submitted for identification of pathogens causing intramammary infection, the individual quarter somatic cell count (SCC) from those samples is most often low. The present study aimed at evaluating the difference in bacteriology results from milk sampled by the standard technique (as recommended by the National Mastitis Council) and by the use of a teat cannula surpassing the teat canal, since C. bovis is often only found in the teat canal. Single quarter milk samples were collected in duplicate from 132 dairy cows on a commercial dairy farm using the standard milk sampling technique and also using a cannula introduced into the teat. Two groups of quarters were sampled: a group that was selected randomly at cow and quarter level and a group that was selected based on having SCC >200,000 cells/ml at the previous milk recording at cow level and on California mastitis test result at quarter level. Bacteriological culture performed on the samples yielded 29 Corynebacterium spp. isolates from the samples collected with the standard technique and 6 isolates from the samples collected with a cannula. Bacteriological culture yielded 73 and 100 culture negative samples respectively with the standard and the alternative sampling technique. A significant difference between the two sampling techniques was observed for recovery of Corynebacterium spp. and for no-growth samples. There was no significant difference in the isolation of Corynebacterium spp. or other bacterial species when using the standard technique before or after sampling with the cannula; thus the observed difference in bacteriology results could not be attributed to a particular sampling order. No significant change was observed overall in individual quarter SCC measured on the sampling day and 7 d later. Our results agree with several studies showing that Corynebacterium bovis often colonizes the teat canal, without causing true intramammary infection.

Antimicrobial resistance in Gram-positive bacteria from Timorese River Buffalo (Bubalus bubalis) skin microbiota.

The Timorese River Buffalo (Bubalus bubalis) plays a major role in the East Timor economy, as it is an important source of animal protein in human nutrition. They are widely spread throughout the country and are in direct contact with the populations. In spite of this proximity, information on their microbiota is scarce. This work aimed at characterizing the skin microbiota of the East Timorese River Buffalo and its antimicrobial resistance profile. Skin swab samples were taken from 46 animals in surveys conducted in three farms located in "Suco de Nairete", Lospalos district, during July and August 2006. Bacteria were isolated and identified according to conventional microbiological procedures. A total of 456 isolates were obtained, including Gram-positive (n = 243) and Gram-negative (n = 213) bacteria. Due to their importance as potential pathogens and as vehicles for antimicrobial resistance transmission, Gram-positive cocci (n = 27) and bacilli (n = 77) isolates were further characterized, and their antimicrobial resistance profile determined by the disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines. This study shows the high bacterial diversity of B. bubalis skin microbiota, representing an important first step towards understanding its importance and epidemiologic role in animal health. It also points out the potential role of these animals as vectors of antimicrobial resistant bacteria dissemination and the importance of antimicrobial resistance monitoring in developing countries.

Human group A streptococci virulence genes in bovine group C streptococci.

Phage-encoded virulence genes of group A streptococci were detected in 10 (55.6%) of 18 isolates of group C streptococci that had caused bovine mastitis. Bovine isolates carried other genetic determinants, such as composite transposon Tn1207.3/F10394.4 (100%) and antimicrobial drug resistance genes erm(B)/erm(A) (22.2%), linB (16.6%), and tet(M)/tet(O) (66.7%), located on mobile elements.

Cutaneous and ocular adverse reactions in a dog following meloxicam administration.

The present report addresses the development of cutaneous and ocular reactions possibly related to meloxicam administration in a dog. Based on clinical signs and absence of laboratory data compatible with the other differential diagnoses considered, the possibility of an adverse drug reaction (ADR) due to meloxicam was considered. Skin biopsy revealed haemorrhage of the superficial and deep dermis, associated with hyperplasia of endothelial cells and epidermal sloughing. Vasculitis in the deep dermis was also noted. Such lesions were considered compatible with an ADR. Although the owner was not aware of any previous allergic reaction to drugs, the animal had a clinical history of atopic dermatitis. Meloxicam is a nonsteroid anti-inflammatory drug (NSAID) in the oxicam family, indicated for the control of inflammation and pain in acute and chronic musculoskeletal disorders in dogs. Although meloxicam is usually well tolerated, the present clinical case represents an alert to practitioners about the potential role of NSAIDS in ADRs in dogs with a history of allergic cutaneous diseases.