The susceptibilities of 12 antimicrobial agents for two collections of Staphylococcus aureus, isolated in the 1970s and in 2006 from poultry, were determined. For eight antibiotics, the percentage of resistance was significantly higher in the recent isolates. Ten recent isolates were methicillin resistant and had spa types t011 and t567, belonging to multilocus sequence type 398. This is the first report of "livestock-associated" methicillin resistant S. aureus from healthy poultry.Antimicrobial agents, including penicillin, erythromycin, and tetracyclines, are widely used for treating staphylococcal and other infections in poultry (1,19,23). The extensive use of antimicrobial agents in animal husbandry contributes to the selection of drug-resistant strains. Recently, the isolation of methicillin-resistant Staphylococcus aureus (MRSA) from animals has been reported at an increasing frequency (14,15,16,18,22,24). This study analyzes the frequency of acquired resistance to 12 antimicrobial agents, ␤-lactamase activity, and the prevalence of the mecA gene between two groups of S. aureus isolates from poultry.Ninety S. aureus isolates were obtained from tendon sheaths of diseased breeder chickens and from the noses and cloacae of healthy broiler breeders between 1970 and 1972 (9) (old isolates), and eighty-one S. aureus isolates were collected from the noses and cloacae of healthy chickens derived from 39 randomly selected industrial broiler farms in 2006 (recent isolates). The old isolates had been lyophilized and stored at Ϫ20°C until used. To collect the recent isolates, the noses and cloacae of five chickens from each flock were sampled. Samples were inoculated on Columbia agar supplemented with sheep blood, colistin, and nalidixic acid (Oxoid, Basingstoke, United Kingdom). Isolates were identified as S. aureus by colony morphology, standard biochemical methods, and growth on modified Baird-Parker medium (10). Multiplex PCR for the femA and mecA genes was performed to confirm the identification and methicillin resistance of S. aureus (17,22). Susceptibility to oxacillin, penicillin, enrofloxacin, erythromycin, tylosin, lincomycin, gentamicin, neomycin, spectinomycin, sulfonamides, tetracycline, and trimethoprim was determined according to CLSI guidelines by using agar dilution tests (6).For interpretation of MICs, European Committee on Antimicrobial Susceptibility Testing (EUCAST; http://www.escmid .org/sites/index_f.aspx?parϭ2.4) wild-type cutoff values were used, except for enrofloxacin and tylosin, for which we used cutoff values from our study, according to the bimodal distribution of MICs (20). ␤-Lactamase production was tested for the penicillin-resistant isolates by using ␤-lactamase diagnostic tablets (Rosco, Taastrup, Denmark) according to the manufacturer's instructions. MRSA isolates (n ϭ 10) were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI macrorestriction analysis and by DNA sequence analysis of the polymorphic repeat region of protein A gene (spa typing) (8, 13). The spa types w...

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The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the ‘Twort-like viruses’. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes.

Despite epidemiological data linking necrotizing skin infections with the production of Panton-Valentine leukocidin (PVL), the contribution of this toxin to the virulence of S. aureus has been highly discussed as a result of inconclusive results of in vivo studies. However, the majority of these results originate from experiments using mice, an animal species which neutrophils - the major target cells for PVL - are highly insensitive to the action of this leukocidin. In contrast, the rabbit neutrophils have been shown to be as sensitive to PVL action as human cells, making the rabbit a better experimental animal to explore the PVL role. In this study we examined whether PVL contributes to S. aureus pathogenicity by means of a rabbit skin infection model. The rabbits were injected intradermally with 108 cfu of either a PVL positive community-associated methicillin-resistant S. aureus isolate, its isogenic PVL knockout or a PVL complemented knockout strain, and the development of skin lesions was observed. While all strains induced skin infection, the wild type strain produced larger lesions and a higher degree of skin necrosis compared to the PVL knockout strain in the first week after the infection. The PVL expression in the rabbits was indirectly confirmed by a raise in the serum titer of anti-LukS-PV antibodies observed only in the rabbits infected with PVL positive strains. These results indicate that the rabbit model is more suitable for studying the role of PVL in staphylococcal diseases than other animal models. Further, they support the epidemiological link between PVL producing S. aureus strains and necrotizing skin infections.

BackgroundIn Europe, methicillin-resistant Staphylococcus aureus (MRSA) belonging to the clonal complex (CC) 398 has become an important pathogen in horses, circulating in equine clinics and causing both colonization and infection. Whether equine MRSA is bound to hospitals or can also circulate in the general horse population is currently unknown. This study, therefore, reports the nasal and perianal MRSA screening of 189 horses on 10 farms in a suspected high prevalence region (East- and West-Flanders, Belgium).ResultsOnly one horse (0.53%) from one farm (10%) tested positive in the nose. It carried a spa type t011-SCCmecV isolate, resistant to β-lactams and tetracycline, which is typical for livestock-associated MRSA CC398.ConclusionIn the region tested here, horses on horse farms seem unlikely to substantially contribute to the large animal associated ST398 MRSA reservoir present at intensive animal production units.

Introduction Staphylococcus aureus sequence type ST398 has recently gained attention due to the spread of methicillin-resistant strains among people exposed to livestock. The aim of this study was to explore temporal changes in the population structure of S. aureus in pigs over the last 40 years with particular reference to the occurrence of ST398.MethodsWe analysed a unique collection of 91 porcine strains isolated in six countries between 1973 and 2009 using a biotyping scheme described in the 1970's in combination with spa typing and multi-locus sequence typing (MLST). The collection comprised 32 historical isolates from 1973–1974 (n = 19) and from 1991–2003 (n = 13), and 59 contemporary isolates from 2004–2009. The latter isolates represented the most common MLST types (ST1, ST9, ST97 and ST433) and spa types isolated from pigs in Europe.Results and Discussion S. aureus sequence type ST398 was not found among old isolates from the 1970's or from 1991–2003, suggesting that this lineage was absent or present at low frequencies in pigs in the past. This hypothesis is supported by the observed association of ST398 with the ovine ecovar, which was not described in pigs by studies carried out in the 1970's. In addition, various phenotypic and genotypic differences were observed between old and contemporary isolates. Some biotypes commonly reported in pigs in the 1970's were either absent (human ecovar) or rare (biotype A) among contemporary isolates. Nine clonal lineages found among old porcine isolates are occasionally reported in pigs today (ST8, ST30, ST97, ST387, ST1092, ST2468) or have never been described in this animal host (ST12, ST133, ST1343). These results indicate that the population structure of porcine S. aureus has changed over the last 40 years and confirm the current theory that S. aureus ST398 does not originate from pigs.

ABSTRACT:The effi cacy of an autogenous vaccine consisting of a whole cell suspension of formalin killed bacteria in sterile buffered saline against Staphylococcus aureus infections was determined, using a well-established rabbit skin infection model. Thirteen 8 wk old rabbits were vaccinated twice subcutaneously with a 2 wk interval while 10 rabbits were injected twice with formalised PBS (sterile phosphate buffered saline). Two weeks after the last injection, 10 vaccinated and all PBS-injected rabbits were inoculated intradermally with 10 8 cfu of a S. aureus strain (KH 171)which had been shown to be highly virulent for rabbits. Three vaccinated animals served as negative controls and were intradermally injected with sterile buffered saline. All rabbits were examined daily for the development of skin lesions until 14 d after the experimental infection when all rabbits were euthanised. All animals experimentally infected with S. aureus developed skin abscesses within 24 h post-inoculation, but in the vaccinated group the maximum abscess diameter was signifi cantly lower than in the non-vaccinated group (P=0.048). This difference between autovaccinated and non-vaccinated groups increased over time (P<0.001). These results indicate that vaccination with an inactivated whole cell bacterin may be useful for control of staphylococcosis in rabbits but does not prevent abscess formation in animals inoculated with a high dose of a highly virulent S. aureus strain.

Thirteen multidrug-resistant MRSA strains from infections of hospitalized horses in an equine clinic were analyzed. They all were indigestible by SmaI restriction, possessed SCCmec type IV and belonged to spa type t011. One isolate was analyzed by MLST and allotted to ST398. The MRSA clonal lineage ST398-SCCmec IV appears to have a high capability of causing clinical infections in an equine hospital environment.