Urszula Lipinska

Antimicrobial Resistance of Old and Recent Staphylococcus aureus Isolates from Poultry: First Detection of Livestock-Associated Methicillin-Resistant Strain ST398

ABSTRACT 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.

High occurrence of methicillin-resistant Staphylococcus aureus ST398 in equine nasal samples

Methicillin-resistant Staphylococcus aureus (MRSA) infections do occur in equine patients. Little is known, however, about their origin and the general equine MRSA colonization status. In West European horses in particular, neither the colonization rate nor the present strains or their antimicrobial susceptibility patterns are known. In the present study, a sample of 110 (Belgian, French, Dutch and Luxemburg) horses presented at a Belgian equine clinic was screened for nasal MRSA carriage. An indirect culturing protocol using a 0.001% colistin and nalidixic acid containing broth was compared to a direct agar method. Phenotypic identification following growth on a chromogenic MRSA screening agar (ChromID MRSA) was combined with genotypic analysis (PCR, PFGE, SCCmec, spa, and MLST typing). Antimicrobial susceptibility was tested through disk diffusion. Twelve (10.9%) horses carried MRSA, with the enrichment protocol resulting in a significantly higher isolation rate. None of the isolated strains were typeable through SmaI PFGE. They all harboured SCCmec type IVa or V and belonged to spa type t011 or t1451 of the ST398 lineage. All isolates were tetracycline resistant and sulfonamide and enrofloxacin susceptible. Macrolide, lincosamide, trimethoprim and aminoglycoside susceptibility varied and in total five different antimicrobial resistance patterns were distinguished. These results show that ST398 is certainly present in West European horses. Due to its known interspecies transmission and the structure of the equine industry, the presence of this clone in horses poses a substantial health hazard for both animals and humans.

Panton-Valentine Leukocidin Does Play a Role in the Early Stage of Staphylococcus aureus Skin Infections: A Rabbit Model

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.

Microbiological and Molecular Assessment of Bacteriophage ISP for the Control of Staphylococcus aureus

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.

Low MRSA prevalence in horses at farm level

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.

Phenotypes and genotypes of old and contemporary porcine strains indicate a temporal change in the S. aureus population structure in pigs

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. Methods We analysed a unique collection of 91 porcine strains isolated in six countries between 1973 and 2009 using a biotyping scheme described in the 1970s 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 1970s 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 1970s. In addition, various phenotypic and genotypic differences were observed between old and contemporary isolates. Some biotypes commonly reported in pigs in the 1970s 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.