Katleen Hermans

Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitis in Belgian cows

Methicillin-resistant Staphylococcus aureus (MRSA) is infrequently reported in mastitis. Yet, as in many other countries, the prevalence of methicillin resistance among S. aureus from mastitis is currently unknown in Belgium. To elucidate this, the presence of mecA was investigated in 118 S. aureus strains originating from diagnostic mastitis milk samples from 118 different farms experiencing S. aureus mastitis. MRSA strains were characterized by disk diffusion susceptibility testing, spa-typing, MLST and SCCmec-typing. In an additional study, four MRSA-positive farms were selected to assess the in-herd prevalence of MRSA, by sampling all cows in lactation. Isolated MRSA strains were similarly characterized. The mecA gene was detected in 11 (9.3%) of the 118 S. aureus isolates, indicating that nearly 10% of the Belgian farms suffering from S. aureus mastitis have an MRSA problem. The in-herd prevalence varied between 0% and 7.4%. Characterization of the MRSA strains showed that they were all resistant to tetracycline. Additional resistances to macrolides, lincosamides and aminoglycosides were frequently detected. The strains were ST398, spa-types t011 or t567 and had SCCmec-type IVa or V, proving that they belong to the emerging livestock-associated MRSA (LA-MRSA) strains of CC398. Our study shows that after detection in Belgian pigs, horses and poultry, LA-MRSA has also attained Belgian cattle. It is the first report on frequent isolation of LA-MRSA from bovine infections. As the in-herd isolation rate resembles that of regular S. aureus in farms experiencing S. aureus mastitis, the multi-resistance of LA-MRSA strains may cause future treatment problems.

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.

Methicillin-resistant Staphylococcus aureus (MRSA) in food production animals

Until recently, reports on methicillin-resistant Staphylococcus aureus (MRSA) in food production animals were mainly limited to occasional detections in dairy cattle mastitis. However, since 2005 a MRSA clone, CC398, has been reported colonizing pigs, veal calves and broiler chickens and infecting dairy cows. Many aspects of its prevalence in pigs remain unclear. In other livestock, colonizing capacity and reservoir status still require elucidation. MRSA CC398 has also been detected in meat, but, as for other MRSA, the risk this poses is somewhat unclear. Currently, the most worrying aspect of MRSA CC398 appears to be its capacity to spread to humans. This might complicate MRSA control measures in human healthcare, urging research into risk factors and transmission routes. Although infections with MRSA CC398 are much less reported than carriage, more investigation into its pathogenic potential is required. Moreover, the origin and evolution of this clone remain unknown.

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.

Methicillin-resistant Staphylococcus aureus in poultry.

Methicillin-resistant Staphylococcus aureus (MRSA) has been detected in several species and animal-derived products. To determine whether MRSA is present in poultry, we sampled 50 laying hens and 75 broiler chickens. MRSA was found in some broiler chickens but no laying hens. In all samples, spa type t1456 was found.

The newly described mecA homologue, mecALGA251, is present in methicillin-resistant Staphylococcus aureus isolates from a diverse range of host species

OBJECTIVES A previously unidentified mecA homologue, mecA(LGA251), has recently been described in methicillin-resistant Staphylococcus aureus (MRSA) from humans and dairy cattle. The origin and epidemiology of this novel homologue are unclear. The objective of this study was to provide basic descriptive information of MRSA isolates harbouring mecA(LGA251) from a range of host animal species. METHODS A number of S. aureus isolates from historical animal isolate collections were chosen for investigation based on their similarity to known mecA(LGA251) MRSA isolates. The presence of mecA(LGA251) was determined using a multiplex PCR and antimicrobial susceptibility testing performed by disc diffusion. RESULTS MRSA harbouring mecA(LGA251) were found in isolates from a domestic dog, brown rats, a rabbit, a common seal, sheep and a chaffinch. All of the isolates were phenotypically MRSA, although this depended on which test was used; some isolates would be considered susceptible with certain assays. All isolates were susceptible to linezolid, rifampicin, kanamycin, norfloxacin, erythromycin, clindamycin, fusidic acid, tetracycline, trimethoprim/sulfamethoxazole and mupirocin. Five multilocus sequence types were represented (2273, 130, 425, 1764 and 1245) and six spa types (t208, t6293, t742, t6594, t7914 and t843). CONCLUSIONS The discovery of MRSA isolates possessing mecA(LGA251) from a diverse range of host species, including different taxonomic classes, has important implications for the diagnosis of MRSA in these species and our understanding of the epidemiology of this novel mecA homologue.

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.

Route of entry and tissue distribution of Yersinia ruckeri in experimentally infected rainbow trout Oncorhynchus mykiss

Yersinia ruckeri is the causative agent of enteric redmouth disease, which leads to significant losses in salmonid aquaculture worldwide. Despite the significance of the disease, little information is available on the pathogenesis. In this study, the portal of entry was investigated using a contact-exposure infection method in rainbow trout Oncorhynchus mykiss with 4 different Y. ruckeri strains. Bacteriological and histological examination revealed the presence of high numbers of bacteria in the gills immediately after infection resulting in a rapid spread of Y. ruckeri in the internal organs. However, only a virulent strain was able to survive and multiply in the host, causing septicaemia and death several days after infection. These findings indicate that gills may be an important site of entry and that Y. ruckeri virulence is related to immune evasion.

Diversity of accessory genome of human and livestock-associated ST398 methicillin resistant Staphylococcus aureus strains

BACKGROUND Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) has documented the diversity of the genetic background of strains associated with healthcare (HA-MRSA), community (CA-MRSA) and livestock (LA-MRSA). The accessory and core-variable genomes of those strains however remain largely unknown. OBJECTIVE To compare the genetic background and accessory and core variable gene content of ST398 LA-MRSA strains with those of HA-and CA-MRSA strains from the same region. METHODS Representative strains of HA- (n=21), CA- (n=13) and ST398 LA-MRSA (n=18) were selected from Belgian National Reference Laboratory collections. The accessory and core-variable genomes of these strains were characterized by a DNA-microarray composed of oligonucleotide probes targeting ~400 resistance, adhesion and virulence associated genes. RESULTS ST398 strains displayed very homogenous hybridization profiles irrespective of their host origin. This ST398 genomic profile was moderately related to that of certain human HA- or CA-lineages but distinctively lacked several virulence- and colonization-associated genes implicated in carriage in humans, such as proteases and adhesins. No enterotoxin gene was found among ST398 strains. Differences were observed in the mobile resistance gene content of ST398 strains, including antibiotic resistance determinants. CONCLUSION LA-MRSA strains represent a homogenous lineage distinct from co-local HA- and CA-MRSA strains, especially in its accessory genome content characterized by a lack of human-associated virulence and adhesion determinants. The absence of detectable enterotoxin gene among ST398 LA-MRSA strains from a wide host range is reassuring regarding their foodborne pathogenic potential.

Rabbit staphylococcosis: difficult solutions for serious problems

Staphylococcus aureus infections are a major problem in rabbitries. The main manifestations are subcutaneous abscesses, mastitis, pododermatitis and septicaemia. Two patterns of infection can be distinguished. In the first type, clinical signs remain limited to a small number of rabbits in a flock. This type has little economic importance and is caused by low-virulence S. aureus strains. In the second type, the disease shows an epidemic spread. Consequences are poor production results, infertility and death. This leads to chronic problems and a subsequent decline in production. The latter type is caused by high-virulence strains. Biotyping, phage typing and RAPD typing contribute to the characterisation of high-virulence S. aureus strains. Administration of antibiotics, disinfection of the environment and vaccination are not able to solve the problems. Therefore, the only effective measure is to cull the entire flock and to restart with a new rabbit population after thorough disinfection. Limiting the introduction of new rabbits in existing rabbitries and reducing contacts between rabbitries to an absolute minimum are currently the only way to face this most difficult problem.