Wannes Vanderhaeghen

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.

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.

Invited review: Effect, persistence, and virulence of coagulase-negative Staphylococcus species associated with ruminant udder health

The aim of this review is to assess the effect of coagulase-negative staphylococci (CNS) species on udder health and milk yield in ruminants, and to evaluate the capacity of CNS to cause persistent intramammary infections (IMI). Furthermore, the literature on factors suspected of playing a role in the pathogenicity of IMI-associated CNS, such as biofilm formation and the presence of various putative virulence genes, is discussed. The focus is on the 5 CNS species that have been most frequently identified as causing bovine IMI using reliable molecular identification methods (Staphylococcus chromogenes, Staphylococcus simulans, Staphylococcus haemolyticus, Staphylococcus xylosus, and Staphylococcus epidermidis). Although the effect on somatic cell count and milk production is accepted to be generally limited or nonexistent for CNS as a group, indications are that the typical effects differ between CNS species and perhaps even strains. It has also become clear that many CNS species can cause persistent IMI, contrary to what has long been believed. However, this trait appears to be quite complicated, being partly strain dependent and partly dependent on the hosts immunity. Consistent definitions of persistence and more uniform methods for testing this phenomenon will benefit future research. The factors explaining the anticipated differences in pathogenic behavior appear to be more difficult to evaluate. Biofilm formation and the presence of various staphylococcal virulence factors do not seem to (directly) influence the effect of CNS on IMI but the available information is indirect or insufficient to draw consistent conclusions. Future studies on the effect, persistence, and virulence of the different CNS species associated with IMI would benefit from using larger and perhaps even shared strain collections and from adjusting study designs to a common framework, as the large variation currently existing therein is a major problem. Also within-species variation should be investigated.

Prevalence, risk factors and genetic diversity of methicillin-resistant Staphylococcus aureus carried by humans and animals across livestock production sectors

OBJECTIVES This study aimed to assess the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in animals and humans on veal, dairy, beef and broiler farms and to compare the risk for human MRSA carriage with that of strictly horticulture farmers. The genetic background, resistance phenotypes and genotypes and toxin gene content of the isolated MRSA strains were compared with MRSA collected on MRSA clonal complex (CC)398-positive pig farms. METHODS MRSA carriage isolates were genotyped (spa, SCCmec and multilocus sequence typing), resistance to 16 antimicrobials was determined and resistance and toxin genes were detected. RESULTS MRSA carriage rates were higher (P<0.01) on veal farms (calves, 64%; farmers, 72%) compared with on dairy (cows, 1%), beef (cows, 5%; farmers, 11%), broiler (pooled broths, 5%; farmers, 3%) and horticulture (farmers, 3%) farms. The intensity of animal contact was identified as a risk factor for human MRSA carriage. The vast majority of MRSA (n=344), including those from pigs, were CC398 (98%). SCCmec V(5C2), V(5C2&5)c, IV(2B) and IV(2B&5) predominated. MRSA CC130 and CC599 carrying mecC were detected in beef and dairy cattle. MRSA from veal calves were significantly more resistant than MRSA from pigs (P<0.01). A few isolates, including mecC-carrying MRSA, harboured pyrogenic superantigen toxins. Human- and animal-derived MRSA from individual farms showed similar characteristics. CONCLUSIONS This systematic cross-sector survey revealed a high prevalence of multiresistant livestock-associated MRSA on Belgian veal calf farms as compared with other farm types. MRSA harbouring mecC was detected at a low frequency in beef and dairy cows, but not in humans.

Identification, typing, ecology and epidemiology of coagulase negative staphylococci associated with ruminants.

Since phenotypic methods to identify coagulase negative staphylococci (CNS) from the milk of ruminants often yield unreliable results, methods for molecular identification based on gene sequencing or fingerprinting techniques have been developed. In addition to culture-based detection of isolates, culture-independent methods may be of interest. On the basis of molecular studies, the five CNS species commonly causing intramammary infections (IMI) are Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus simulans and Staphylococcus xylosus. Current knowledge suggests that S. chromogenes is a bovine-adapted species, with most cases of IMI due to this bacterium being opportunistic. S. haemolyticus also appears to be an opportunistic pathogen, but this bacterium occupies a variety of habitats, the importance of which as a source of IMI remains to be elucidated. S. xylosus appears to be a versatile species, but little is known of its epidemiology. S. epidermidis is considered to be a human-adapted species and most cases of IMI appear to arise from human sources, but the organism is capable of residing in other habitats. S. simulans typically causes contagious IMI, but opportunistic cases also occur and the ecology of this bacterium requires further study. Further studies of the ecology and epidemiology of CNS as a cause of IMI in cattle are required, along with careful attention to classification of these bacteria and the diseases they cause.

High genetic diversity of methicillin-susceptible Staphylococcus aureus (MSSA) from humans and animals on livestock farms and presence of SCCmec remnant DNA in MSSA CC398

OBJECTIVES To investigate the genetic diversity of methicillin-susceptible Staphylococcus aureus (MSSA) carriage isolates from animals and humans on pig, veal, dairy, beef and broiler farms. METHODS S. aureus isolates were genotyped using spa typing and multilocus sequence typing (MLST). Antimicrobial susceptibility phenotypes and genotypes were determined. The presence of staphylococcal cassette chromosome mec (SCCmec)-associated DNA was characterized by PCR and sequencing among isolates of clonal complex (CC) 398. RESULTS Overall, 41 MSSA isolates in humans and 141 in animals were found, originating from all farm types. These MSSA were mainly assigned to CC398, CC1, CC5, CC9, CC30, CC97, CC133 and CC705/151. MSSA CC398 showed resistance to tetracycline, trimethoprim, macrolides and/or lincosamides, aminoglycosides, ciprofloxacin, spectinomycin and quinupristin/dalfopristin, whereas non-CC398 MSSA showed considerably less resistance. Three porcine MSSA CC398-t011 isolates harboured remnant DNA of a composite SCCmec V(5C2&5)c element that lacked the mec gene complex. This resulted from an MRSA-to-MSSA conversion due to recombination between the ccrC genes flanking the mec gene complex. The SCC remnant still contained an intact J1 region harbouring czrC and tet(K), encoding zinc and tetracycline resistance, respectively, thereby illustrating the capacity of S. aureus CC398 to adapt to different antibiotic selection pressures in the farming environment. Processes such as mec gene complex deletion probably contribute to the enormous diversity of SCC(mec) elements observed in staphylococci. CONCLUSIONS MSSA CC398 precursors from which MRSA CC398 might (re)emerge were present on pig, veal and broiler farms, all of which are livestock sectors commonly known to be affected by MRSA CC398. The multiresistance phenotype of S. aureus CC398 appears to be independent of methicillin resistance.

Transmission Dynamics of Methicillin-Resistant Staphylococcus aureus in Pigs

From the mid-2000s on, numerous studies have shown that methicillin-resistant Staphylococcus aureus (MRSA), renowned as human pathogen, has a reservoir in pigs and other livestock. In Europe and North America, clonal complex (CC) 398 appears to be the predominant lineage involved. Especially worrisome is its capacity to contaminate humans in close contact with affected animals. Indeed, the typical multi-resistant phenotype of MRSA CC398 and its observed ability of easily acquiring genetic material suggests that MRSA CC398 strains with an increased virulence potential may emerge, for which few therapeutic options would remain. This questions the need to implement interventions to control the presence and spread of MRSA CC398 among pigs. MRSA CC398 shows a high but not fully understood transmission potential in the pig population and is able to persist within that population. Although direct contact is probably the main route for MRSA transmission between pigs, also environmental contamination, the presence of other livestock, the herd size, and farm management are factors that may be involved in the dissemination of MRSA CC398. The current review aims at summarizing the research that has so far been done on the transmission dynamics and risk factors for introduction and persistence of MRSA CC398 in farms.

Comparison of fingerprinting methods for typing methicillin-resistant Staphylococcus aureus sequence type 398.

ABSTRACT This study evaluates the multiple-locus variable-number tandem-repeat assay (MLVA) and pulsed-field gel electrophoresis (PFGE) when using restriction enzymes BstZI, SacII, and ApaI to fingerprint a diverse collection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) sequence type 398 (ST398) isolates. These isolates had been characterized previously by multilocus sequence typing, spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Typeability and discriminatory power were analyzed, and the concordance between the various methods was determined. All MRSA ST398 isolates were typeable by the MLVA and PFGE using BstZI, SacII, and ApaI. With each method, the MRSA ST398 isolates formed a separate group from the two non-ST398 MRSA strains. PFGE, performed with any of the three restriction enzymes, had the most discriminatory power, followed by MLVA, spa typing, and SCCmec typing. The MLVA showed the highest concordance with PFGE using ApaI and spa typing. As further expressed by the Wallace coefficient, the MLVA type was poorly predicted by spa typing, whereas the spa type was well predicted by MLVA. PFGE, using a combination of all three restriction enzymes, had the highest concordance with the MLVA but had a low probability of being predicted by MLVA. PFGE, using a combination of all three restriction enzymes, was able to predict SCCmec type and MLVA type completely and had a high probability of predicting spa type. Both the MLVA and PFGE could be used to discriminate among the MRSA ST398 isolates. Although the MLVA is a faster technique, PFGE had more discriminatory power than the MLVA, especially when a combination of restriction enzymes was used.

Species and staphylococcal cassette chromosome mec (SCCmec) diversity among methicillin-resistant non-Staphylococcus aureus staphylococci isolated from pigs.

While methicillin-resistant Staphylococcus aureus (MRSA) ST398 is known to be widespread in pig farms, few studies have investigated the species diversity and SCCmec types of methicillin-resistant non-S. aureus staphylococci (MRNAS) residing in the nose of pigs. We examined nasal swab samples of 200 pigs originating from 10 Belgian pig farms previously found positive for MRSA ST398. Suspected staphylococcal isolates were subjected to a 16S rRNA-mecA-nuc PCR. Confirmed MRNAS were genotypically identified to the species level and investigated with a SCCmec typing PCR. MRNAS (n=72) were detected on all 10 farms and were carried by 29.5% of the pigs. Seven MRNAS species were found: Staphylococcus epidermidis (38.9%), Staphylococcus sciuri (18.1%), Staphylococcus pasteuri (18.1%), Staphylococcus rostri (12.5%), Staphylococcus warneri (8.3%), Staphylococcus haemolyticus (2.7%) and Staphylococcus hominis (1.4%). SCCmec cassettes were of type IVa (29.2%), type IVc (25%), type III (22.2%), type V (5.6%) or could not be assigned to any of the known types (NT types) (18.1%). Five distinct NT types were found. The predominance of methicillin-resistant S. epidermidis (MRSE) in our samples is remarkable, as MRSE is mainly associated with humans. The finding of three different SCCmec elements (IVa, V, NT type 1) in MRNAS that also prevail or predominate in MRSA ST398 shows that MRNAS might be an important SCCmec reservoir for MRSA in pigs. Yet, the occurrence of multiple other SCCmec types illustrates that further studies are required to understand the presence and spread of SCCmec in methicillin-resistant staphylococci from animals.

Colonization and Transmission of Methicillin-Resistant Staphylococcus aureus ST398 in Nursery Piglets

ABSTRACT A transmission experiment was performed to evaluate the spread of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in nursery piglets. Reproduction ratios (R0) in three experimental groups were found to vary between 3.92 and 52.54, indicating that after introduction, MRSA ST398 will spread easily among weaned piglets, with a tendency to become established.