Birgit Walther

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) in different animal species

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in animals such as horses, pet animals and productive livestock has raised questions of a probable human origin and in more general of host specificity of S. aureus. Particular clonal lineages are obviously specific for humans (e.g. ST15, ST25, ST45) and other for ruminants (e.g. ST151). MRSA associated with veterinary nosocomial infections (e.g. ST8 and ST254 in horses, ST22 in small animals) very likely have their origin in health care facilities. MRSA ST398 which became first known from widespread colonization in industrially raised pigs seems to have a limited host specificity and is able to colonize and to cause infections in various hosts. Mechanisms of host adaptation and their genomic background are poorly understood so far.

Prevalence of Methicillin-resistant Staphylococcus pseudintermedius isolated from clinical samples of companion animals and equidaes.

In this study the prevalence of Methicillin-resistant Staphylococcus pseudintermedius (MRSP) in clinical specimens of different animal species was defined by investigating a total of 16,103 clinical samples originating from veterinary facilities of five German federal states in 2007. Of all samples examined, 72 were positive for MRSP, giving an overall prevalence of 0.45%. In clinical specimens originating from small animals the prevalence was 0.58% (n=67; dogs n=61 and cats n=6), while samples from equidaes revealed a prevalence of 0.10% (n=5; horses n=4, donkey n=1). Forty-six representative phenotypically identified MRSP were further differentiated by DNA-based species assignment, PCR detection of mecA, SCCmec-typing and MIC determination. As expected, all 46 isolates were unambiguously proven to be MRSP by sequencing of housekeeping genes pta and cpn60 and being positive for mecA. Furthermore, all isolates harboured the mobile staphylococcal cassette chromosome mec (SCCmec) SCCmecIII. Antibiotic susceptibility testing for 20 different conventional antimicrobial agents disclosed a high rate of multidrug-resistant isolates (45 of 46) displaying an identical or at least similar resistance pattern for non-beta-lactam antimicrobials. The recognized prevalence of MRSP, which have already been shown to be potential zoonotic agents, reflects the recently emerging development of these serious and often multidrug-resistant pathogens in Germany.

Methicillin-resistant staphylococci (MRS) and extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae in companion animals: Nosocomial infections as one reason for the rising prevalence of these potential zoonotic pathogens in clinical samples

The ongoing change in the relationship between humans and companion animals is hallmarked by the increasing intensive care provided to companion animals in veterinary medicine, resulting in growing numbers of high-risk animal patients. The emergence of nosocomial infections in small animal clinics is one of the major drawbacks of this development, especially in terms of multidrug-resistance and potentially zoonotic pathogens. This mini-review therefore addresses recent findings regarding the increasing prevalence of multi-resistant bacterial pathogens like methicillin-resistant staphylococci (MRS), including Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) as well as extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae in companion animals. Along with the steady increase of nosocomial infection rates in veterinary clinics, particular attention has recently been drawn to the genetic background of multi-resistant strains, resulting in the identification of certain genetic lineages which frequently appear in both, human and animal samples. These sequence types (ST), included ST254, ST8 and ST22 in terms of MRSA and ST131, ST405 and ST648 for ESBL-producing E. coli. The interspecies distribution of these STs resulted in the assumption that certain extended-host spectrum genotypes (EHSG) might exist both for MRS and ESBL-producing E. coli. These initial findings underline the necessity to investigate the major molecular or functional driving forces facilitating interspecies transferability of such EHSG strains. Due to the zoonotic potential of these multi-resistant bacteria, another aspect of the changing social role of companion animals needs to be addressed: the close contact of pets with their owners, resulting in presumptive new transmission and infection routes. We therefore envision retaliatory actions like initial surveillance and monitoring programs not only in livestock, but also particularly in companion animals. Interdisciplinary approaches including human and veterinary experts should be implemented to develop reliable investigation procedures with respect to the current reality of animal owners and their pets. Additionally, consequent basic hygienic measures, prudent use of antimicrobials in companion animals and efforts regarding implementation of antibiotic stewardships should be fostered.

Comparative Molecular Analysis Substantiates Zoonotic Potential of Equine Methicillin-Resistant Staphylococcus aureus

Despite the increasing importance of methicillin-resistant Staphylococcus aureus (MRSA) in veterinary medicine, knowledge about the epidemiology of the pathogen in horses is still poor. The phylogenetic relationship of strains of human and equine origins has been addressed before, usually by analyzing results of common standard classification methods for MRSA. This work intends to go beyond the baseline of typing procedures in order to comparatively characterize equine and human MRSA strains with similar phylogenetic backgrounds. In addition to multilocus sequence typing, pulsed-field gel electrophoresis, spa typing, staphylococcal cassette chromosome mec typing, and a PCR for Panton-Valentine leukocidin gene detection, a microarray analysis of a total of 185 structural, virulence-associated, and resistance loci was applied. The results showed that clonal complex 8 (CC8) was absolutely predominant (16 strains) in 19 investigated equine MSRA strains. Of the CC8 strains, 13 belonged to sequence type 254 (ST254) and the other 3 to ST8. This genotype has been isolated from different equine patients in various regions over several years, substantiating the apparent predominance of CC8 STs in MRSA strains of horses worldwide. Furthermore, comparatively investigated human strains of ST254 displayed molecular-typing results indistinguishable from those for strains of equine origin. Two further equine strains (ST22 and ST1117) showed similarity to ST22 human strains (CC22). One equine strain belonged to ST398, a genotype recently described as being frequently isolated from specimens from pigs and pig farmers. These data provide evidence for the adaptation of certain MRSA genotypes to more than one mammalian species, reflecting their extended host spectra.

Widespread rapid emergence of a distinct methicillin- and multidrug-resistant Staphylococcus pseudintermedius (MRSP) genetic lineage in Europe

In order to gain a deeper insight into the phylogenetic background and diversity of methicillin-resistant S. pseudintermedius (MRSP) of animal origin, genetic relationships and clonal distribution among 146 European MRSP were examined using different molecular and phenotypical typing approaches. MRSP strains were derived from clinical microbiological specimens (mainly of small animal origin) sent in for diagnostic purposes from various veterinary facilities between 2005 and 2008. Pulsed-field gel electrophoresis (PFGE) of SmaI-macrorestriction fragments allowed differentiation of five PFGE-clusters that were subdivided into further distinct subtypes. Representatives of each PFGE subtype were analyzed by multilocus sequence typing (MLST) for assignment of sequence types (ST). With one exception (ST5), all these MRSP strains belonged to ST71. Furthermore, assessment of spa-typing results revealed that the majority of all strains harboured spa type t02. Further sporadically detected spa types t05 and t06 as well as two new types (t15 and t23), were found to be closely related to t02. According to PCR-based SCCmec-typing, SCCmecIII was the most prevalent type (n=138), and solely one non-typeable variant was identified in several strains (n=8). In addition, all strains were tested positive by PCR for the leukotoxin encoding operon LukI and the Staphylococcus intermedius-exfoliative toxin (SIET), respectively. Our cumulative data indicate a recent emergence of a certain multidrug-resistant MRSP-lineage (ST71) in central and southern European countries during the last few years.

Sharing More than Friendship — Nasal Colonization with Coagulase-Positive Staphylococci (CPS) and Co-Habitation Aspects of Dogs and Their Owners

Background Since the relationship between dogs and their owners has changed, and dogs moved from being working dogs to family members in post-industrial countries, we hypothesized that zoonotic transmission of opportunistic pathogens like coagulase positive staphylococci (CPS) is likely between dogs and their owners. Methodology/Principal Findings CPS- nasal carriage, different aspects of human-to-dog relationship as well as potential interspecies transmission risk factors were investigated by offering nasal swabs and a questionnaire to dog owners (108) and their dogs (108) at a dog show in 2009. S. aureus was found in swabs of 20 (18.5%) humans and two dogs (1.8%), and spa types which correspond to well known human S. aureus lineages dominated (e.g. CC45, CC30 and CC22). Multilocus sequence typing (MLST) of the two canine strains revealed ST72 and ST2065 (single locus variant of ST34). Fifteen dogs (13.9%) and six owners (5.6%) harboured S. pseudintermedius, including one mecA-positive human isolate (MRSP). Pulsed field gel electrophoresis (PFGE) revealed that one dog/owner pair harboured indistinguishable S. pseudintermedius- isolates of ST33. Ten (48%) of the 21 S. pseudintermedius-isolates showed resistance towards more than one antimicrobial class. 88.9% of the dog owners reported to allow at least one dog into the house, 68.5% allow the dog(s) to rest on the sofa, 39.8% allow their dogs to come onto the bed, 93.5% let them lick their hands and 52.8% let them lick their face. Bivariate analysis of putative risk factors revealed that dog owners who keep more than two dogs have a significantly higher chance of being colonized with S. pseudintermedius than those who keep 1–2 dogs (p<0.05). Conclusions/Recommendations In conclusion, CPS transmission between dog owners and their dogs is possible. Further investigation regarding interspecies transmission and the diverse adaptive pathways influencing the epidemiology of CPS (including MRSA and MRSP) in different hosts is needed.

Alarming proportions of methicillin-resistant Staphylococcus aureus (MRSA) in wound samples from companion animals, Germany 2010-2012.

Staphylococcus (S.) aureus is an important cause of wound infections in companion animals, and infections with methicillin-resistant S. aureus (MRSA) are of particular concern due to limited treatment options and their zoonotic potential. However, comparable epidemiological data on MRSA infections in dogs, cats and horses is scarce, also limiting the knowledge about possible links to MRSA isolates from human populations. To gain more knowledge about the occurrence and genotypic variation of MRSA among wound swabs of companion animal origin in Germany we performed a survey (2010–2012) including 5,229 samples from 1,170 veterinary practices. S. aureus was identified in 201 (5.8%) canine, 140 (12.2%) feline and 138 (22.8%) equine swabs from a total of 3,479 canine, 1,146 feline and 604 equine wounds, respectively. High MRSA rates were identified with 62.7%, 46.4% and 41.3% in S. aureus of canine, feline and equine origin, respectively. Further genotyping including spa typing and multilocus sequence typing (MLST) revealed a comparable distribution of spa types among canine and feline MRSA with CC22 (47.6%; 49.2%) and CC5 (30.2%; 29.2%) as predominant lineages followed by CC398 (13.5%; 7.7%) and CC8 (4.0%; 9.2%). In contrast, the majority of equine MRSA belonged to CC398 (87.7%). Our data highlight the importance of S. aureus and MRSA as a cause of wound infections, particularly in cats and horses in Germany. While “human-associated” MRSA lineages were most common in dogs and cats, a remarkable number of CC398-MRSA was detected in horses, indicating a replacement of CC8-MRSA as the predominant lineage within horses in Germany. These data enforce further longitudinal epidemiological approaches to examine the diversity and temporal relatedness of MRSA populations in humans and animals to assess probable sources of MRSA infections. This would enable a sound risk assessment and establishment of intervention strategies to limit the additional spread of MRSA.

MRSA Variant in Companion Animals

Methicillin-resistant Staphylocoocus aureus (MRSA) harboring mecALGA251 has been isolated from humans and ruminants. Database screening identified this MRSA variant in cats, dogs, and a guinea pig in Germany during 2008–2011. The novel MRSA variant is not restricted to ruminants or humans, and contact with companion animals might pose a zoonotic risk.

Species differentiation within the Staphylococcus intermedius group using a refined MALDI-TOF MS database.

Among coagulase-positive staphylococci of animal origin, the members of the Staphylococcus intermedius-group (SIG: S. intermedius, Staphylococcus pseudintermedius and Staphylococcus delphini) are important opportunistic pathogens in different animal hosts and occasionally in humans. However, the unambiguous species diagnosis of SIG is often challenging. Therefore, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) -based SIG-identification with Bruker Microflex LT in combination with Biotyper 3.0 software (Bruker Daltonics, Bremen, Germany) was evaluated using (i) the original database content and (ii) the database after extension with distinct hierarchical clustered reference spectra for 60 SIG. A convenience sample comprising 200 isolates was used to compare both database performances. As a result, 17 isolates initially diagnosed as S. intermedius with the current content of the Bruker database were identified as S. pseudintermedius by applying the in-house reference spectra extended version. Furthermore, a significant improvement (average rise of log score value: 0.24) of the SIG identification score values was achieved, emphasizing that further sequence-based refinement of the Bruker database content allows improvement of MALDI-TOF MS-based identification.

Phylogenetic Analysis of Staphylococcus aureus CC398 Reveals a Sub-Lineage Epidemiologically Associated with Infections in Horses

In the early 2000s, a particular MRSA clonal complex (CC398) was found mainly in pigs and pig farmers in Europe. Since then, CC398 has been detected among a wide variety of animal species worldwide. We investigated the population structure of CC398 through mutation discovery at 97 genetic housekeeping loci, which are distributed along the CC398 chromosome within 195 CC398 isolates, collected from various countries and host species, including humans. Most of the isolates in this collection were received from collaborating microbiologists, who had preserved them over years. We discovered 96 bi-allelic polymorphisms, and phylogenetic analyses revealed that an epidemic sub-clone within CC398 (dubbed ‘clade (C)’) has spread within and between equine hospitals, where it causes nosocomial infections in horses and colonises the personnel. While clade (C) was strongly associated with S. aureus from horses in veterinary-care settings (p = 2×10−7), it remained extremely rare among S. aureus isolates from human infections.