JAC Advance Access originally published online on June 16, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):428-431; doi:10.1093/jac/dkl253
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Molecular typing of methicillin-resistant staphylococci isolated from cats and dogs
1 Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia GPO Box 2471, Adelaide, 5000 SA, Australia 2 Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine WA Royal Perth Hospital, Perth GPO Box X2213, Perth, 6847 WA, Australia 3 Gram-Positive Bacteria Typing and Research Unit, Molecular Genetics Research Unit, School of Biomedical Sciences, Curtin University of Technology GPO Box U1987, Perth, 6845 WA, Australia
*Corresponding author. Tel: +61-8-83022933; Fax: +61-8-83022389; E-mail: mary.barton{at}unisa.edu.au
Received 4 April 2006; returned 25 April 2006; revised 27 May 2006; accepted 27 May 2006
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Abstract |
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Objectives: Methicillin-resistant staphylococci (MRS) isolates from healthy and diseased cats and dogs were characterized by staphylococcal cassette chromosome mec (SCCmec), multilocus sequence typing (MLST) and cassette chromosome recombinase gene (ccrAB) sequencing.
Methods: PCR-directed SCCmec typing was carried out for all MRS isolates and two Staphylococcus aureus and two Staphylococcus epidermidis strains were analysed by MLST. Strains belonging to SCCmec type III and IV were sequenced for their ccrAB gene of allotypes 3 and 2, respectively.
Results: Five types of SCCmec, types I, III, IV, IV (paediatric) and V SCCmec, were found. The S. aureus strains belonged to sequence type (ST) 239 and the two S. epidermidis belonged to ST43 and ST60 respectively. High sequence conservation was observed for the ccrAB gene of allotypes 2 and 3.
Conclusions: MRS isolates from cats and dogs demonstrate a similar diversity of SCCmec types to those found in human staphylococci and ST239-MRSA-III, a widely dispersed strain in human hospitals, was identified in diseased dogs.
Keywords: ccrAB gene , C-MRSA, MLST , SCCmec
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Introduction |
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Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infections in health care settings worldwide and in recent years community-acquired MRSA has emerged simultaneously in a number of countries. MRSA produces penicillin-binding protein 2A (PBP 2A), which has reduced affinity for ß-lactam antibiotics and is encoded by the mecA gene.1 Recent studies of the genetic basis of methicillin-resistant staphylococci (MRS) have elucidated the structure of the mobile genetic element staphylococcal cassette chromosome mec (SCCmec). SCCmec consists of the mec locus which carries mecA and its regulatory genes, and cassette chromosome recombinase (ccr) gene complexes. The ccr complex consists of the ccr genes ccrA and ccrB in combination (ccrAB) or alone (ccrC), which encode recombinases necessary for site- and orientation-specific integration and excision of the SCCmec element. Six SCCmec types with five ccr allotypes have been described in staphylococci (SCCmec I, II, III, IV, IV paediatric and V).2–5 The ccrA2B2 and ccrC are the only allotypes to date shown to mediate precise excision activity of the SCCmec element.3,6 SCCmec typing technique has become an essential tool in investigating MRSA epidemiology in humans and has led to successful classification of healthcare-associated MRSA (H-MRSA) and community-acquired MRSA (C-MRSA).
There are a limited number of reports on MRSA infections in pet animals and therefore few typing studies assessing SCCmec elements and multilocus sequence typing (MLST) have been reported.7–11 In the face of rapidly emerging MRSA in the community and the fact that cats and dogs are in close contact with their owners, the risk of transmission of such bacteria between animals and humans must be considered.
In this study, we report the results of molecular typing of MRS strains isolated from healthy and diseased cats and dogs using SCCmec typing, ccrAB gene sequencing and MLST.
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Materials and methods |
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Methicillin-resistant strains
MRS were isolated from 10 animals (5 healthy dogs, 2 healthy cats and 3 diseased dogs) amongst a total of 252 animals sampled over a 6 month period as previously reported.12 In brief, two S. aureus strains were recovered from each of two dogs (from one veterinary clinic) with skin infections, one Staphylococcus epidermidis from a dog with an infected wound, one S. epidermidis from a healthy cat, three Staphylococcus haemolyticus (two from healthy dogs, one from a healthy cat), two Staphylococcus warneri (both from healthy dogs) and one Staphylococcus hominis from a healthy dog were investigated in this study.
DNA isolation
Chromosomal DNA was prepared using the QIAGEN DNeasy kit system (QIAGEN, GmbH Hilden, Germany) according to the manufacturer's instructions. DNA was amplified using a Mycycler thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA).
SCCmec typing
SCCmec elements were determined for all MRS isolates using the method described by Lim and co-workers.13 Methods as described by Oliveira and de Lancastre 14 and Ma et al.,4 were used to distinguish variants among the SCCmec type III and IV.
CcrAB gene sequencing
The entire nucleotide sequence of the ccrAB gene of allotypes 2 and 3 from 6 isolates (Table 1) was determined using primers as listed in Table 2. The overlapping primers were designed to amplify the entire ccrAB region by using previously published ccrAB nucleotide sequences with accession numbers D86934 [GenBank] and AB037671 [GenBank] .2 PCR was performed in a 50 µL volume containing 1 µL of DNA product, 0.2 mM of each deoxynucleotide triphosphate (Promega, Madison, USA), 2.5 U of Taq polymerase (Promega), 1.5 mM magnesium chloride (Promega), 1 x buffer (Promega) and 25 pmol each of forward and reverse primer. The reaction mixture was subjected to pre-denaturation at 95°C for 5 min; 30 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 1 min; post-extension for 5 min at 72°C. The PCR products were separated on 1% agarose gels in 1x Tris-acetate-EDTA buffer stained with ethidium bromide and visualized with UV light.
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The PCR products were purified using UltraClean PCR clean-up kit (Mo Bio Laboratories Inc, Solena Beach, CA, USA) according to the manufacturer's instructions and were sequenced using Big Dye Terminator v3.1 cycle sequencing ready reactions (Applied Biosystems, Foster City, CA, USA) at the DNA Sequencing Facility, Flinders Medical Centre, Adelaide. The nucleotide sequences and the deduced amino acids were edited by using Chromas software (version 2.3). Homology searches and sequence alignments were performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and a computer program from Australian National Genomic Information Services (ANGIS) (http://www.angis.org.au).
MLST
MLST was performed on S. aureus isolates A-44K9S and A-47K9A and S epidermidis isolates A-53DS and CS8 by PCR amplification of seven housekeeping genes using published primer sequences and protocols (http://www.mlst.net). The alleles at each of the seven housekeeping loci were identified by comparing the sequences obtained from the test isolates with sequences held in the MLST database (http://www.saureus.mlst.net, http://www.sepidermidis.mlst.net). These databases were used to identify the allelic profile and the sequence type (ST) of each isolate.
Nucleotide sequences of ccrAB genes were submitted to GenBank under accession numbers DQ196432, DQ196433, DQ196434 and DQ225180.
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Results and discussion |
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Despite the fact that cloxacillin or other antistaphylococcal penicillins are rarely used in the treatment of staphylococcal infections in cats and dogs, MRS have been isolated from cats and dogs in recent years albeit at low frequency.11 However, until recently, little attention has been paid to this emerging problem and hence the types of SCCmec harboured by MRS of cat and dog origin are poorly understood. In this study only a small number of MRS were isolated (10/252 animals sampled) and were mostly derived from healthy animals. These isolates were confirmed as having the mecA gene by partial mecA nucleotide sequencing and assessed for resistance to other antibiotics by agar dilution methods as previously reported.7 The SCCmec typing strategy we used in this study detected five SCCmec elements (SCCmec type I, III, IV, IV paediatric and V) (Table 1). SCCmec types I and III (associated with H-MRSA) were identified in S. hominis and S. aureus strains respectively. SCCmec types IV and V associated with C-MRSA were obtained from S. epidermidis, S. haemolyticus and S. warneri. Another SCCmec type classified as IV paediatric5 was identified in two isolates of S. haemolyticus. This study has clearly demonstrated that MRS from cats and dogs can carry diverse SCCmec mobile genetic elements.
Strong sequence conservation among the ccrAB2 and ccrAB3 alleles was observed and was independent of species; S. aureus (A-44K9S and A-47K9A), S. warneri (DM35* and DM36) and S. epidermidis (A-53DS and CS8) contained ccrAB genes that were identical to ccrAB genes of human MRSA strains. This was further confirmation that these isolates belong to type IV and III SCCmec because the combination of ccr genes and mec gene complexes defines the SCCmec type.3,6 It is worth noting that the ccrA2B2 and ccrC genes have been shown in human MRSA to be most active with regard to precise excision.3,6 Similarly, SCCmec IV which harbours the ccrA2B2 allotype has been postulated to be highly transmissible because of its size and extreme heterogeneity in its junkyard region.3 It has also been suggested that S. epidermidis with SCCmec type IV isolated from healthy human individuals are responsible for the conversion of commensal S. aureus to MRSA.15 Although so far actual horizontal transfer of these mobile genetic elements between different companion animal staphylococcal species has not been demonstrated, the identification of SCCmec IV with active ccrA2B2 genes in coagulase-negative staphylococci from healthy cats and dogs is significant. These species are likely to be missed in routine microbiological testing where methods are focussed on detecting more pathogenic species such as S. aureus and S. intermedius through use of more selective medium or molecular markers specific for these organisms.
The small size and high polymorphism of type IV SCCmec, may render this element highly transmissible to other staphylococcal species by an as yet unidentified mechanism and therefore might lead to cats and dogs becoming a potential reservoir for community-MRS.
Of the ten MRS isolates investigated in this study, two S. aureus and two S. epidermidis strains were examined by MLST (Table 1) due to the availability of a comparative database. The two S. aureus strains had allelic profile 2-3-1-1-4-4-3 with ST239 and belonged to Clonal Complex (CC) 8. S. epidermidis strain A-53DS had allelic profile 1-6-3-6-2-1-1 as described for ST60 and CS8 had 2-1-3-2-1-1-1 with ST43. ST239-MRSA-III is a well-recognized human MRSA lineage that is mainly confined to health care settings and one can only speculate that the dogs became infected through household or other close contact with a human MRSA carrier. On the other hand, S. epidermidis strains had unique STs in the MLST database (S. J. O'Hanlon, Department of Infectious Disease Epidemiology, Faculty of Medicine, Imperial College London, personal communication) suggesting possible specificity of these strains to cat and dog.
In conclusion, MRS from cats and dogs can harbour SCCmec elements encoding determinants for the expression of the methicillin-resistance phenotype and some strains can share their clonal lineage with human staphylococci. Due to lack of information about the MRS colonization status of the owners of these animals, the source of these strains can not be confirmed and therefore further studies in this area are required to investigate the epidemiology of MRS in companion animals.
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None to declare.
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Acknowledgements |
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We wish to thank Associate Professor Teruyo Ito, Juntendo University, Japan, and Dr. Michael Heuzenroeder of the Institute for Medical and Veterinary Science, Adelaide for their useful discussion on the ccrAB gene sequencing. We also wish to thank Simon O’Hanlon, Faculty of Medicine, Imperial College London for providing allelic profile and STs for the S. epidermidis strains. This study was funded by Australian Companion Animal Health Foundation.
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References |
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1 Chambers HF. (1997) Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin Microbiol Rev 10:781–91.[Abstract]
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14
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