Partial Nucleotide Sequencing of the mecA Genes of
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微生物临床杂志 2006年第2期
ABSTRACT
Methicillin-resistant staphylococci (MRS) pose a challenge to clinicians and health administrators in human medicine, but MRS infections in cats and dogs are not perceived as a problem in veterinary medicine. Ten methicillin-resistant staphylococcal isolates obtained from healthy and diseased cats and dogs were subjected to partial DNA sequencing of the mecA gene. Sequence analysis shows that MRS isolates from both healthy and diseased cats and dogs can harbor the mecA gene. The mecA genes of animal isolates were identical to that found in human MRS strains, and therefore the possibility of zoonotic transfer must be considered.
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) isolates have been identified as a serious cause of nosocomial infections, and more recently, community-acquired MRSA has been recognized as an emerging problem in a number of countries. The resultant infections include a range of conditions from minor skin infections to severe life-threatening conditions. Staphylococcal resistance to methicillin is attributable to multiple mechanisms (6), but the most important mechanism from the standpoint of nosocomial infections and treatment appears to be intrinsic resistance and is due to the expression of low-affinity penicillin-binding protein 2A (11, 22, 30). Penicillin-binding protein 2A is highly conserved among staphylococci and is encoded by the chromosomal gene mecA (3).
Cats and dogs have become an integral part of modern society, especially in developed countries, and attention is given to their care and welfare. A large proportion of the human population is in contact with cats and dogs on a daily basis, and so there is the potential for transfer of bacteria or resistance genes between companion animals and humans (5, 9). Studies conducted in recent years have shown that staphylococci isolated from cats and dogs have become resistant to methicillin (8, 16, 19, 20, 24, 25, 26, 31, 33). However, none of these studies on methicillin-resistant staphylococci have compared the mecA gene from these animals' isolates with that from human MRSA strains. Although methicillin or other antistaphylococcal penicillins such as oxacillin and cloxacillin are not commonly used antibiotics in the treatment of staphylococcal infections in cats and dogs, there are clear public health implications related to the presence of methicillin-resistant staphylococci on the skin of cats and dogs. In this study, we report the isolation, antibiotic susceptibility, and partial nucleotide sequence determination of 2 kbp of the mecA gene of staphylococci isolated from diseased and healthy cats and dogs.
MATERIALS AND METHODS
Isolation of staphylococci. A total of 55 healthy cats and 51 healthy dogs undergoing desexing or boarding at two veterinary clinics in Adelaide were sampled. One hundred forty-one diseased dogs and five diseased cats with various skin infections were also sampled. Staphylococci were isolated from swabs taken from the skin and lesions of the animals. A selective medium, Gioliti-Cantoni broth (Oxoid, Basingstoke, England) containing 0.035% potassium tellurite, was used as an enrichment broth without addition of sterile paraffin wax. This broth was used for isolation because it contains mannitol and sodium pyruvate, which act as growth stimulants for staphylococci and can therefore improve their detection when the organisms are present in small numbers.
Swabs were incubated at 37°C in Gioliti-Cantoni broth for 48 h. Broths showing complete blackening were subcultured onto mannitol salt agar (Oxoid) and incubated at 37°C for 48 h. Yellow or white colonies were picked onto blood agar and incubated at 37°C for 24 h. Catalase-positive, oxidase-negative colonies were gram stained. Bacitracin (0.05-U disk; Oxoid) and furazolidone (100-μg disk; Oxoid) sensitivity tests were performed to differentiate staphylococci from micrococci. Coagulase tests were performed with rabbit plasma, and the ID 32 Staph identification system (Bio Mérieux S.A., Marcy l'Etoile, France) was used to fully identify the isolates.
Antimicrobial susceptibility test. The agar dilution method described in the National Committee for Clinical Laboratory Standards (NCCLS) guidelines (23) was used to determine in vitro susceptibilities. The antimicrobial drugs included were oxacillin, tetracycline, gentamicin, amoxicillin-clavulanic acid, clindamycin, ceftiofur, ampicillin, ciprofloxacin, cephalothin, erythromycin, chloramphenicol, rifampin, and vancomycin (Sigma, St. Louis, Mo.). Briefly, for each organism, a single colony from an overnight culture on a blood agar plate was suspended in a 0.85% saline solution. The suspensions were adjusted to a 0.5 McFarland standard with a Vitek colorimeter (Bio Mérieux S.A., Marcy l'Etoile, France). The 0.5 McFarland suspensions were diluted 1:10 in saline solution. Mueller-Hinton agar (Oxoid) supplemented with 0%, 2%, or 4% salt and various concentration of antibiotics was inoculated with the final suspensions with a replicator to deliver approximately 104 CFU in each spot. Vancomycin was tested with brain heart infusion agar (Oxoid) supplemented with 2% salt (7). Plates were read after 24 h at 35°C. S. aureus ATCC 29213 and ATCC 43300, Escherichia coli ATCC 25922, and Enterococcus faecalis ATCC 29212 were included in each run as control organisms.
DNA extraction. Chromosomal DNA of the 10 methicillin-resistant staphylococcal isolates was extracted by the simple lysis method as previously described (1). The isolates were first grown overnight at 37°C in 10 ml of brain heart infusion broth (Oxoid), and then a 1-ml volume was microcentrifuged. The pellets were washed in 1 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and microcentrifuged. The pellets were resuspended in 200 μl of TE buffer, and the suspension was boiled at 95°C for 20 min and microcentrifuged for 2 min. The supernatants were kept on ice, and 5 μl was used for PCR amplification.
PCR and sequencing. The list of PCR primers used to amplify mecA, the expected amplicon sizes, and the locations of the genes on S. aureus chromosome strain TK784 (31) are shown in Table 1. The sequencing primers used were thesame as the PCR primers used to amplify mecA. The designed primers wereobtained from GeneWorks Pty. Ltd., Adelaide, Australia. Mutations within mecA genes have been reported to occur at nucleotide positions 503, 510, 593, 642, 737, 907 to 912, 1721, and 1834 (32), and hence we used these overlapping primers to compensate for any potential mutation occurring in these regions. The nucleotide positions of the primers are based on the sequence submitted to GenBank under accession number Y00688 (30). A 50-μl PCR mixture consisted of 5 μl of template DNA, 2.5 U of Taq DNA polymerase (Roche Diagnostic GmbH, Mannheim, Germany), 10x PCR buffer plus Mg (5 μl) (Roche Diagnostic), 0.25 mM each deoxynucleoside triphosphate (Promega, Madison, Wis.), and 25 pmol each of the forward and reverse primers. The reaction mixture was subjected to predenaturation at 95°C for 5 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and extension for 5 min at 72°C. PCR products (6 μl) were resolved on a 2% agarose gel (Promega, Madison, Wis.) in 1x Tris-acetic EDTA buffer at 120 V for 1 h. The gel was stained with ethidium bromide and visualized under UV (Gel doc; Bio-Rad Laboratories, Hercules, Calif.).
Nucleotide sequencing. The DNA fragments for sequencing were obtained by PCR amplification of each chromosomal DNA as the template. After purification with the QIAquick PCR purification kit (QIAGEN GmbH, Hilden, Germany), the fragments of each PCR products were sequenced with the set of primers listed in Table 2 with Big Dye Terminator v3.1 (Applied Biosystems, Foster City, Calif.). Sequence alignment was carried out by using the Australian National Genomic Information Service (ANGIS) program (http://www.angis.org.au).
RESULTS
Isolation of staphylococci. Of the 252 animals (192 dogs and 60 cats) sampled, methicillin-resistant staphylococci were found in 5 healthy dogs, 3 diseased dogs, and 2 healthy cats. Methicillin-resistant staphylococci were identified as S. aureus (2), S. epidermidis (2), S. haemolyticus (3), S. warneri (2), and S. hominis (1) (Table 2). MRSA was isolated from skin lesions of two infected dogs, and none was isolated from healthy animals. However, one methicillin-resistant S. epidermidis strain was recovered from a wound infection of a dog and was the only coagulase-negative staphylococcus infecting a diseased animal. Healthy cats and dogs in this study had predominately coagulase-negative, methicillin-resistant staphylococci comprising mostly S. haemolyticus (isolated from two dogs and one cat), S. warneri (two dogs), S. hominis (one dog), and S. epidermidis (one cat) strains.
Antimicrobial susceptibility. The antibiotic susceptibility patterns are shown in Table 2. The 10 isolates were resistant to methicillin (oxacillin), and various MICs were determined. For two MRSA and two S. haemolyticus strains isolated from healthy cats and dogs, the oxacillin MIC was 128 μg/ml. These four isolates were resistant to all of the antibiotics tested except vancomycin and rifampin. Another S. haemolyticus strain, dubbed DN12 (Table 2), also showed multiresistance properties, but the MIC of oxacillin was low (4 μg/ml) and the strain was susceptible to ciprofloxacin, chloramphenicol, vancomycin, and rifampin. The remaining five isolates had borderline resistance to methicillin (MIC, 4 μg/ml) and had various patterns of resistance to other antibiotics. Generally, they were susceptible to vancomycin, rifampin, chloramphenicol, and ciprofloxacin. Susceptibility to the ?-lactam antibiotics, such as ampicillin, amoxicillin-clavulanic acid, and ceftiofur, used in this study differed among the isolates, but NCCLS standards recommend reporting these organisms as resistant due to reported treatment failure in vivo with these antibiotics. S. hominis and S. warneri were resistant to methicillin (?-lactam antibiotics) only but were susceptible to all of the other classes of antibiotics. S. epidermidis strains from a diseased dog and a healthy cat, however, were both susceptible to ciprofloxacin and gentamicin but resistant to tetracycline and erythromycin, respectively.
Nucleotide sequencing. Each primer pair yielded a single major amplicon of the expected size, ranging from 454 bp to 714 bp, as shown in Table 1 and Fig. 1. Sequencing results obtained from the 10 isolates and analysis with the ANGIS global alignment program resulted in 100% homology to the fractions of Y00688 particularly targeted for amplification (result not shown). The overlapping sets of primers developed for amplification and sequencing facilitated the alignment due to the usual difficulties of getting the beginning and end of a sequenced product.
DISCUSSION
Most of the methicillin-resistant staphylococci in this study were coagulase-negative species, in agreement with previously reports (8, 16), and were predominantly isolated from healthy cats and dogs. The MRSA strains in this study were derived from diseased dogs, and none were from healthy animals. This probably reflects the virulent nature of S. aureus compared to nonpathogenic or opportunistic coagulase-negative staphylococci that are normal resident bacteria on the skin and other anatomical sites of healthy and diseased individuals (13, 18). In human medicine, methicillin-resistant, coagulase-negative staphylococci such as S. epidermidis and S. haemolyticus are a leading cause of nosocomial infections in neonates, immunocompromised patients, and patients with indwelling catheters (15, 37). However, in dogs, staphylococcal infections are mainly due to S. intermedius, with S. aureus causing only occasional infections. The predominant pathogenic species in cats is S. aureus (13); hence, the buildup of methicillin-resistant, coagulase-negative species in these animals could be missed in routine laboratory testing for recognized pathogenic species and could serve as a gene pool or reservoir of a methicillin resistance gene.
Nucleotide sequence analyses of several mecA genes from different species such as S. aureus (DDBJ/EMBL/GenBank accession numbers Y00688 [30] and X52593 [28]), S. epidermidis (accession no. X52592) (28), S. sciuri (accession no. Y13096) (35), and S. aureus strain N315 (14) have revealed that the mecA gene is much more conserved among staphylococcal species of human origin. However, little is known about the mecA gene from companion animal sources. There is concern that cats and dogs are a reservoir or source of MRSA infections in humans (10, 29) due to their close contact with humans in the household.
The source of the mecA gene in these animals is unclear. In human medicine, it has been demonstrated that horizontal transfer from a primitive staphylococci species, S. sciuri, into an S. aureus chromosome may have occurred (36). The discovery of the mobile genetic element staphylococcal chromosome cassette mec (SCCmec) in MRSA strains of human origin further advances the theory that mecA could be shared among staphylococcal species (17). SCCmec ranges in size from approximately 20 to about 68 kbp and is composed of the mec gene complex (mecA gene and its regulators) and the ccr gene complex, which encodes site-specific recombinases responsible for the mobility of the element.
Methicillin and other antistaphylococcal penicillins such as oxacillin and cloxacillin are rarely used in the treatment of staphylococcal infections in cats and dogs (27), and therefore selective pressure resulting from their use is not likely to occur. In this study, 80% of the methicillin-resistant staphylococcal isolates were coagulase-negative strains and were mostly isolated from healthy cats and dogs. These isolates are likely to be normal bacterial flora of healthy animals and were probably not exposed to methicillin or any other antistaphylococcal penicillins that could have driven the development of methicillin resistance and/or acquisition of the mecA gene. Thus, the mecA genes in these isolates were probably derived from their owners, a view supported because the nucleotide sequence was identical to that of a human MRSA strain. The same assumption could hold true for the two MRSA strains isolated from diseased dogs. This is because staphylococci are species specific in nature but human S. aureus strains have been implicated in infections of other animal species (4, 12). The S. aureus strains in this study could be human-derived MRSA due to the 100% sequence homology between them. Identical chromosomal patterns from pulsed-field gel electrophoresis and SCCmec typing (2, 21, 34) have led to the conclusion that cats and dogs and their owners might carry the same MRSA strains, and this study confirms these findings. This preliminary molecular study on methicillin-resistant staphylococci from cats and dogs shows that humans and their companion animals are likely to share methicillin-resistant staphylococci and other resistance genes as a result of close contact. The dispersal of these resistance genes is likely to be facilitated by the fact that staphylococci are normal resident bacterial flora of the skin and the mucosal surfaces of the respiratory tracts of humans and animals and hence could easily be spread by skin-to-skin contact, saliva, and aerosols from sneezing and coughing.
In conclusion, we have demonstrated that the mecA gene which encodes methicillin resistance in staphylococci of human origin is the same gene responsible for methicillin resistance in staphylococci isolated from cats and dogs. Further studies on SCCmec and multilocus sequence typing of these isolates will improve our understanding of the mobile genetic element carrying the mecA gene and the clonal relationship of these isolates to human MRSA. This will aid in the understanding and management of potential transmission of methicillin-resistant staphylococci between human and their pets.
ACKNOWLEDGMENTS
We thank Gregory Parkins (Plympton Veterinary Clinic, Adelaide, Australia), Mike Lawley, and Darren Hanshaw (Black Forest Veterinary Surgery, Adelaide, Australia) for given us access to their practice for sampling of healthy animals. We also thank Daniela Signoriello (IDEXX, Adelaide, Australia) for supplying the clinical isolates from diseased animals. ATCC 43300 was a kind gift from Jan Bell (Women's and Children's Hospital, Adelaide, Australia).
This work was supported by a University of South Australia President's Scholarship.
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School of Pharmacy and Medical Sciences, University of South Australia, Adelaide 5001, South Australia, Australia(Seidu Malik, Haihong Peng)
Methicillin-resistant staphylococci (MRS) pose a challenge to clinicians and health administrators in human medicine, but MRS infections in cats and dogs are not perceived as a problem in veterinary medicine. Ten methicillin-resistant staphylococcal isolates obtained from healthy and diseased cats and dogs were subjected to partial DNA sequencing of the mecA gene. Sequence analysis shows that MRS isolates from both healthy and diseased cats and dogs can harbor the mecA gene. The mecA genes of animal isolates were identical to that found in human MRS strains, and therefore the possibility of zoonotic transfer must be considered.
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) isolates have been identified as a serious cause of nosocomial infections, and more recently, community-acquired MRSA has been recognized as an emerging problem in a number of countries. The resultant infections include a range of conditions from minor skin infections to severe life-threatening conditions. Staphylococcal resistance to methicillin is attributable to multiple mechanisms (6), but the most important mechanism from the standpoint of nosocomial infections and treatment appears to be intrinsic resistance and is due to the expression of low-affinity penicillin-binding protein 2A (11, 22, 30). Penicillin-binding protein 2A is highly conserved among staphylococci and is encoded by the chromosomal gene mecA (3).
Cats and dogs have become an integral part of modern society, especially in developed countries, and attention is given to their care and welfare. A large proportion of the human population is in contact with cats and dogs on a daily basis, and so there is the potential for transfer of bacteria or resistance genes between companion animals and humans (5, 9). Studies conducted in recent years have shown that staphylococci isolated from cats and dogs have become resistant to methicillin (8, 16, 19, 20, 24, 25, 26, 31, 33). However, none of these studies on methicillin-resistant staphylococci have compared the mecA gene from these animals' isolates with that from human MRSA strains. Although methicillin or other antistaphylococcal penicillins such as oxacillin and cloxacillin are not commonly used antibiotics in the treatment of staphylococcal infections in cats and dogs, there are clear public health implications related to the presence of methicillin-resistant staphylococci on the skin of cats and dogs. In this study, we report the isolation, antibiotic susceptibility, and partial nucleotide sequence determination of 2 kbp of the mecA gene of staphylococci isolated from diseased and healthy cats and dogs.
MATERIALS AND METHODS
Isolation of staphylococci. A total of 55 healthy cats and 51 healthy dogs undergoing desexing or boarding at two veterinary clinics in Adelaide were sampled. One hundred forty-one diseased dogs and five diseased cats with various skin infections were also sampled. Staphylococci were isolated from swabs taken from the skin and lesions of the animals. A selective medium, Gioliti-Cantoni broth (Oxoid, Basingstoke, England) containing 0.035% potassium tellurite, was used as an enrichment broth without addition of sterile paraffin wax. This broth was used for isolation because it contains mannitol and sodium pyruvate, which act as growth stimulants for staphylococci and can therefore improve their detection when the organisms are present in small numbers.
Swabs were incubated at 37°C in Gioliti-Cantoni broth for 48 h. Broths showing complete blackening were subcultured onto mannitol salt agar (Oxoid) and incubated at 37°C for 48 h. Yellow or white colonies were picked onto blood agar and incubated at 37°C for 24 h. Catalase-positive, oxidase-negative colonies were gram stained. Bacitracin (0.05-U disk; Oxoid) and furazolidone (100-μg disk; Oxoid) sensitivity tests were performed to differentiate staphylococci from micrococci. Coagulase tests were performed with rabbit plasma, and the ID 32 Staph identification system (Bio Mérieux S.A., Marcy l'Etoile, France) was used to fully identify the isolates.
Antimicrobial susceptibility test. The agar dilution method described in the National Committee for Clinical Laboratory Standards (NCCLS) guidelines (23) was used to determine in vitro susceptibilities. The antimicrobial drugs included were oxacillin, tetracycline, gentamicin, amoxicillin-clavulanic acid, clindamycin, ceftiofur, ampicillin, ciprofloxacin, cephalothin, erythromycin, chloramphenicol, rifampin, and vancomycin (Sigma, St. Louis, Mo.). Briefly, for each organism, a single colony from an overnight culture on a blood agar plate was suspended in a 0.85% saline solution. The suspensions were adjusted to a 0.5 McFarland standard with a Vitek colorimeter (Bio Mérieux S.A., Marcy l'Etoile, France). The 0.5 McFarland suspensions were diluted 1:10 in saline solution. Mueller-Hinton agar (Oxoid) supplemented with 0%, 2%, or 4% salt and various concentration of antibiotics was inoculated with the final suspensions with a replicator to deliver approximately 104 CFU in each spot. Vancomycin was tested with brain heart infusion agar (Oxoid) supplemented with 2% salt (7). Plates were read after 24 h at 35°C. S. aureus ATCC 29213 and ATCC 43300, Escherichia coli ATCC 25922, and Enterococcus faecalis ATCC 29212 were included in each run as control organisms.
DNA extraction. Chromosomal DNA of the 10 methicillin-resistant staphylococcal isolates was extracted by the simple lysis method as previously described (1). The isolates were first grown overnight at 37°C in 10 ml of brain heart infusion broth (Oxoid), and then a 1-ml volume was microcentrifuged. The pellets were washed in 1 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and microcentrifuged. The pellets were resuspended in 200 μl of TE buffer, and the suspension was boiled at 95°C for 20 min and microcentrifuged for 2 min. The supernatants were kept on ice, and 5 μl was used for PCR amplification.
PCR and sequencing. The list of PCR primers used to amplify mecA, the expected amplicon sizes, and the locations of the genes on S. aureus chromosome strain TK784 (31) are shown in Table 1. The sequencing primers used were thesame as the PCR primers used to amplify mecA. The designed primers wereobtained from GeneWorks Pty. Ltd., Adelaide, Australia. Mutations within mecA genes have been reported to occur at nucleotide positions 503, 510, 593, 642, 737, 907 to 912, 1721, and 1834 (32), and hence we used these overlapping primers to compensate for any potential mutation occurring in these regions. The nucleotide positions of the primers are based on the sequence submitted to GenBank under accession number Y00688 (30). A 50-μl PCR mixture consisted of 5 μl of template DNA, 2.5 U of Taq DNA polymerase (Roche Diagnostic GmbH, Mannheim, Germany), 10x PCR buffer plus Mg (5 μl) (Roche Diagnostic), 0.25 mM each deoxynucleoside triphosphate (Promega, Madison, Wis.), and 25 pmol each of the forward and reverse primers. The reaction mixture was subjected to predenaturation at 95°C for 5 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and extension for 5 min at 72°C. PCR products (6 μl) were resolved on a 2% agarose gel (Promega, Madison, Wis.) in 1x Tris-acetic EDTA buffer at 120 V for 1 h. The gel was stained with ethidium bromide and visualized under UV (Gel doc; Bio-Rad Laboratories, Hercules, Calif.).
Nucleotide sequencing. The DNA fragments for sequencing were obtained by PCR amplification of each chromosomal DNA as the template. After purification with the QIAquick PCR purification kit (QIAGEN GmbH, Hilden, Germany), the fragments of each PCR products were sequenced with the set of primers listed in Table 2 with Big Dye Terminator v3.1 (Applied Biosystems, Foster City, Calif.). Sequence alignment was carried out by using the Australian National Genomic Information Service (ANGIS) program (http://www.angis.org.au).
RESULTS
Isolation of staphylococci. Of the 252 animals (192 dogs and 60 cats) sampled, methicillin-resistant staphylococci were found in 5 healthy dogs, 3 diseased dogs, and 2 healthy cats. Methicillin-resistant staphylococci were identified as S. aureus (2), S. epidermidis (2), S. haemolyticus (3), S. warneri (2), and S. hominis (1) (Table 2). MRSA was isolated from skin lesions of two infected dogs, and none was isolated from healthy animals. However, one methicillin-resistant S. epidermidis strain was recovered from a wound infection of a dog and was the only coagulase-negative staphylococcus infecting a diseased animal. Healthy cats and dogs in this study had predominately coagulase-negative, methicillin-resistant staphylococci comprising mostly S. haemolyticus (isolated from two dogs and one cat), S. warneri (two dogs), S. hominis (one dog), and S. epidermidis (one cat) strains.
Antimicrobial susceptibility. The antibiotic susceptibility patterns are shown in Table 2. The 10 isolates were resistant to methicillin (oxacillin), and various MICs were determined. For two MRSA and two S. haemolyticus strains isolated from healthy cats and dogs, the oxacillin MIC was 128 μg/ml. These four isolates were resistant to all of the antibiotics tested except vancomycin and rifampin. Another S. haemolyticus strain, dubbed DN12 (Table 2), also showed multiresistance properties, but the MIC of oxacillin was low (4 μg/ml) and the strain was susceptible to ciprofloxacin, chloramphenicol, vancomycin, and rifampin. The remaining five isolates had borderline resistance to methicillin (MIC, 4 μg/ml) and had various patterns of resistance to other antibiotics. Generally, they were susceptible to vancomycin, rifampin, chloramphenicol, and ciprofloxacin. Susceptibility to the ?-lactam antibiotics, such as ampicillin, amoxicillin-clavulanic acid, and ceftiofur, used in this study differed among the isolates, but NCCLS standards recommend reporting these organisms as resistant due to reported treatment failure in vivo with these antibiotics. S. hominis and S. warneri were resistant to methicillin (?-lactam antibiotics) only but were susceptible to all of the other classes of antibiotics. S. epidermidis strains from a diseased dog and a healthy cat, however, were both susceptible to ciprofloxacin and gentamicin but resistant to tetracycline and erythromycin, respectively.
Nucleotide sequencing. Each primer pair yielded a single major amplicon of the expected size, ranging from 454 bp to 714 bp, as shown in Table 1 and Fig. 1. Sequencing results obtained from the 10 isolates and analysis with the ANGIS global alignment program resulted in 100% homology to the fractions of Y00688 particularly targeted for amplification (result not shown). The overlapping sets of primers developed for amplification and sequencing facilitated the alignment due to the usual difficulties of getting the beginning and end of a sequenced product.
DISCUSSION
Most of the methicillin-resistant staphylococci in this study were coagulase-negative species, in agreement with previously reports (8, 16), and were predominantly isolated from healthy cats and dogs. The MRSA strains in this study were derived from diseased dogs, and none were from healthy animals. This probably reflects the virulent nature of S. aureus compared to nonpathogenic or opportunistic coagulase-negative staphylococci that are normal resident bacteria on the skin and other anatomical sites of healthy and diseased individuals (13, 18). In human medicine, methicillin-resistant, coagulase-negative staphylococci such as S. epidermidis and S. haemolyticus are a leading cause of nosocomial infections in neonates, immunocompromised patients, and patients with indwelling catheters (15, 37). However, in dogs, staphylococcal infections are mainly due to S. intermedius, with S. aureus causing only occasional infections. The predominant pathogenic species in cats is S. aureus (13); hence, the buildup of methicillin-resistant, coagulase-negative species in these animals could be missed in routine laboratory testing for recognized pathogenic species and could serve as a gene pool or reservoir of a methicillin resistance gene.
Nucleotide sequence analyses of several mecA genes from different species such as S. aureus (DDBJ/EMBL/GenBank accession numbers Y00688 [30] and X52593 [28]), S. epidermidis (accession no. X52592) (28), S. sciuri (accession no. Y13096) (35), and S. aureus strain N315 (14) have revealed that the mecA gene is much more conserved among staphylococcal species of human origin. However, little is known about the mecA gene from companion animal sources. There is concern that cats and dogs are a reservoir or source of MRSA infections in humans (10, 29) due to their close contact with humans in the household.
The source of the mecA gene in these animals is unclear. In human medicine, it has been demonstrated that horizontal transfer from a primitive staphylococci species, S. sciuri, into an S. aureus chromosome may have occurred (36). The discovery of the mobile genetic element staphylococcal chromosome cassette mec (SCCmec) in MRSA strains of human origin further advances the theory that mecA could be shared among staphylococcal species (17). SCCmec ranges in size from approximately 20 to about 68 kbp and is composed of the mec gene complex (mecA gene and its regulators) and the ccr gene complex, which encodes site-specific recombinases responsible for the mobility of the element.
Methicillin and other antistaphylococcal penicillins such as oxacillin and cloxacillin are rarely used in the treatment of staphylococcal infections in cats and dogs (27), and therefore selective pressure resulting from their use is not likely to occur. In this study, 80% of the methicillin-resistant staphylococcal isolates were coagulase-negative strains and were mostly isolated from healthy cats and dogs. These isolates are likely to be normal bacterial flora of healthy animals and were probably not exposed to methicillin or any other antistaphylococcal penicillins that could have driven the development of methicillin resistance and/or acquisition of the mecA gene. Thus, the mecA genes in these isolates were probably derived from their owners, a view supported because the nucleotide sequence was identical to that of a human MRSA strain. The same assumption could hold true for the two MRSA strains isolated from diseased dogs. This is because staphylococci are species specific in nature but human S. aureus strains have been implicated in infections of other animal species (4, 12). The S. aureus strains in this study could be human-derived MRSA due to the 100% sequence homology between them. Identical chromosomal patterns from pulsed-field gel electrophoresis and SCCmec typing (2, 21, 34) have led to the conclusion that cats and dogs and their owners might carry the same MRSA strains, and this study confirms these findings. This preliminary molecular study on methicillin-resistant staphylococci from cats and dogs shows that humans and their companion animals are likely to share methicillin-resistant staphylococci and other resistance genes as a result of close contact. The dispersal of these resistance genes is likely to be facilitated by the fact that staphylococci are normal resident bacterial flora of the skin and the mucosal surfaces of the respiratory tracts of humans and animals and hence could easily be spread by skin-to-skin contact, saliva, and aerosols from sneezing and coughing.
In conclusion, we have demonstrated that the mecA gene which encodes methicillin resistance in staphylococci of human origin is the same gene responsible for methicillin resistance in staphylococci isolated from cats and dogs. Further studies on SCCmec and multilocus sequence typing of these isolates will improve our understanding of the mobile genetic element carrying the mecA gene and the clonal relationship of these isolates to human MRSA. This will aid in the understanding and management of potential transmission of methicillin-resistant staphylococci between human and their pets.
ACKNOWLEDGMENTS
We thank Gregory Parkins (Plympton Veterinary Clinic, Adelaide, Australia), Mike Lawley, and Darren Hanshaw (Black Forest Veterinary Surgery, Adelaide, Australia) for given us access to their practice for sampling of healthy animals. We also thank Daniela Signoriello (IDEXX, Adelaide, Australia) for supplying the clinical isolates from diseased animals. ATCC 43300 was a kind gift from Jan Bell (Women's and Children's Hospital, Adelaide, Australia).
This work was supported by a University of South Australia President's Scholarship.
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School of Pharmacy and Medical Sciences, University of South Australia, Adelaide 5001, South Australia, Australia(Seidu Malik, Haihong Peng)