Open Access

Antimicrobial resistance and characterisation of staphylococci isolated from healthy Labrador retrievers in the United Kingdom

  • Vanessa M Schmidt1, 6Email author,
  • Nicola J Williams2,
  • Gina Pinchbeck2,
  • Caroline E Corless3,
  • Stephen Shaw4,
  • Neil McEwan1, 6,
  • Susan Dawson2 and
  • Tim Nuttall5
Contributed equally
BMC Veterinary Research201410:17

https://doi.org/10.1186/1746-6148-10-17

Received: 19 April 2013

Accepted: 6 January 2014

Published: 14 January 2014

Abstract

Background

Coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci are normal commensals of the skin and mucosa, but are also opportunist pathogens. Meticillin-resistant (MR) and multidrug-resistant (MDR) isolates are increasing in human and veterinary healthcare. Healthy humans and other animals harbour a variety of staphylococci, including MR-CoPS and MR-CoNS. The main aims of the study were to characterise the population and antimicrobial resistance profiles of staphylococci from healthy non-vet visiting and non-antimicrobial treated Labrador retrievers in the UK.

Results

Nasal and perineal samples were collected from 73 Labrador retrievers; staphylococci isolated and identified using phenotypic and biochemical methods. They were also confirmed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), PCR of the nuc gene and PCR and sequencing of the tuf gene. Disc diffusion and minimum inhibitory concentration (MIC) susceptibility tests were determined for a range of antimicrobials. In total, 102 CoPS (S. pseudintermedius n = 91, S. aureus n = 11) and 334 CoNS isolates were detected from 99% of dogs in this study. In 52% of dogs CoNS only were detected, with both CoNS and CoPS detected in 43% dogs and CoPS only detected in 4% of dogs. Antimicrobial resistance was not common among CoPS, but at least one MDR-CoNS isolate was detected in 34% of dogs. MR-CoNS were detected from 42% of dogs but no MR-CoPS were isolated. S. epidermidis (52% of dogs) was the most common CoNS found followed by S. warneri (30%) and S. equorum (27%), with another 15 CoNS species isolated from ≤ 15% of dogs. S. pseudintermedius and S. aureus were detected in 44% and 8% of dogs respectively.

Conclusions

MR- and MDR-CoPS were rare. However a high prevalence of MR- and MDR-CoNS were found in these dogs, even though they had no prior antimicrobial treatment or admission to veterinary premises. These findings are of concern due to the potential for opportunistic infections, zoonotic transmission and transmission of antimicrobial resistant determinants from these bacteria to coagulase positive staphylococci.

Keywords

Coagulase-postive staphylococci Coagulase-negative staphylococci Meticillin-resistant Dogs MALDI-TOF-MS Tuf gene Nuc gene Antimicrobial-susceptibility

Background

Staphylococci are normal commensal bacteria of the skin and mucous membranes of humans and other animals. They can be differentiated by their ability to produce coagulase, with coagulase positive (CoPS) staphylococci regarded as more pathogenic than coagulase negative (CoNS) species [15].

Healthy humans and other animals may harbour multiple species and strains of staphylococci. Staphylococcus aureus is the main human commensal CoPS species and is carried in the nasal cavity of approximately 30% of healthy people [6]. S. epidermidis is the most common CoNS isolated from the nares, perineum, inguinal skin, axillae and interdigital skin in man [2, 7]. The main commensal CoPS of dogs, S. pseudintermedius[8], has been isolated from 37% to 92% of healthy dogs [914], while S. aureus is carried by 4.3% to 12% of healthy dogs [10, 12, 1520]. Other species isolated from the mucosa and skin of healthy dogs include the CoPS S. schleiferi subspecies coagulans[10, 21] and numerous CoNS (S. schleiferi subspecies schleiferi, S. epidermidis, S. haemolyticus, S. saprophyticus, S. devriesei, S. warneri, S. simulans, S. xylosus, S.capitus, S. caprae, and S. sciuri) [12, 15, 2226]. The carriage rate of CoNS isolated from the nasal mucosae of healthy dogs was reported to be 38% in one large cross-sectional study [15].

Staphylococci are frequent opportunistic pathogens and commensal isolates are the most common source of infection in humans [3] and dogs [12, 16, 27]. Antimicrobial resistance can increase the morbidity, mortality and treatment cost of staphylococcal infections. Meticillin (oxacillin) resistance associated with carriage of the mecA gene confers resistance to all β-lactam antimicrobials [28]. The mecA gene is located on a large mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec), enabling horizontal transmission between staphylococcal isolates [29]. Meticillin resistant staphylococci (MRS) are important pathogens in human and veterinary healthcare and are often multi-drug resistant (MDR; resistant to three or more classes of antimicrobial) [3035], extremely limiting therapeutic options. MRSP clones with a broader resistance spectrum than MRSA or MR-CoNS are increasingly reported in domestic animals throughout Europe, USA and Canada [32, 34]. MR-CoNS are associated with infections in humans and animals [31, 3638]. In humans the most prevalent species is MR S. epidermidis (MRSE), which may be a reservoir of MR for S. aureus[39, 40]. In addition, the SCCmec cassette of the major European MRSP clone (ST71-J-t02-II–III) [34] consists of a combination of SCCmec II from MRSE and SCCmec III from MRSA [41].

The prevalence of MRSA and MRSP carriage in healthy humans and dogs in the community is low [11, 18, 36, 4247]. However, human community-based surveys report a wider range of carriage rates for MR-CoNS (11–50%) [39, 48, 49]. MR-CoNS have also been isolated from the carriage sites of 13% of healthy dogs [23, 50]. The reported prevalence of MRS is higher in animals exposed to veterinary healthcare environments and antimicrobial therapy [47, 5153] suggesting that these are risk factors for colonisation.

Previous studies looking at the commensal staphylococci in dogs have concentrated on CoPS species, particularly MR-CoPS species, the CoNS group or MR-CoNS species [911, 13, 14, 17, 23, 50], but no study has characterised the complete canine commensal staphylococcal population. Moreover, reporting of the antimicrobial treatment history of dogs in these studies have been inconsistent. The aim of this study was to characterise the mucosal staphylococcal population structure and antimicrobial resistance profiles in healthy Labrador retrievers in the UK in the absence of antimicrobial pressure. This will be important in understanding changes in staphylococcal populations and their antimicrobial susceptibility patterns in dogs exposed to antimicrobials and other risk factors.

Methods

Study population

Labrador retriever dogs were recruited for the study from dog shows in the UK between November 2010 and June 2011. One healthy dog was enrolled from each household if the dog had not received topical or systemic antimicrobial therapy, or had not been admitted to a veterinary clinic within the last 12 months. All dog owners gave written informed consent before enrolment in this study and completed a questionnaire regarding potential risk factors for the carriage of antimicrobial resistant bacteria. The University of Liverpool School of Veterinary Science ethics committee approved the study protocol.

Staphylococci

Specimen collection and bacterial isolation

One nasal swab and one perineal swab were collected from each dog (Copan Eswab LQ Amies Minitip Nylon Flocked Applicator, Appleton Woods, Birmingham, UK). A sterile swab was either inserted 5 mm into one nostril or rubbed on the skin of the perineum for 3–5 seconds before being placed in Amies transport media, stored at 4°C and processed within 36 hours. Swabs were incubated aerobically overnight at 37°C in nutrient broth with 6.5% sodium chloride. The broth was streaked onto mannitol salt agar (MSA), oxacillin resistance screening agar (ORSA) supplemented with 2 μg/ml of oxacillin and Columbia 5% horse blood agar (CAB), and incubated aerobically overnight at 37°C. Where present, isolates typical of staphylococci were selected from all plates, sub-cultured onto CAB and incubated aerobically overnight at 37°C. Fresh staphylococcal cultures on CAB were subject to Gram stain (Sigma-Aldrich Company Ltd., Gillingham, UK), tested for catalase (Sigma-Aldrich Company Ltd., Gillingham, UK) and free coagulase production (Rabbit plasma, Pro-Lab, Bromborough, UK) according the manufacturer’s instructions and stored at − 80°C in Microbank vials (Pro-Lab, Bromborough, UK). All media were obtained from LabM Ltd, Bury, UK.

Antimicrobial susceptibility testing

Disc diffusion testing was performed on all staphylococcal isolates in accordance with the Clinical and Laboratory Standards Institute (CLSI) and the following panel of ten antimicrobial discs were applied: 1 μg oxacillin (OX), 1 μg ciprofloxacin (CIP), 10 μg gentamicin (GM), 10 μg fusidic acid (FA), 30 μg cefalexin (CFX), 30 μg cefovecin (CVN), 25 μg trimethoprim-sulfamethoxazole (TS), 10 μg tetracycline (Tet), 2 μg clindamycin (CD) and 5 μg vancomycin (Va) [54]. All the discs were purchased from MAST Group Ltd., Liverpool, UK, except for CVN, which were obtained from Oxoid, Basingstoke, UK. Micro-dilution susceptibility testing (Trek Diagnostic Systems, Cleveland, Ohio, USA) was performed on a subset of the CoNS isolates using the same antimicrobial panel, except vancomycin [54]. Interpretation was based on the CLSI guidelines for animal species-specific zone diameter (mm) interpretive standards and minimal inhibitory concentration (MIC; mg/l) breakpoints for veterinary pathogens or human-derived interpretive standards when available. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) zone diameter interpretive standards and MIC breakpoints were used for CIP and FA [55]. The breakpoints used for interpretation of OX resistance were a zone of inhibition of ≤ 17 mm and MIC ≥ 0.5 mg/l for S. pseudintermedius and CoNS, and ≤ 10 mm and MIC ≥ 4 mg/l for S. aureus[56, 57]. The breakpoints used for interpretation of resistance to CVN as a zone of inhibition of ≤ 19 mm and MIC ≥ 8 mg/l in accordance with the manufacturer’s recommendations. The reference strain S. aureus ATCC®25923 (LGC Standards, Teddington, UK) was used for quality control for MIC and zone diameter determinations.

DNA extraction

Three colonies of each staphylococcal isolate were homogenised in 90 μl of sterile distilled water (SDW) and 10 μl of lysostaphin (1 mg/ml; Sigma-Aldrich Company Ltd., Gillingham, UK) and vortexed for 5 seconds. The suspensions were then incubated at 37°C for 10 minutes and heated at 100°C for 10 minutes before adding 400 μl of SDW. Samples were stored at 4°C.

Characterisation of antimicrobial resistance genes

PCR assays were performed to detect the presence of mecA gene (Table 1) in staphylococcal isolates that were phenotypically resistant to oxacillin. All the PCR assays were performed with 0.5 μl of each primer (10 pmol/μl), 1 μl of DNA and 1.1x PCR master mix (ReddyMix™, Thermo Fisher Scientific Inc., Surrey, UK) made up to a total reaction volume of 25 μl. Molecular grade water (Sigma-Aldrich Company Ltd., Gillingham, UK) was used as the negative control in all PCR assays. PCR products were analysed by agarose gel (1.5%) electrophoresis and the DNA fragments were visualised under UV light after ethidium bromide staining.
Table 1

Details of PCR assays used in this study for nuc , tuf and mec A gene identification

Primer

Sequence (5′-3′)

Amplicon size (bp)

Annealing Temperature (°C)

Control strain

Reference

au-F3

TCGCTTGCTATGATTGTGG

359

57

S. aureus ATCC®25923 (LGC Standards, Teddington, UK)

[77]

au-nucR*

GCCAATGTTCTACCATAGC

pse-F2

TRGGCAGTAGGATTCGTTAA

926

57

S. pseudintermedius (clinical isolate)

 

pse-R5*

CTTTTGTGCTYCMTTTTGG

SSnucF

AATGGCTACAATGATAATCACTAA

526

57

S. schleiferi subspecies coagulans ATCC®49545

 

SSnucR*

CATATCTGTCTTTCGGCGCG

tuf-F

GCCAGTTGAGGACGTATTCT

412

55

S. epidermidis ATCC®12228

[105]

tuf-R

CCATTTCAGTACCTTCTGGTAA

mecAF

TGGCTATCGTGTCACAATCG

310

55

MRSA (clinical isolate)

[103, 104]

mecAR

CTGGAACTTGTTGAGCAGAG

  

mA1

TGCTATCCACCCTCAAACAGG

286

57

mA2

AACGTTGTAACCACCCCAAGA

    

(*multiplex assay).

Species identification

Genotypic species identification

PCR assays to detect the presence of the nuc genes of S. pseudintermedius, S. aureus and S. schleiferi were performed on all CoPS isolates using Qiagen® Multiplex PCR Mix (Qiagen, Crawley, UK), according to the manufacturer’s instructions with minor modifications. In short, the PCR assays were performed in a reaction volume of 25 μl, consisting of 5 μl of bacterial DNA extract, 12.5 μl of master mix, 2.5 μl of 10x primer mix (2 μM of each primer) and 5 μl of RNase-free water. The cycling conditions consisted of an initial activation step at 95°C for 15 minutes, followed by 30 cycles of 95°C for 30 seconds, 57°C for 90 seconds and 72°C for 60 seconds, and a final extension step at 72°C for 10 minutes (Table 1).

MALDI-TOF-MS

All isolates were subjected to matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) according to the manufacturer’s protocol. Raw spectra were analysed by the MALDI Biotyper 2.0 software programme with default settings (Bruker Daltonics, Bremen, Germany). The extraction method was performed as previously described [58] on overnight colonies grown on CAB at 37°C and all isolates were tested in duplicate. The bacterial test standard (E. coli DH5 alpha, Bruker, Bremen, Germany) was used for calibration before each experiment and included in duplicate on each target plate. The mass peak profiles were matched to the reference database and a score generated based on similarity [59].

Sequencing

Two subsets of isolates detected from our group of dogs underwent sequencing following PCR amplification of the tuf gene [59, 60]; a control group of CoNS isolates (n = 27) identified by MALDI-TOF-MS and a test group of isolates (n = 52) that had not been identified by MALDI-TOF-MS. Initial PCR assays were performed using HotStarTaq® Master Mix Kit (Qiagen, Crawley, UK) in a 25 μl reaction volume with an initial activation step at 95°C for 15 minutes followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds, with a final extension step of 72°C for 5 minutes according to the manufacturer’s protocol. The resulting amplicons were sequenced using BigDye Terminator version 1.1 cycle sequencing (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol on the ABI3131 genetic analyser at the Department of Microbiology, Royal Liverpool University Hospital. The sequences were aligned using the ABI Sequencing analysis software, with contiguous sequences matched to the GenBank database using the Basic Local Alignment Search Tool (BLAST) [61] and positively identified if there was ≥ 98% sequence similarity with a reference sequence. S. epidermidis ATCC®12228 was used as the control strain.

Statistical analysis

Data were analysed using SPSS software package (SPSS 20.0 for Mac, SPSS Inc, Chicago, Illinois).

To examine the association between isolation of S. pseudintermedius with each of the 16 different CoNS species Pearson’s chi-square was calculated (P < 0.003; Bonferroni correction). To examine the association between MR and MDR with potential risk factors (previous antimicrobial therapy or hospitalisation within 12 months of enrolment, health-care or large animal-association by in-contact people or pets) identified from the questionnaires Pearson’s chi-square was calculated (P < 0.0125; Bonferroni correction). To examine the agreement between antimicrobial susceptibility tests by disc diffusion and MIC a kappa statistic was calculated [62] and an independent t-test was conducted to compare the MIC of oxacillin resistant CoNS isolates that were either positive or negative for the mecA gene.

Results

Staphylococci

Specimen collection

Seventy-three Labrador retriever dogs were recruited. Twenty-one dogs were aged between 3 to 12 months, 25 dogs were aged between 12 months to 2 years, and 27 dogs were > 2 years old, with 35 female dogs and 38 male dogs in total.

Bacterial isolation

Staphylococci were isolated from in 72 out of 73 dogs (99%; 95% confidence interval (CI): 99.6-95.8) and from both sample sites in the majority of dogs (78%; 95% CI: 67.3, 86.0). Isolation of staphylococci from the nasal mucosae (16%, 95% CI 9.7, 26.6) or perineum (4%, 95% CI 1.4, 11.4) only occurred in a small number of dogs. If only the nasal mucosae had been sampled, CoPS (all S. pseudintermedius) would not have been detected in seven dogs (10%, 95% CI 4.7, 18.5) and CoNS in six dogs (8%, 95% CI 3.8, 16.8). CoNS were detected in the majority of dogs (95%, 95% CI 86.7, 97.8) either alone (52%, 95% CI 40.8, 63.1) or with CoPS (43%, 95% CI 31.8, 53.9). Detection of CoPS alone was significantly less common (4%, 95% CI 1.4, 11.4). In total, there were 436 staphylococcal isolates; 102 of which were CoPS and 334 were CoNS isolates.

Antimicrobial susceptibility testing by disc diffusion

The overall prevalence of antimicrobial resistance among the isolates detected in this study appeared high, with at least one MDR isolate detected in 34% of dogs. Antimicrobial resistant CoNS isolates were detected in more dogs than antimicrobial resistant CoPS isolates for OX, GM, FA, CFX, CVN and CD and MDR (Figure 1). At least one OX resistant isolate was detected in 58% dogs (n = 126 oxacillin resistant isolates), but resistance to the other tested β-lactam antimicrobials, CVN (25%) and CFX (29%), was less common. Few CoPS demonstrated antimicrobial resistance; isolates from twelve dogs had Tet resistance (all S. pseudintermedius), seven with FA resistance (S. pseudintermedius = 5, S. aureus = 3); two with TS resistance (both S. pseudintermedius); two with CD resistance (S. pseudintermedius = 1, S. aureus = 1) and two with CIP resistance (both S. pseudintermedius). MDR CoPS was detected from only one dog (S. pseudintermedius with FA, Tet and CD resistance) (Figure 1).
Figure 1

The proportion of dogs (n = 73) carrying at least one staphylococcal isolate with resistance to each antimicrobial, MDR and mec A gene positive oxacillin resistance by the disc diffusion method. Total = CoNS and CoPS.

MIC compared to disc diffusion testing for antimicrobial resistance

Micro-dilution susceptibility testing (Trek Diagnostic Systems, Cleveland, Ohio, USA) was performed on 172 CoNS isolates, of which 52 were OX susceptible and 120 were OX resistant by disc diffusion. The OX resistant isolates were further divided into those found to be positive (n = 74) or negative (n = 46) for carriage of the mecA gene by PCR. The strength of agreement between antimicrobial resistance detected by MIC and disc diffusion was very good for OX, GM, CVN, Tet and CD resistance, good for CFX and TS resistance and moderate for CIP (Kappa = 0.593) and FC resistance (Kappa = 0.589). MIC testing identified more isolates as resistant to OX, GM, CFX, CVN and Tet compared to disc diffusion, and disc diffusion identified more isolates as resistant to CIP, FA, TS and CD compared to MIC testing (Table 2).
Table 2

Cross tabulation of the results of 172 staphylococcal isolates classified as resistant or susceptible to the antimicrobials tested in this study by both MIC and disc diffusion testing

Antimicrobial resistance

  

MIC

  
   

No

Yes

Total

Oxacillin (OX)

Disc diffusion

No

50

10

60

  

Yes

2

110

112

  

Total

52

120

172

  

Kappa = 0.842

   
   

No

Yes

Total

Oxacillin mecA positive

 

No

115

0

115

  

Yes

1

56

57

  

Total

116

56

172

  

Kappa = 0.987

   
   

No

Yes

Total

Ciprofloxacin (CIP)

 

No

146

0

157

  

Yes

14

12

15

  

Total

160

12

172

  

Kappa = 0.593

   
   

No

Yes

Total

Gentamicin (GM)

 

No

156

1

157

  

Yes

0

15

15

  

Total

156

16

172

  

Kappa = 0.965

   
   

No

Yes

Total

Fusidic acid (FA)

 

No

36

5

41

  

Yes

25

106

131

  

Total

61

111

172

  

Kappa = 0.589

   
   

No

Yes

Total

Cefalexin (CFX)

 

No

117

15

132

  

Yes

0

40

40

  

Total

117

55

172

  

Kappa = 0.784

   
   

No

Yes

Total

Cefovecin (CVN)

 

No

130

11

141

  

Yes

0

31

31

  

Total

130

42

172

  

Kappa = 0.810

   
   

No

Yes

Total

Tetracycline (T10)

 

No

148

1

149

  

Yes

0

23

23

  

Total

148

24

172

  

Kappa = 0.975

   
    

Yes

Total

Trimethoprim-sulfamethoxazole (TS)

 

No

156

2

158

  

Yes

3

11

14

  

Total

159

13

172

  

Kappa = 0.799

   
   

No

Yes

Total

Clindamycin (CD)

 

No

148

2

150

  

Yes

4

18

22

  

Total

152

20

172

  

Kappa = 0.837

   

Characterisation of antimicrobial resistance genes

Of the 126 OX resistant CoNS isolates detected by disc diffusion, 75 isolates (60%, 95% CI 51, 68) from 31 dogs (42%, 95% CI 32, 54) were positive for the mecA gene (Figure 1). Nine additional oxacillin resistant isolates were detected by MIC and two of these were positive for the mecA gene, resulting in two additional dogs with MR-CoNS and one additional dog with phenotypic oxacillin resistant CoNS. There was a significant difference between the MIC of mecA positive (M = 3.84, SD = 0.18) and mecA negative isolates (M = 0.97, SD = 0.12, P < 0.001). In addition the epidemiological breakpoint for OX resistant CoNS isolates with mecA gene carriage isolated in this study was consistent with the clinical CLSI breakpoint (≥ 0.5 mg/l) (Figure 2). Eleven different CoNS species (S. epidermidis, S. warneri, S. sciuri, S. equorum, S. fleurettii, S. vitulinus, S. saprophyticus, S. haemolyticus, S. lentus, S. succinus and S. pettenkoferi) were found to carry the mecA gene. Among oxacillin resistant CoNS species, S epidermidis and S. sciuri were more likely to carry the mecA gene than S. saprophyticus, S. equorum, S. vitulinus and S. succinus (Figure 3). MRSE isolates were detected in 18 dogs (25%, 95% CI 14.8, 34.5), meticillin-resistant S. warneri were detected in 7 dogs (10%, 95% CI 2.8, 16.3) and meticillin-resistant S. sciuri were detected in 5 dogs (7%, 95% CI 1.1, 12.6). The remaining species were only isolated from one or two dogs. MDR mecA positive CoNS were detected in 19 dogs (26%, 95% CI 17.3, 37.1). There was no significant association between detection of MR-CoNS or MDR isolates and potential risk factors tested in this study (Pearson’s chi-square; P < 0.0125).
Figure 2

The MIC (μg/ml) data for staphylococcal isolates (n = 172). The isolates consisted of 52 oxacillin susceptible isolates, 46 oxacillin resistant mecA negative isolates and 74 oxacillin resistant mecA positive isolates.

Figure 3

The percentage of each oxacillin-resistant staphylococcal species by disc diffusion and MIC that was either positive ( mec A+) or negative ( mec A-) for the mec A gene.

Species identification

Phenotypic and biochemical methods identified 436 isolates as Staphylococcus species. Using a combination of nuc gene PCR, MALDI-TOF-MS and sequencing of the tuf gene, 399 isolates (92%, 95% CI 88.5, 93.8) were identified to the species level. MALDI-TOF-MS identified 345 isolates to the species level including 264 of 334 CoNS isolates (79%, 95% CI 74.4, 83.1). Amplification and sequencing of the tuf gene identified 33 out of 51 CoNS isolates (65%, 95% CI 51, 76.4) to the species level (n = 11 species; ≥ 98% sequence similarity) and an additional control group (n = 27) of CoNS isolates that had also been identified by MALDI-TOF-MS. There was 100% agreement between the two methods for the identification of the control group. In particular, sequencing of the tuf gene identified all of the S. fleurettii, S. arlettae and S. pettenkoferi isolates, 12 isolates closely related to S. felis (96% sequence similarity) and an additional 15 isolates to the genus level (Staphylococcus spp. ≥ 98% sequence similarity). PCR amplification of the nuc gene detected all of the S. aureus n = 11 (100%, 95% CI 74.1, 100) and S. pseudintermedius isolates n = 91 (100%, 95% CI 96.0, 100). There was 100% agreement of this assay with MALDI-TOF-MS for the identification of S. aureus isolates, however MALDI-TOF-MS only identified 69 out of 91 S. pseudintermedius isolates.

Overall from the combined results using PCR amplification of the nuc gene, MALDI-TOF-MS and sequencing of the tuf gene we detected S. epidermidis in 52% (95% CI 41, 63) and S. pseudintermedius in 44% (95% CI 33, 55) of the dogs. S. warneri and S. equorum were the next most common species, isolated from 30% and 27% of dogs respectively, and the remaining staphylococcal species were carried by no more than 15% of the dogs. S. aureus was detected in 6 of the dogs, exclusively from the nasal mucosae, and usually with S. pseudintermedius (88%, 95% CI 52.9, 97.8). S. pseudintermedius was concurrently isolated with 16 different CoNS species, although there was no significant association between the presence of S. pseudintermedius and any CoNS species (Pearson’s chi-square; P < 0.003) (Figure 4 and Table 3).
Figure 4

The percentage of dogs (n = 73) carrying each staphylococcal species identified in this study by MALDI-TOF-MS, PCR of the nuc gene and sequencing of the tuf gene (CoNS grey and CoPS black).

Table 3

The number of staphylococcal isolates identified to species level by MALDI-TOF-MS, nuc gene PCR (CoPS), and tuf gene sequencing

Staphylococcal species

Number of isolates

Number (%) of positive dogs

Number (%) identified by MALDI-TOF-MS

Number (%) of CoPS identified by nucPCR

Number (%) identified by tufgene sequencing

S. pseudintermedius

91

32 (44)

70 (77)

91 (100)

0

S. aureus

11

6 (8)

11 (100)

11 (100)

0

S. epidermidis

67

38 (52)

64 (96)

N/A

3 (4)

S. warneri

35

22 (30)

35 (100)

N/A

0

S. equorum

39

20 (27)

36 (92)

N/A

3 (8)

S. saprophyticus

19

11 (15)

15 (79)

N/A

4 (21)

S. sciuri

27

11 (15)

21 (78)

N/A

6 (22)

S. succinus

19

10 (14)

16 (84)

N/A

3 (16)

S. simulans

15

9 (12)

15 (100)

N/A

0

S. capitus

7

8 (11)

7 (100)

N/A

0

S. pasteuri

8

6 (8)

8 (100)

N/A

0

S. xylosus

17

6 (8)

17 (100)

N/A

0

S. lentus

9

5 (7)

4 (44)

N/A

5 (56)

S. hominis

6

4 (5)

6 (100)

N/A

0

S. cohnii

5

3 (4)

3 (60)

N/A

2 (40)

S. vitulinus

14

3 (4)

13 (93)

N/A

1 (7)

S. haemolyticus

4

3 (4)

4 (100)

N/A

0

S. fleurettii

4

2 (3)

0

N/A

4 (100)

S. arlettae

1

1 (1)

0

N/A

1 (100)

S. pettenkoferi

1

1 (1)

0

N/A

1 (100)

Total ID

399d

72e

345f

101f

33f

Staphylococcus spp.

3

N/A

N/A

N/A

3

Species related to S. felis

12

N/A

N/A

N/A

12

No ID

22

N/A

N/A

2

3

Total

436a

73b

436c

102c

51c

Values in the table are expressed as total numbers and percentage in parenthesis where applicable. aTotal number of isolates in study, btotal number of dogs in study, ctotal number of isolates tested by each method, dtotal number of isolates with positive identification (ID), enumber of dogs with staphylococcal detection, fnumber of isolates with positive ID from each method.

Discussion

This is the first study incorporating MALDI-TOF-MS to successfully characterise commensal staphylococcal populations in a group of healthy dogs in the absence of antimicrobial pressure. We isolated staphylococci from 99% of our dogs, with 95% carrying CoNS and 47% carrying CoPS. The relative prevalence of the staphylococci concurs with other published studies in humans [2, 3], horses [6367] and dogs [15, 17, 68], although the overall staphylococcal prevalence was double that reported for healthy vet visiting dogs [15]. This could be related to the study population and techniques, as we sampled both the nose and the perineum to increase detection of CoPS [12, 13, 68, 69].

We were able to assign 92% of the staphylococcal isolates to 20 different species, including 18 CoNS. This is the first study to demonstrate such diversity in dogs, and carriage of this number of different species has only been previously reported for humans [2, 10, 12, 15, 2126, 70]. The most common species was S. epidermidis, which was detected in 52% of the dogs, mainly from the nasal cavity. This is similar to human reports [71], but apart from one canine study [23], S. epidermidis has not been commonly reported in different animal species [67, 72, 73]. S. pseudintermedius was the second most common species and the most common CoPS detected, also in agreement with previous reports [911, 13]. Unlike S. epidermidis, S. pseudintermedius was carried equally in the nose and on the perineum, suggesting that this species may have a wider range of mucosal niches. Very few dogs carried S. aureus (8%), which is comparable to other studies that reported carriage rates of approximately 7% from healthy vet visiting dogs [12, 15]. The majority of the CoNS in our study were human-associated and included S. epidermidis, S. hominis, S. haemolyticus, S. capitus, S. saprophyticus, S. warneri, S. cohnii, S. simulans, S. pettenkoferi and S. pasteuri. Human associated CoNS species have previously been isolated from dogs, horses, cows and pigs [23, 67, 72, 7476]. The other CoNS species isolated from our dogs are reported as indigenous to animals (S. equorum, S. vitulinus, S. arlettae S. sciuri, S. lentus and S. fleurettii) [2].

We used several methods to identify staphylococcal isolates to species level. Multiplex PCR for the nuc gene is an accurate, rapid and cost efficient method to speciate CoPS [77], which identified 100% of our S. pseudintermedius (n = 91) and 100% of our S. aureus isolates (n = 11). Recently MALDI-TOF-MS has been reported as a rapid and reliable method to characterise CoNS, S. aureus and S. intermedius group (SIG) strains [59, 72, 7880]. MALDI-TOF-MS identified all of our S. aureus isolates, 77% of our S. pseudintermedius isolates and 79% of our CoNS isolates, identified by phenotypic and biochemical characteristics, to the species level. Similar results for the identification of S. aureus, S. pseudintermedius and CoNS by MALDI-TOF-MS, in comparison to molecular methods, have been reported [7981]. The overall performance of MALDI-TOF-MS to speciate the staphylococcal isolates in this study, similar to other reports [80], is likely to be directly related to the database, which at the time of analysis consisted mainly of common human-derived species and only one S. pseudintermedius strain. However species level identification will improve as more highly characterised reference isolates are added to the database. Amplification and sequencing of the tuf gene is regarded as the gold standard to speciate CoNS isolates [59, 60]. This method identified 77% of the tested staphylococcal isolates (n = 79) to the species level. The performance of this method in our study may have been affected by the lack of certain-animal derived isolates representing different species in the database. Additionally, we may have improved identification by sequencing a larger region of the tuf gene. We sequenced a previously described 412 base pair region of the tuf gene that was reported to have successfully identified 88% of human-derived staphylococcal strains [60]. However, a more recent publication that sequenced a 660 bp region of the tuf gene, reported 98.9% identification of 186 human and animal-derived staphylococcal strains.

We did not detect any MR-CoPS isolates. Other studies of healthy dogs have similarly reported a low prevalence [15, 82, 83]. In contrast, 58% of the dogs in our study carried at least one CoNS isolate with phenotypic meticillin resistance and 42% carried a meticillin resistant mecA positive isolate. Other studies have also reported high levels of meticillin resistance among CoNS isolates from humans [31, 35, 84], horses [23, 6466] and livestock [72, 85]. However, the prevalence of MR-CoNS carriage in our study is markedly higher than the levels reported in other community canine studies [15, 23, 50, 74, 83]. High community carriage rates of MR-CoNS are of concern for animals and humans, as these organisms may not only be reservoirs of resistance genes for CoPS [39, 40, 86], but also act as pathogens [31, 3638, 8789]. Cross-transmission is reported to be an important mechanism for dissemination of MRS [49, 90], and transmission between dogs and in-contact humans may occur in the community and in veterinary premises [36, 83].

Nine different CoNS species carried the mecA gene in our study with MRSE detected in 25% of our dogs. MRSE is the predominant MR-CoNS species in humans both in hospital and community settings [39, 48, 49], and has been reported in one study investigating nasal carriage of MRS in dogs [23]. Other canine studies have isolated meticillin resistant S. sciuri and meticillin resistant S. warneri[23, 74]. Our research found that the majority of the S. sciuri and S. fleurettii isolates were mecA positive, which is consistent with other studies in humans, livestock and horses [35, 64, 66, 67, 72].

MDR CoNS (n = 38) were isolated from 34% of dogs in this study. MDR was generally associated with resistance to β-lactams, FA and additional antimicrobials. In particular MDR-MRSE were resistant to at least four antimicrobial classes tested in our study. A similar finding was reported in a study of hospitalised animals, medical equipment and veterinary staff [68]. MDR among CoNS isolates is widely reported [15, 49, 72, 73, 91] and may be associated with the carriage of multiple antimicrobial resistance genes on SCCmec cassettes [40]. In contrast, the majority of our commensal CoPS isolates were susceptible to a broad range of antimicrobials (apart from Tet), in line with previous reports for clinical isolates [9294] and isolates from healthy vet-visiting dogs [15]. There was good to very good agreement between disc and MIC antimicrobial susceptibility testing apart for FC and CIP. These two antimicrobials were the only ones where human breakpoints were applied and emphasises potential species differences in pharmacokinetic and pharmacodynamic data for individual antimicrobials.

The mecA gene was not identified in 40% of the phenotypic oxacillin resistant isolates in this study and may include some isolate duplication due to our sampling methods. Other studies have reported phenotypic meticillin resistance with absence of the mecA gene in staphylococci [9598]. Our OX-resistant mecA negative isolates may be truly negative for the mecA gene as they were less likely to be resistant to the other antimicrobials tested in this study, including CVN and CFX, and had significantly lower MICs compared to the OX resistant mecA positive isolates. It is possible that they had low-level resistance associated with other mechanisms such as hyperproduction of β-lactamases [99], or production of an oxacillin-specific β-lactamases [100]. There are bovine mastitis CoNS isolates with oxacillin MICs of 0.5 – 1 mg/l that lack the mecA gene [97], and the CLSI guidelines state that ‘oxacillin interpretive criteria may overcall resistance for these CoNS strains’ [57]. In addition, many of the published PCR assays to identify and characterise the mecA gene have been developed for MRSA [101104] and may therefore lack sensitivity for some CoNS isolates. However, other authors have successfully employed the same methods for mecA detection among CoNS isolates as used in our study [68, 98, 105]. Nevertheless it is possible that additional PCR assay [106], or latex agglutination for PBP2a [107] may have improved the sensitivity of mecA detection or detected phenotypic mecA-associated resistance in our oxacillin resistant mecA negative isolates.

Our study had some limitations, including the small sample size. Still, these dogs yielded 436 staphylococcal isolates and a high prevalence of resistance was identified among the CoNS isolates even in the absence of antimicrobial exposure. Another weakness was that the study population was limited to one breed (Labrador retrievers) and the dogs were recruited at dog shows. Kennelled dogs have been shown to have higher levels of antimicrobial resistance in faecal E. coli than individually owned and non-kennelled dogs [108]. Kennelling was transient in our dogs, but this may have affected the results. Many of the dogs came from multi-dog households but only one dog from each household was sampled to avoid cluster effects.

Conclusions

This is the first comprehensive study of commensal staphylococcal populations in a group of healthy dogs. Staphylococci, particularly CoNS, form a normal part of the canine commensal population and were detected from almost all the dogs. The most commonly isolated staphylococcal species in this group of dogs was S. epidermidis, although a wide variety of other human- and animal-associated CoNS were found. CoPS were less common, and the major species was S. pseudintermedius. Antimicrobial resistance among the CoPS was uncommon, and no MRSP or MRSA were isolated, however the sample size was small. Antimicrobial resistance (including MDR and meticillin resistance) was common among the CoNS isolates, even though this was a community population of healthy dogs in the absence of direct-antimicrobial pressure or veterinary contact. The clinical significance of commensal CoNS and MR-CoNS is unclear, but S. epidermidis carries a number of virulence factors and is an increasing cause of nosocomial and community-acquired infections in humans. The possibility of similar infections escalating in companion animals cannot be excluded. In addition, there is potential for cross-species transmission of antimicrobial resistant bacteria and exchange of resistance determinants between bacterial species. In particular, MR- and MDR-CoNS may provide a reservoir of antimicrobial resistance genes that could rapidly spread within bacterial populations under the selection pressure exerted by antimicrobial therapy. Further longitudinal studies in healthy dogs and in dogs receiving antimicrobials are required to assess the population diversity, antimicrobial resistance profiles and persistence of antimicrobial resistant staphylococci in dogs.

Notes

Abbreviations

CoPS: 

Coagulase positive staphylococci

CoNS: 

Coagulase negative staphylococci

MDR: 

Multidrug resistance

MR: 

Meticillin resistance

MIC: 

Minimum inhibitory concentration

MRSA: 

Meticillin resistant Staphylococcus aureus

MRSP: 

Meticillin resistant staphylococcus pseudintermedius

MRSE: 

Meticillin resistant staphylococcus epidermidis

OX: 

Oxacillin

CIP: 

Ciprofloxacin

CFX: 

Cefalexin

CVN: 

Cefovecin

TS: 

Cotrimazole

Tet: 

Tetracycline

CD: 

Clindamycin.

Declarations

Acknowledgements

The authors would like to thank Zoetis (previously Pfizer Animal Health) for funding this project, and Maureen D’Arcy and the North West and Midland Counties Labrador Retriever Clubs for allowing us to obtain samples. We would like to thank Erica Tranfield from Bruker (Liverpool, UK) for training and access to MALDI-TOF-MS Biotyper. We would also like to thank Ruth Ryvar and Gill Hutchinson for their technical support, Dorina Timofte and Andy Wattret for their technical advice, and Clara MacFarlane, Atina Unwin and Camilla Brena for their help in sample collection.

Authors’ Affiliations

(1)
Department of Infection Biology, The University of Liverpool
(2)
Department of Epidemiology and Population Health, The University of Liverpool
(3)
Infection and Immunity, Royal Liverpool University Hospital
(4)
UK VetDerm
(5)
The Royal (Dick) School of Veterinary Studies, Easter Bush Campus, University of Edinburgh
(6)
The University of Liverpool School of Veterinary Science

References

  1. Kloos WE: Natural populations of the genus Staphylococcus. Annu Rev Microbiol. 1980, 34: 559-592.PubMedGoogle Scholar
  2. Kloos WE, Bannerman TL: Update on clinical significance of coagulase-negative staphylococci. Clin Microbiol Rev. 1994, 7 (1): 117-140.PubMed CentralPubMedGoogle Scholar
  3. von Eiff C, Peters G, Heilmann C: Pathogenesis of infections due to coagulase-negative staphylococci. Lancet Infect Dis. 2002, 2 (11): 677-685.PubMedGoogle Scholar
  4. Rich M: Staphylococci in animals: prevalence, identification and antimicrobial susceptibility, with an emphasis on methicillin-resistant Staphylococcus aureus. Br J Biomed Sci. 2005, 62 (2): 98-105.PubMedGoogle Scholar
  5. Devriese LA, Vancanneyt M, Baele M, Vaneechoutte M, De Graef E, Snauwaert C, Cleenwerck I, Dawyndt P, Swings J, Decostere A, et al: Staphylococcus pseudintermedius sp. nov., a coagulase-positive species from animals. Int J Syst Evol Microbiol. 2005, 55 (Pt 4): 1569-1573.PubMedGoogle Scholar
  6. Mainous AG, Hueston WJ, Everett CJ, Diaz VA: Nasal carriage of Staphylococcus aureus and methicillin-resistant S aureus in the United States, 2001–2002. Ann Fam Med. 2006, 4 (2): 132-137.PubMed CentralPubMedGoogle Scholar
  7. Huebner J, Goldmann DA: Coagulase-negative staphylococci: role as pathogens. Annu Rev Med. 1999, 50: 223-236.PubMedGoogle Scholar
  8. Mason IS, Mason KV, Lloyd DH: A review of the biology of canine skin with respect to the commensals Staphylococcus intermedius, Demodex canis and Malassezia pachydermatis. Vet Dermatol. 1996, 7 (3): 119-132.Google Scholar
  9. Devriese LA, De Pelsmaecker K: The anal region as a main carrier site of Staphylococcus intermedius and Streptococcus canis in dogs. Vet Rec. 1987, 121 (13): 302-303.PubMedGoogle Scholar
  10. Griffeth GC, Morris DO, Abraham JL, Shofer FS, Rankin SC: Screening for skin carriage of methicillin-resistant coagulase-positive staphylococci and Staphylococcus schleiferi in dogs with healthy and inflamed skin. Vet Dermatol. 2008, 19 (3): 142-149.PubMedGoogle Scholar
  11. Hanselman BA, Kruth SA, Rousseau J, Weese JS: Coagulase positive staphylococcal colonization of humans and their household pets. Can Vet J. 2009, 50 (9): 954-958.PubMed CentralPubMedGoogle Scholar
  12. Fazakerley J, Williams N, Carter S, McEwan N, Nuttall T: Heterogeneity of Staphylococcus pseudintermedius isolates from atopic and healthy dogs. Vet Dermatol. 2010, 21 (6): 578-585.PubMedGoogle Scholar
  13. Rubin JE, Ball KR, Chirino-Trejo M: Antimicrobial susceptibility of Staphylococcus aureus and Staphylococcus pseudintermedius isolated from various animals. Can Vet J. 2011, 52 (2): 153-157.PubMed CentralPubMedGoogle Scholar
  14. Paul NC, Bargman SC, Moodley A, Nielsen SS, Guardabassi L: Staphylococcus pseudintermedius colonisation patterns and strain diversity in healthy dogs: a cross-sectional and longitudinal study. Vet Microbiol. 2012, 160 (3–4): 420-427.PubMedGoogle Scholar
  15. Wedley AL: Prevalence of Staphyococcus spp. carriage in dogs. Department of Epidemiology and Population Health, Institute of Infection and Global Health, University of Liverpool; 2012.Google Scholar
  16. Pinchbeck LR, Cole LK, Hillier A, Kowalski JJ, Rajala-Schultz PJ, Bannerman TL, York S: Genotypic relatedness of staphylococcal strains isolated from pustules and carriage sites in dogs with superficial bacterial folliculitis. Am J Vet Res. 2006, 67 (8): 1337-1346.PubMedGoogle Scholar
  17. Loeffler A, Boag AK, Sung J, Lindsay JA, Guardabassi L, Dalsgaard A, Smith H, Stevens KB, Lloyd DH: Prevalence of methicillin-resistant Staphylococcus aureus among staff and pets in a small animal referral hospital in the UK. J Antimicrob Chemother. 2005, 56 (4): 692-697.PubMedGoogle Scholar
  18. Boost MV, O’Donoghue MM, Siu KH: Characterisation of methicillin-resistant Staphylococcus aureus isolates from dogs and their owners. Clin Microbiol Infect. 2007, 13 (7): 731-733.PubMedGoogle Scholar
  19. Sasaki T, Kikuchi K, Tanaka Y, Takahashi N, Kamata S, Hiramatsu K: Reclassification of phenotypically identified Staphylococcus intermedius strains. J Clin Microbiol. 2007, 45 (9): 2770-2778.PubMed CentralPubMedGoogle Scholar
  20. Kottler S, Middleton JR, Perry J, Weese JS, Cohn LA: Prevalence of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus carriage in three populations. J Vet Intern Med. 2010, 24 (1): 132-139.PubMedGoogle Scholar
  21. Yamashita K, Shimizu A, Kawano J, Uchida E, Haruna A, Igimi S: Isolation and characterization of staphylococci from external auditory meatus of dogs with or without otitis externa with special reference to Staphylococcus schleiferi subsp. coagulans isolates. J Vet Med Sci. 2005, 67 (3): 263-268.PubMedGoogle Scholar
  22. May ER, Hnilica KA, Frank LA, Jones RD, Bemis DA: Isolation of Staphylococcus schleiferi from healthy dogs and dogs with otitis, pyoderma, or both. J Am Vet Med Assoc. 2005, 227 (6): 928-931.PubMedGoogle Scholar
  23. Bagcigil FA, Moodley A, Baptiste KE, Jensen VF, Guardabassi L: Occurrence, species distribution, antimicrobial resistance and clonality of methicillin- and erythromycin-resistant staphylococci in the nasal cavity of domestic animals. Vet Microbiol. 2007, 121 (3–4): 307-315.PubMedGoogle Scholar
  24. Kania SA, Williamson NL, Frank LA, Wilkes RP, Jones RD, Bemis DA: Methicillin resistance of staphylococci isolated from the skin of dogs with pyoderma. Am J Vet Res. 2004, 65 (9): 1265-1268.PubMedGoogle Scholar
  25. Medleau L, Long RE, Brown J, Miller WH: Frequency and antimicrobial susceptibility of Staphylococcus species isolated from canine pyodermas. Am J Vet Res. 1986, 47 (2): 229-231.PubMedGoogle Scholar
  26. Cox HU, Hoskins JD, Newman SS, Foil CS, Turnwald GH, Roy AF: Temporal study of staphylococcal species on healthy dogs. Am J Vet Res. 1988, 49 (6): 747-751.PubMedGoogle Scholar
  27. Bannoehr J, Guardabassi L: Staphylococcus pseudintermedius in the dog: taxonomy, diagnostics, ecology, epidemiology and pathogenicity. Vet Dermatol. 2012, 23 (4): 253-266. e251-252PubMedGoogle Scholar
  28. Hartman BJ, Tomasz A: Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus. J Bacteriol. 1984, 158 (2): 513-516.PubMed CentralPubMedGoogle Scholar
  29. Black CC, Solyman SM, Eberlein LC, Bemis DA, Woron AM, Kania SA: Identification of a predominant multilocus sequence type, pulsed-field gel electrophoresis cluster, and novel staphylococcal chromosomal cassette in clinical isolates of mecA-containing, methicillin-resistant Staphylococcus pseudintermedius. Vet Microbiol. 2009, 139 (3–4): 333-338.PubMedGoogle Scholar
  30. Hryniewicz W: Epidemiology of MRSA. Infection. 1999, 27 (Suppl 2): S13-S16.PubMedGoogle Scholar
  31. Diekema DJ, Pfaller MA, Schmitz FJ, Smayevsky J, Bell J, Jones RN, Beach M: Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin Infect Dis. 2001, 32 (Suppl 2): S114-S132.PubMedGoogle Scholar
  32. Loeffler A, Linek M, Moodley A, Guardabassi L, Sung JM, Winkler M, Weiss R, Lloyd DH: First report of multiresistant, mecA-positive Staphylococcus intermedius in Europe: 12 cases from a veterinary dermatology referral clinic in Germany. Vet Dermatol. 2007, 18 (6): 412-421.PubMedGoogle Scholar
  33. Weese JS, van Duijkeren E: Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in veterinary medicine. Vet Microbiol. 2010, 140 (3–4): 418-429.PubMedGoogle Scholar
  34. Perreten V, Kadlec K, Schwarz S, Gronlund Andersson U, Finn M, Greko C, Moodley A, Kania SA, Frank LA, Bemis DA, et al: Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study. J Antimicrob Chemother. 2010, 65 (6): 1145-1154.PubMedGoogle Scholar
  35. Garza-Gonzalez E, Morfin-Otero R, Martinez-Vazquez MA, Gonzalez-Diaz E, Gonzalez-Santiago O, Rodriguez-Noriega E: Microbiological and molecular characterization of human clinical isolates of Staphylococcus cohnii, Staphylococcus hominis, and Staphylococcus sciuri. Scand J Infect Dis. 2011, 43 (11–12): 930-936.PubMedGoogle Scholar
  36. van Duijkeren E, Kamphuis M, van der Mije IC, Laarhoven LM, Duim B, Wagenaar JA, Houwers DJ: Transmission of methicillin-resistant Staphylococcus pseudintermedius between infected dogs and cats and contact pets, humans and the environment in households and veterinary clinics. Vet Microbiol. 2011, 150 (3–4): 338-343.PubMedGoogle Scholar
  37. Hauschild T, Wojcik A: Species distribution and properties of staphylococci from canine dermatitis. Res Vet Sci. 2007, 82 (1): 1-6.PubMedGoogle Scholar
  38. Kern A, Perreten V: Clinical and molecular features of methicillin-resistant, coagulase-negative staphylococci of pets and horses. J Antimicrob Chemother. 2013, 68 (6): 1256-1266.PubMedGoogle Scholar
  39. Barbier F, Ruppe E, Hernandez D, Lebeaux D, Francois P, Felix B, Desprez A, Maiga A, Woerther PL, Gaillard K, et al: Methicillin-resistant coagulase-negative staphylococci in the community: high homology of SCCmec IVa between Staphylococcus epidermidis and major clones of methicillin-resistant Staphylococcus aureus. J Infect Dis. 2010, 202 (2): 270-281.PubMedGoogle Scholar
  40. Smyth DS, Wong A, Robinson DA: Cross-species spread of SCCmec IV subtypes in staphylococci. Infect Genet Evol. 2011, 11 (2): 446-453.PubMed CentralPubMedGoogle Scholar
  41. Descloux S, Rossano A, Perreten V: Characterization of new staphylococcal cassette chromosome mec (SCCmec) and topoisomerase genes in fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius. J Clin Microbiol. 2008, 46 (5): 1818-1823.PubMed CentralPubMedGoogle Scholar
  42. Abudu L, Blair I, Fraise A, Cheng KK: Methicillin-resistant Staphylococcus aureus (MRSA): a community-based prevalence survey. Epidemiol Infect. 2001, 126 (3): 351-356.PubMed CentralPubMedGoogle Scholar
  43. Shopsin B, Mathema B, Martinez J, Ha E, Campo ML, Fierman A, Krasinski K, Kornblum J, Alcabes P, Waddington M, et al: Prevalence of methicillin-resistant and methicillin-susceptible Staphylococcus aureus in the community. J Infect Dis. 2000, 182 (1): 359-362.PubMedGoogle Scholar
  44. Maudsley J, Stone SP, Kibbler CC, Iliffe SR, Conaty SJ, Cookson BD, Duckworth GJ, Johnson A, Wallace PG: The community prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in older people living in their own homes: implications for treatment, screening and surveillance in the UK. J Hosp Infect. 2004, 57 (3): 258-262.PubMedGoogle Scholar
  45. Sa-Leao R, Sanches IS, Couto I, Alves CR, de Lencastre H: Low prevalence of methicillin-resistant strains among Staphylococcus aureus colonizing young and healthy members of the community in Portugal. Microb Drug Resist. 2001, 7 (3): 237-245.PubMedGoogle Scholar
  46. Zanelli G, Sansoni A, Zanchi A, Cresti S, Pollini S, Rossolini GM, Cellesi C: Staphylococcus aureus nasal carriage in the community: a survey from central Italy. Epidemiol Infect. 2002, 129 (2): 417-420.PubMed CentralPubMedGoogle Scholar
  47. Loeffler A, Pfeiffer DU, Lindsay JA, Magalhaes RJ, Lloyd DH: Prevalence of and risk factors for MRSA carriage in companion animals: a survey of dogs, cats and horses. Epidemiol Infect. 2010, 1-10.Google Scholar
  48. Lebeaux D, Barbier F, Angebault C, Benmahdi L, Ruppe E, Felix B, Gaillard K, Djossou F, Epelboin L, Dupont C, et al: Evolution of nasal carriage of methicillin-resistant coagulase-negative staphylococci in a remote population. Antimicrob Agents Chemother. 2012, 56 (1): 315-323.PubMed CentralPubMedGoogle Scholar
  49. Silva FR, Mattos EM, Coimbra MV, Ferreira-Carvalho BT, Figueiredo AM: Isolation and molecular characterization of methicillin-resistant coagulase-negative staphylococci from nasal flora of healthy humans at three community institutions in Rio de Janeiro City. Epidemiol Infect. 2001, 127 (1): 57-62.PubMed CentralPubMedGoogle Scholar
  50. Vengust M, Anderson ME, Rousseau J, Weese JS: Methicillin-resistant staphylococcal colonization in clinically normal dogs and horses in the community. Lett Appl Microbiol. 2006, 43 (6): 602-606.PubMedGoogle Scholar
  51. Nienhoff U, Kadlec K, Chaberny IF, Verspohl J, Gerlach GF, Kreienbrock L, Schwarz S, Simon D, Nolte I: Methicillin-resistant Staphylococcus pseudintermedius among dogs admitted to a small animal hospital. Vet Microbiol. 2011, 150 (1–2): 191-197.PubMedGoogle Scholar
  52. Bergstrom A, Gustafsson C, Leander M, Fredriksson M, Gronlund U, Trowald-Wigh G: Occurrence of methicillin-resistant staphylococci in surgically treated dogs and the environment in a Swedish animal hospital. J Small Anim Pract. 2012, 53 (7): 404-410.PubMedGoogle Scholar
  53. Huerta B, Maldonado A, Ginel PJ, Tarradas C, Gomez-Gascon L, Astorga RJ, Luque I: Risk factors associated with the antimicrobial resistance of staphylococci in canine pyoderma. Vet Microbiol. 2011, 150 (3–4): 302-308.PubMedGoogle Scholar
  54. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals; Approved Standard—Third Edition. CLSI document M31-A3. Edited by: CLSI. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.Google Scholar
  55. EUCAST: Breakpoint tables for interpretation of MICs and zone diameters. Version 3.1, 2013. http://www.eucast.org.
  56. Bemis DA, Jones RD, Frank LA, Kania SA: Evaluation of susceptibility test breakpoints used to predict mecA-mediated resistance in Staphylococcus pseudintermedius isolated from dogs. J Vet Diagn Invest. 2009, 21 (1): 53-58.PubMedGoogle Scholar
  57. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; Approved Standard -- Fourth Edition. CLSI document VET01-A. Edited by: CLSI. Wayne, PA: Clinical and Laboratory Standards Institute; 2013.Google Scholar
  58. Alatoom AA, Cunningham SA, Ihde SM, Mandrekar J, Patel R: Comparison of direct colony method versus extraction method for identification of gram-positive cocci by use of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2011, 49 (8): 2868-2873.PubMed CentralPubMedGoogle Scholar
  59. Carpaij N, Willems RJ, Bonten MJ, Fluit AC: Comparison of the identification of coagulase-negative staphylococci by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tuf sequencing. Eur J Clin Microbiol Infect Dis. 2011, 30 (10): 1169-1172.PubMed CentralPubMedGoogle Scholar
  60. Heikens E, Fleer A, Paauw A, Florijn A, Fluit AC: Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci. J Clin Microbiol. 2005, 43 (5): 2286-2290.PubMed CentralPubMedGoogle Scholar
  61. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215 (3): 403-410.PubMedGoogle Scholar
  62. Landis JR, Koch GG: Measurement of observer agreement for categorical data. Biometrics. 1977, 33 (1): 159-174.PubMedGoogle Scholar
  63. Yasuda R, Kawano J, Onda H, Takagi M, Shimizu A, Anzai T: Methicillin-resistant coagulase-negative staphylococci isolated from healthy horses in Japan. Am J Vet Res. 2000, 61 (11): 1451-1455.PubMedGoogle Scholar
  64. Busscher JF, van Duijkeren E, Sloet van Oldruitenborgh-Oosterbaan MM: The prevalence of methicillin-resistant staphylococci in healthy horses in the Netherlands. Vet Microbiol. 2006, 113 (1–2): 131-136.PubMedGoogle Scholar
  65. Moodley A, Guardabassi L: Clonal spread of methicillin-resistant coagulase-negative staphylococci among horses, personnel and environmental sites at equine facilities. Vet Microbiol. 2009, 137 (3–4): 397-401.PubMedGoogle Scholar
  66. De Martino L, Lucido M, Mallardo K, Facello B, Mallardo M, Iovane G, Pagnini U, Tufano MA, Catalanotti P: Methicillin-resistant staphylococci isolated from healthy horses and horse personnel in Italy. J Vet Diagn Invest. 2010, 22 (1): 77-82.PubMedGoogle Scholar
  67. Karakulska J, Fijalkowski K, Nawrotek P, Pobucewicz A, Poszumski F, Czernomysy-Furowicz D: Identification and methicillin resistance of coagulase-negative staphylococci isolated from nasal cavity of healthy horses. J Microbiol. 2012, 50 (3): 444-451.PubMedGoogle Scholar
  68. Moon BY, Youn JH, Shin S, Hwang SY, Park YH: Genetic and phenotypic characterization of methicillin-resistant staphylococci isolated from veterinary hospitals in South Korea. J Vet Diagn Invest. 2012, 24 (3): 489-498.PubMedGoogle Scholar
  69. Windahl U, Reimegard E, Holst BS, Egenvall A, Fernstrom L, Fredriksson M, Trowald-Wigh G, Andersson UG: Carriage of methicillin-resistant Staphylococcus pseudintermedius in dogs–a longitudinal study. BMC Vet Res. 2012, 8: 34.PubMed CentralPubMedGoogle Scholar
  70. Widerstrom M, Wistrom J, Sjostedt A, Monsen T: Coagulase-negative staphylococci: update on the molecular epidemiology and clinical presentation, with a focus on Staphylococcus epidermidis and Staphylococcus saprophyticus. Eur J Clin Microbiol Infect Dis. 2012, 31 (1): 7-20.PubMedGoogle Scholar
  71. Rogers KL, Fey PD, Rupp ME: Coagulase-negative staphylococcal infections. Infect Dis Clin North Am. 2009, 23 (1): 73-98.PubMedGoogle Scholar
  72. Huber H, Ziegler D, Pfluger V, Vogel G, Zweifel C, Stephan R: Prevalence and characteristics of methicillin-resistant coagulase-negative staphylococci from livestock, chicken carcasses, bulk tank milk, minced meat, and contact persons. BMC Vet Res. 2011, 7: 6.PubMed CentralPubMedGoogle Scholar
  73. Garbacz K, Zarnowska S, Piechowicz L, Haras K: Staphylococci isolated from carriage sites and infected sites of dogs as a reservoir of multidrug resistance and methicillin resistance. Curr Microbiol. 2013, 66 (2): 169-173.PubMedGoogle Scholar
  74. Malik S, Coombs GW, O’Brien FG, Peng H, Barton MD: Molecular typing of methicillin-resistant staphylococci isolated from cats and dogs. J Antimicrob Chemother. 2006, 58 (2): 428-431.PubMedGoogle Scholar
  75. Gillespie BE, Headrick SI, Boonyayatra S, Oliver SP: Prevalence and persistence of coagulase-negative Staphylococcus species in three dairy research herds. Vet Microbiol. 2009, 134 (1–2): 65-72.PubMedGoogle Scholar
  76. Tulinski P, Fluit AC, Wagenaar JA, Mevius D, van de Vijver L, Duim B: Methicillin-resistant coagulase-negative staphylococci on pig farms as a reservoir of heterogeneous staphylococcal cassette chromosome mec elements. Appl Environ Microbiol. 2012, 78 (2): 299-304.PubMed CentralPubMedGoogle Scholar
  77. Sasaki T, Tsubakishita S, Tanaka Y, Sakusabe A, Ohtsuka M, Hirotaki S, Kawakami T, Fukata T, Hiramatsu K: Multiplex-PCR method for species identification of coagulase-positive staphylococci. J Clin Microbiol. 2010, 48 (3): 765-769.PubMed CentralPubMedGoogle Scholar
  78. Dubois D, Leyssene D, Chacornac JP, Kostrzewa M, Schmit PO, Talon R, Bonnet R, Delmas J: Identification of a variety of Staphylococcus species by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2010, 48 (3): 941-945.PubMed CentralPubMedGoogle Scholar
  79. Szabados F, Woloszyn J, Richter C, Kaase M, Gatermann S: Identification of molecularly defined Staphylococcus aureus strains using matrix-assisted laser desorption/ionization time of flight mass spectrometry and the Biotyper 2.0 database. J Med Microbiol. 2010, 59 (Pt 7): 787-790.PubMedGoogle Scholar
  80. Decristophoris P, Fasola A, Benagli C, Tonolla M, Petrini O: Identification of Staphylococcus intermedius Group by MALDI-TOF MS. Syst Appl Microbiol. 2011, 34 (1): 45-51.PubMedGoogle Scholar
  81. Bergeron M, Dauwalder O, Gouy M, Freydiere AM, Bes M, Meugnier H, Benito Y, Etienne J, Lina G, Vandenesch F, et al: Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Eur J Clin Microbiol Infect Dis. 2011, 30 (3): 343-354.PubMedGoogle Scholar
  82. Onuma K, Tanabe T, Sato H: Antimicrobial resistance of Staphylococcus pseudintermedius isolates from healthy dogs and dogs affected with pyoderma in Japan. Vet Dermatol. 2012, 23 (1): 17-22. e15PubMedGoogle Scholar
  83. Vanderhaeghen W, Van de Velde E, Crombe F, Polis I, Hermans K, Haesebrouck F, Butaye P: Screening for methicillin-resistant staphylococci in dogs admitted to a veterinary teaching hospital. Res Vet Sci. 2012, 93 (1): 133-136.PubMedGoogle Scholar
  84. Hanssen AM, Ericson Sollid JU: SCCmec in staphylococci: genes on the move. FEMS Immunol Med Microbiol. 2006, 46 (1): 8-20.PubMedGoogle Scholar
  85. Zhang Y, Agidi S, LeJeune JT: Diversity of staphylococcal cassette chromosome in coagulase-negative staphylococci from animal sources. J Appl Microbiol. 2009, 107 (4): 1375-1383.PubMedGoogle Scholar
  86. Tsubakishita S, Kuwahara-Arai K, Sasaki T, Hiramatsu K: Origin and molecular evolution of the determinant of methicillin resistance in staphylococci. Antimicrob Agents Chemother. 2010, 54 (10): 4352-4359.PubMed CentralPubMedGoogle Scholar
  87. Duval X, Selton-Suty C, Alla F, Salvador-Mazenq M, Bernard Y, Weber M, Lacassin F, Nazeyrolas P, Chidiac C, Hoen B, et al: Endocarditis in patients with a permanent pacemaker: a 1-year epidemiological survey on infective endocarditis due to valvular and/or pacemaker infection. Clin Infect Dis. 2004, 39 (1): 68-74.PubMedGoogle Scholar
  88. Moran E, Masters S, Berendt AR, McLardy-Smith P, Byren I, Atkins BL: Guiding empirical antibiotic therapy in orthopaedics: the microbiology of prosthetic joint infection managed by debridement, irrigation and prosthesis retention. J Infect. 2007, 55 (1): 1-7.PubMedGoogle Scholar
  89. Piette A, Verschraegen G: Role of coagulase-negative staphylococci in human disease. Vet Microbiol. 2009, 134 (1–2): 45-54.PubMedGoogle Scholar
  90. Cimiotti JP, Wu F, Della-Latta P, Nesin M, Larson E: Emergence of resistant staphylococci on the hands of new graduate nurses. Infect Control Hosp Epidemiol. 2004, 25 (5): 431-435.PubMed CentralPubMedGoogle Scholar
  91. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB: Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis. 2004, 39 (3): 309-317.PubMedGoogle Scholar
  92. Lloyd DH, Lamport AI, Feeney C: Sensitivity to antibiotics amongst cutaneous and mucosal isolates of canine pathogenic staphylococci in the UK, 1980–96. Vet Dermatol. 1996, 7: 171-175.Google Scholar
  93. Kruse H, Hofshagen M, Thoresen SI, Bredal WP, Vollset I, Soli NE: The antimicrobial susceptibility of Staphylococcus species isolated from canine dermatitis. Vet Res Commun. 1996, 20 (3): 205-214.PubMedGoogle Scholar
  94. Hoekstra KA, Paulton RJ: Clinical prevalence and antimicrobial susceptibility of Staphylococcus aureus and Staphylococcus intermedius in dogs. J Appl Microbiol. 2002, 93 (3): 406-413.PubMedGoogle Scholar
  95. Suzuki E, Hiramatsu K, Yokota T: Survey of methicillin-resistant clinical strains of coagulase-negative staphylococci for mecA gene distribution. Antimicrob Agents Chemother. 1992, 36 (2): 429-434.PubMed CentralPubMedGoogle Scholar
  96. Bignardi GE, Woodford N, Chapman A, Johnson AP, Speller DC: Detection of the mec-A gene and phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillinresistance. J Antimicrob Chemother. 1996, 37 (1): 53-63.PubMedGoogle Scholar
  97. Fessler AT, Billerbeck C, Kadlec K, Schwarz S: Identification and characterization of methicillin-resistant coagulase-negative staphylococci from bovine mastitis. J Antimicrob Chemother. 2010, 65 (8): 1576-1582.PubMedGoogle Scholar
  98. Eckholm NG, Outerbridge CA, White SD, Sykes JE: Prevalence of and risk factors for isolation of meticillin-resistant Staphylococcus spp. from dogs with pyoderma in northern California, USA. Vet Dermatol. 2013, 24 (1): 154-161. e134PubMedGoogle Scholar
  99. Rosdahl VT, Rosendal K: Correlation of penicillinase production with phage type and susceptibility to antibiotics and heavy metals in Staphylococcus aureus. J Med Microbiol. 1983, 16 (4): 391-399.PubMedGoogle Scholar
  100. Jones RD, Kania SA, Rohrbach BW, Frank LA, Bemis DA: Prevalence of oxacillin- and multidrug-resistant staphylococci in clinical samples from dogs: 1,772 samples (2001–2005). J Am Vet Med Assoc. 2007, 230 (2): 221-227.PubMedGoogle Scholar
  101. Mehrotra M, Wang G, Johnson WM: Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin resistance. J Clin Microbiol. 2000, 38 (3): 1032-1035.PubMed CentralPubMedGoogle Scholar
  102. Zhang K, McClure JA, Elsayed S, Louie T, Conly JM: Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 2005, 43 (10): 5026-5033.PubMed CentralPubMedGoogle Scholar
  103. Kondo Y, Ito T, Ma XX, Watanabe S, Kreiswirth BN, Etienne J, Hiramatsu K: Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob Agents Chemother. 2007, 51 (1): 264-274.PubMed CentralPubMedGoogle Scholar
  104. Francois P, Pittet D, Bento M, Pepey B, Vaudaux P, Lew D, Schrenzel J: Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay. J Clin Microbiol. 2003, 41 (1): 254-260.PubMed CentralPubMedGoogle Scholar
  105. Ruppe E, Barbier F, Mesli Y, Maiga A, Cojocaru R, Benkhalfat M, Benchouk S, Hassaine H, Maiga I, Diallo A, et al: Diversity of staphylococcal cassette chromosome mec structures in methicillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus strains among outpatients from four countries. Antimicrob Agents Chemother. 2009, 53 (2): 442-449.PubMed CentralPubMedGoogle Scholar
  106. Murakami K, Minamide W, Wada K, Nakamura E, Teraoka H, Watanabe S: Detection of methicillin-resistant Staphylococcus aureus by polymerase chain reaction. Rinsho Byori. 1991, 39 (12): 1325-1330.PubMedGoogle Scholar
  107. Baddour MM, AbuElKheir MM, Fatani AJ: Comparison of mecA polymerase chain reaction with phenotypic methods for the detection of methicillin-resistant Staphylococcus aureus. Curr Microbiol. 2007, 55 (6): 473-479.PubMedGoogle Scholar
  108. De Graef EM, Decostere A, Devriese LA, Haesebrouck F: Antibiotic resistance among fecal indicator bacteria from healthy individually owned and kennel dogs. Microb Drug Resist. 2004, 10 (1): 65-69.PubMedGoogle Scholar

Copyright

© Schmidt et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement