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Article

Methicillin-Resistant Coagulase Negative Staphylococci and Their Antibiotic Susceptibility Pattern from Healthy Dogs and Their Owners from Kathmandu Valley

by
Muna Khanal
1,
Prabhu Raj Joshi
2,
Saroj Paudel
2,
Mahesh Acharya
2,
Komal Raj Rijal
1,
Prakash Ghimire
1 and
Megha Raj Banjara
1,*
1
Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu 44618, Nepal
2
Nepalese Farming Institute, Maitidevi, Kathmandu 44605, Nepal
*
Author to whom correspondence should be addressed.
Trop. Med. Infect. Dis. 2021, 6(4), 194; https://doi.org/10.3390/tropicalmed6040194
Submission received: 14 September 2021 / Revised: 2 October 2021 / Accepted: 5 October 2021 / Published: 2 November 2021
Versions Notes

Abstract

:
This cross-sectional study was designed to identify information on the frequency, antimicrobial resistance and species diversity of methicillin-resistant coagulase negative staphylococci (MRCoNS) among pet dogs and humans within households. Fifty five nasal swabs each from dogs and their owners were collected. MRCoNS were identified based on gram staining, culture on mannitol salt agar, biochemical tests, and mecA gene amplification. The antibiotic susceptibility of the isolates was assessed by a disc diffusion test. Uniplex and multiplex polymerase chain reaction (PCR) were employed for the species identification of MRCoNS and SCCmec typing, respectively. Species were further confirmed by MALDI-TOF-MS. The prevalence of MRCoNS was 29% in dog owners and 23.6% in dogs. Four different species of MRCoNS, Staphylococci saprophyticus (48.3%), S. haemolyticus (24.1%), S. warneri (17.2%), and S. epidermidis (10.3%), were detected. Two isolates each from dog owners and dogs showed a constitutive resistance to macrolide-lincosamide-streptogramin B (cMLSB) resistance, eight isolates each from dogs and their owners showed a macrolide-streptogramin B (MSB) resistance, and only two isolates from dog owners revealed an inducible resistance to macrolide-lincosamide-streptogramin B (iMLSB) resistance. SCCmec types were SCCmec type IV (55.2%), SCCmec type V (24.1%), SCCmec III (10.3%), SCCmec II (3.4%); two isolates were non-typable. MRCoNS are prevalent and genetically diverse in companion animals and humans. Different species of MRCoNS were found in dogs and their owners.

1. Introduction

Staphylococcus epidermidis, a common coagulase negative Staphylococci (CoNS), has been implicated in human infections, and a majority of the strains circulating in hospitals has been estimated to be methicillin-resistant and additionally resistant to several classes of antibiotics [1]. Staphylococci acquire methicillin resistance by the acquisition of staphylococcal chromosomal cassette mec (SCCmec), the mobile genetic element carrying mecA, which encodes altered penicillin binding protein (PBP2a) that mediates resistance to virtually all beta-lactam antibiotics [2]. Besides S. epidermidis, other methicillin-resistant CoNS (MRCoNS), including S. capitis, S. sciuri, S. hominis, S. haemolyticus, S. saprophyticus and S. warneri, have also been reported from human clinical infections [3,4].
The use of many different classes of antibiotics in veterinary medicine for the treatment of infections has probably selected for an antibiotic-resistant commensal flora, including the colonization of healthy dogs and horses with MRCoNS [5,6]. Regardless of the pathogenic potential, MRCoNS have been identified as an important source of antibiotic resistance determinants; for instance, some strains of S. xylosus have been regarded as a potential source of the mecC gene encoded in methicillin-resistant S. aureus (MRSA) [7].
Antimicrobial resistance (AMR) in Nepal has increased rapidly since the past few decades [8]. In Nepal, companion animals such as dogs, which may be the source of antimicrobial-resistant bacteria or the corresponding resistance genes, live in close contact with humans. Additionally, stray dogs, which are more numerous in major towns of Nepal, including districts of Kathmandu, Bhaktapur, and Lalitpur, are also indirectly in contact with humans; close contact allows the transmission of bacterial agents including staphylococci. It is not clear yet to what extent Staphylococci may be shared between dogs and humans, which makes it important to investigate the potential colonization of human body sites with Staphylococci as well as to study the possible transfer of antibiotic resistance genes between dog and human Staphylococci. To test the association of these exchanges, we characterized MRCoNS from dogs and dog owners, and their antimicrobial resistance genes in this study.

2. Materials and Methods

2.1. Ethics Statement

This study received ethical approval from the Nepal Health Research Council (Regd. 457/2019 MT). Before the collection of nasal swab samples from humans and dogs, a written informed consent form was completed from humans.

2.2. Research Design

This was a cross-sectional quantitative study, and primary data were collected from February to July 2019. Samples were collected from pet dogs and their owners in the community and processed in the laboratory.

2.3. Study Sites

The study was carried out in different communities of the Kathmandu, Bhaktapur and Lalitpur districts: these districts are highly populated, and human-dog contact is frequent.

2.4. Sample Size and Sampling Technique

A total of 55 pairs (n = 110) of nasal swabs were collected from dogs and their owners, including 20 pairs each from the Kathmandu and Bhaktapur districts, and 15 pairs from the Lalitpur district. The nonduplicate random samples were collected from each population unit.

2.5. Specimen Collection, Transport, and Identification of MRCoNS

Nasal swabs collected from dogs and their owners were kept in vials with M-Staph broth (HiMedia, India) enriched with a final concentration of 75 mg/L of polymyxin B, 0.01% potassium tellurite and 12.5 mg/L nystatin (Sigma-Aldrich, Massachusetts, MA, USA), screw-capped, clearly labeled and carried to the laboratory. Specimens were incubated inside a candle jar at 35 °C for 48 h. The broth was streaked in Mannitol salt agar plates and incubated at 35 °C for 24 h. Both mannitol fermenter and nonfermenter, at 2–3 colonies per sample, were further sub-cultured on nutrient agar. The bacteria were identified based on colony morphology, mannitol fermentation, Gram staining and conventional biochemical tests including catalase, oxidase, coagulase tests and DNAase activity. Isolates were further tested for methicillin resistance by growth on Mueller–Hinton Agar (MHA) containing 4 mcg/mL oxacillin, a cefoxitin (30 mcg) disc diffusion test and the PCR amplification of the mecA gene [9,10].

2.6. Antibiotic Susceptibility Test

MRCoNS isolates were subjected to an in-vitro antimicrobial susceptibility test by the disc diffusion method with the following antibiotics: gentamicin (10 µg), erythromycin (15 µg), ciprofloxacin (5 µg), tetracycline (30 µg), clindamycin (2 µg), co-trimoxazole (25 mcg), linezolid (30 µg) and chloramphenicol (50 µg) (HiMedia, India). The results were interpreted following the Clinical and Laboratory Standard Institute guidelines M100 (CLSI 2019). The minimum inhibitory concentration (MIC) of oxacillin (0.125 to 64 mg/L) to the bacterial isolates was determined by agar dilution method. Briefly, colonies isolated from the overnight growth of each organism were selected to prepare direct suspensions in tryptic soy broth. The suspensions were adjusted to a 0.5 McFarland standard. Control Mueller–Hinton agar and oxacillin Mueller–Hinton agar plates were inoculated with the final suspensions of bacteria. Plates were incubated at 35 °C for 24 h. The MIC was determined as the lowest concentration of oxacillin that completely inhibited growth. Erythromycin-resistant and clindamycin-susceptible isolates were also tested for inducible clindamycin resistance by D zone test [9].

2.7. Species Identification of MRCoNS and SCCmec Typing

The species identification of MRCoNS was carried out by uniplex PCR using primer sequences and optimization conditions, as described previously (Table 1). A multiplex PCR assay was employed to characterize the isolates by SCCmec typing [10]. The primer sets are listed in Table 2. The amplified PCR products were analyzed through electrophoresis on a 1.5% agarose gel.
The species of MRCoNS were further confirmed by MALDI-TOF-MS, as described by Kitti et al [11]. Briefly, several colonies from MHA were harvested and collected in 100 μL of sterile water. One microliter of this aliquot was deposited on two replicated target plates (Bruker Daltonics, Germany), being allowed to dry at room temperature. Then, one microliter of absolute ethanol (Merck, Darmstadt, Germany) was added to each well. Following drying at room temperature, 1 μL of α-cyano-4-hydroxycinnamic acid (Bruker Daltonics, Germany) dissolved in a solution of 50% acetonitrile, 2.5% trifluoroacetic acid, and 47.5% water (Sigma-Aldrich, Fluka, MO, USA) was added as a matrix. Both MALDI-TOF-MS Spectrometer Autoflex speed (Bruker Daltonics, Germany) and FlexControl software (version 3.4.135, Bruker Daltonics, Germany) were processed for the detection of protein and identification of the difference between species. The scores were used for the genus- and species-level identification: a score of 2000–3000 specified a species-level identification, while a score ranging from 1700–1999 indicated a genus-level identification, and those strains with a score below 1700 had an unreliable identification. The strains which could not be identified by MALDI-TOF MS were not processed further.

2.8. Data Analysis

Data were entered into SPSS version 21.0. A chi-square test was used to determine the association of human isolates and dog isolates. A p-value less than 0.05 was considered significant.

3. Results

3.1. Nasal Carriage Rate of MRCoNS

From the 55 human nasal samples, 16 (29%) were positive for MRCoNS; nine and seven isolates were from Kathmandu and Bhaktapur, respectively. Similarly, out of 55 nasal samples from dogs, 13 (23.6%) MRCoNS were isolated, and the distribution was: Kathmandu (n = 2), Bhaktapur (n = 5) and Lalitpur (n = 6).

3.2. Antibiotic Susceptibility of MRCoNS

The antibiotic susceptibility of MRCoNS isolated from dog owners (n = 16) and dogs (n=13) revealed an almost similar resistance pattern to erythromycin (75% vs. 77%), co-trimoxazole (31.2% vs. 38.5%), and ciprofloxacin (18.8% vs. 23.1%). MRCoNS isolates from dog owners were also resistant to clindamycin (18.8%), gentamicin (12.5%) and chloramphenicol (6.3%). None of the MRCoNS isolates from dog owners showed resistance to tetracycline and linezolid. Likewise, MRCoNS isolated from dogs were resistant to clindamycin (15.4%) and tetracycine (15.4%), and were fully susceptible to linezolid (Table 3).
The MIC of oxacillin to MRCoNS were determined and found to be 4 µg/mL to 32 µg/mL; isolates from dog owners had higher MIC of oxacillin than isolates from dogs (Table 4).
Isolates were also tested for whether they had constitutive and/or inducible clindamycin resistance. Four MRCoNS isolates, two each from dog owners and dogs, showed a constitutive resistance to macrolide-lincosamide-streptogramin B (cMLSB) resistance. Likewise, 16 isolates, eight each from dogs and their owners, revealed MSB resistance. Only two isolates from dog owners showed an inducible resistance to macrolide-lincosamide-streptogramin B (iMLSB) resistance (Table 5).

3.3. mecA Gene in MRCoNS, Species and SCCmec Types of MRCoNS from Dog Owners and Dogs

The mecA gene was detected among all the 29 isolates of MRCoNS that were resistant to cefoxitin (30 µg) and oxacillin (MIC > 4 µg/mL). The species distribution of 16 MRCoNS from dog owners were S. saprophyticus (n = 9; 56.3%), S. haemolyticus (n = 3; 18.8%), S. epidermidis (n = 2; 12.5%) and S. warneri (n = 2; 12.5%). Similarly, four different species of MRCoNS were detected from dogs, including S. saprophyticus (n = 5; 38.5%), S. haemolyticus (n = 4; 30.8%), S. warneri (n = 3; 23.1%), and S. epidermidis (n = 1; 7.7%) (Table 6).
The MRCoNS isolates were further characterized by SCCmec typing. Most strains were SCCmec type IV (55.2% n = 16), seven (43.7%) from dog owners and nine (69.2%) from dogs. Similarly, SCCmec type V were 24.1% (n = 7), five (31.3%) from dog owners and two (15.4%) from dogs. In total, SCCmec III were 10.3% (n = 3), SCCmec II were 3.4% (n = 1), and two isolates were non-typable (Table 6).

3.4. Coexistence of MRCoNS in Dogs and Their Owners

The same species of MRCoNS was not found to coexist in pairs; however, one sample from Bhaktapur had MRCoNS from both the pets and their owners, but from different species: S. warneri was found in dogs and S. saprophyticus in humans.

4. Discussion

This study provides information on the frequency, antimicrobial resistance and species diversity of MRCoNS within households, as well as on MRCoNS pet–human interaction. From this study, we found that the prevalence of MRCoNS was slightly higher in dog owners than in dogs. S. saprophyticus was found to be the dominant species in both dogs and dogs’ owners. MRCoNS isolates, both from dogs and their owners, showed resistance to ciprofloxacin, co-trimoxazole, chloramphenicol, clindamycin, erythromycin and gentamicin in varying proportions, and all the MRCoNS were susceptible to linezolid among the tested antibiotics. The molecular characterization of the isolates revealed the presence of the mecA gene among all MRCoNS, and most isolates carried the SCCmec IV element. The same strains of MRCoNS were not found to be shared among dogs and their owners in this study.
The carriage rate of MRCoNS detected among healthy owners (29%) was higher than those reported in previous studies among healthy individuals (7–28%) in non-healthcare settings [16,17,18,19,20,21]. Jamaluddin et al. detected a higher rate of MRCoNS carriage (30%) in Japanese children in day-care centers and kindergartens [22], and one study also reported a 47–51% carriage of MRCoNS among a remote population in French Guiana [23]. The data from all of these studies show that the types of cohort markedly determine the nasal carriage of MRCoNS. Very few data are available on the nasal MRCoNS carriage rate and associated risk factors. In one study, the MRCoNS prevalence in healthy humans that were in daily contact with a companion animal was 54.2%, which suggested a direct pet–human contact as a risk factor of a higher MRCoNS carriage [24]. The carriage of MRCoNS among pets is also in the variable range: lower rates (1–15%) than those detected in our study (23.6%) have been reported among healthy dogs from nasal, rectal, oral, anal and belly sites [6,25,26,27,28,29], and a very high rate (42%) has been reported in nasal and perineal samples taken from healthy non-vet-visiting and non-antibiotic-treated Labrador retrievers [30], indicating that the observed prevalence may be affected by different sampling methodologies.
In contrast with the former studies, which have detected S. epidermidis as a predominant staphylococcal species colonizing human body sites [1,16,30], we detected four species of staphylococci, S. saprophyticus S. haemolyticus, S. epidermidis and S. warneri, in decreasing proportions. The species distribution of human MRCoNS reported in our study seems diverse, even in similar geographic locations. Different species of MRCoNS have been detected among the dogs, including S. sciuri, S. warneri, S. lentus, S. vitulinus, or S. fleurettii and S. epidermidis [1,16,26,27,29,30]. In agreement with these findings, our study also detected diverse species of MRCoNS in dogs. The exact cause of the dominance of S. saprophyticus over other species of MRCoNS in both humans and dogs, however, needs further investigation.
SCCmec types having a high diversity were detected, most being SCCmec IV (55.2%), which was in agreement with former studies that also detected SCCmec IV as a prevalent cassette among companion animals and humans [1,17,20,23,31,32,33,34,35,36,37]. This study observed two mecA positive S. warneri non-typable for the SCCmec element, and this is common in MRCoNS strains because of the high variability of the SCCmec element and lack of primers that target all ccr and mec genes. The whole genome sequencing is definitely needed for an in-depth analysis [16]. The lack of typeability, highly diverse SCCmec, and presence of novel mec and ccr genes’ complex combination reveal an increasing complexity in the SCCmec typing of MRCoNS from humans and companion animals. Such mobile segments may be an important source of resistance determinants to other staphylococci that reside in the same niche.
Erythromycin, an important antimicrobial used for staphylococcal infections in both human and animals, was the drug for which most MRCoNS from humans and dogs exhibited resistance. The macrolides-lincosamides-streptogramins (MLS) resistance in staphylococci is not surprising [25,28,32,37,38]. The resistance to both erythromycin and clindamycin is common in S. aureus, but most MRCoNS strains exhibit resistance to either erythromycin or clindamycin, as shown in our study and in another previous report [39]. This shows the differential ability of S. aureus and MRCoNS to acquire resistance genes. We also detected MRCoNS isolates resistant to co-trimoxazole and ciprofloxacin in almost equal proportion, and to the other antibiotics including gentamicin and chloramphenicol in varying proportions. These antibiotics are used in both human as well as veterinary medicine in Nepal, and resistance to these drugs may be attributed to their frequent use. We detected only two isolates of MRCoNS from dogs that were resistant to tetracycline, and staphylococci resistance to this drug is low. The linezolid-resistant strain of methicillin-resistant S. aureus (MRSA) has been reported in one study carried out by Roberts et al in Nepalese swines [40]. This study did not report linezolid-resistant MRCoNS, which shows that strains of staphylococci resistant to this drug are uncommon in Nepal.
Studies targeting the transmission dynamics of MRCoNS between dogs and their owners have found that the same strain of CoNS is shared among these two hosts, even though the occurrence is very low [16,41]. However, this study did not find any sharing of the same strain to occur between dogs and their owners. This may be attributed to the low sample size and limited sample types; a large-scale study with multiple sample types should be done in order to warrant the conclusion. We could not perform pulse-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), spa typing, and whole genome sequencing (WGS) of the strains, which could provide an epidemiological pattern of the transmission of MRCoNS. All the dogs that were included in this study were kept in the house; however, data regarding their age, breeds, and captivity were not available for comparison.

Author Contributions

Conceptualization, M.K., P.R.J., S.P., M.A., K.R.R., P.G. and M.R.B.; Data curation, M.K.; Formal analysis, M.K. and M.R.B.; Investigation, M.K., S.P. and M.A.; Methodology, M.K. and P.R.J.; Project administration, M.K., P.R.J. and M.A.; Supervision, P.R.J., K.R.R., P.G. and M.R.B.; Writing of the original draft, M.K., P.R.J. and M.R.B.; Writing–review & editing, P.R.J., S.P., K.R.R., P.G. and M.R.B. All authors have read and agreed to the published version of the manuscript.

Funding

This study has not received funding.

Institutional Review Board Statement

The study received ethical approval from the Nepal Health Research Council (Regd. 457/2019 MT).

Informed Consent Statement

Written consent was obtained from the dog owners before the collection of data and nasal samples.

Data Availability Statement

All relevant data has been presented in the manuscript.

Acknowledgments

The authors would like to thank all the participants who agreed to be involved in this study.

Conflicts of Interest

The authors declare that they have no competing interest.

References

  1. Becker, K.; Heilmann, C.; Peters, G. Coagulase-negative staphylococci. Clin. Microbiol. Rev. 2014, 274, 870–926. [Google Scholar] [CrossRef] [Green Version]
  2. Tulinski, P.; Fluit, A.C.; Wagenaar, J.A.; 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, 299–304. [Google Scholar] [CrossRef] [Green Version]
  3. Garza, G.E.; Morfı, O.R.; Llaca, D.J.M. Staphylococcal cassette chromosome mec (SCCmec) in methicillin-resistant coagulase-negative Staphylococci. A review and the experience in a tertiary-care setting. Epidemiol. Infect. 2010, 138, 645–654. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Widerstrom, M.; Wistrom, J.; Sjostedt, A.; Monsenet, 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, 7–20. [Google Scholar] [CrossRef] [PubMed]
  5. Bagcigil, A.F.; Taponen, S.; Koort, J.; Bengtsson, B.; Myllyniemi, A.L.; Pyorala, S. Genetic basis of Penicillin resistance of S. aureus isolated in bovine mastitis. Acta Vet. Scand. 2012, 54, 69. [Google Scholar] [CrossRef] [Green Version]
  6. Vengust, M.; Anderson, M.E.; Rousseau, J.; Weese, J.S. Methicillin-resistant staphylococcal colonization in clinically normal dogs and horses in the community. Lett. Appl. Microbiol. 2006, 43, 602–606. [Google Scholar] [CrossRef]
  7. Harrison, E.M.; Paterson, G.K.; Holden, M.T.; Morgan, F.J.; Larsen, A.R.; Petersen, A.; Leroy, S.; Vliegher, S.D.; Perretten, V.; Fox, L.K.; et al. A Staphyloccocus xylosus isolate with a new mecC allotype. Antimicrob. Agents Chemother. 2013, 57, 1524–1528. [Google Scholar] [CrossRef] [Green Version]
  8. Acharya, K.P.; Wilson, R.T. Antimicrobial Resistance in Nepal. Front. Med. 2019, 6, 105. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Clinical and Laboratory Standards Institute (CLSI). M100—Performance Standards for Antimicrobial Susceptibility Testing, 31st ed.; CLSI: Wayne, PA, USA, 2019. [Google Scholar]
  10. Boye, K.; Bartels, M.D.; Andersen, I.S.; Moller, J.A.; Westh, H. A new multiplex PCR for easy screening of methicillin-resistant Staphylococcus aureus SCCmec types I–V. Clin. Microbiol. Infect. 2007, 3, 725–727. [Google Scholar] [CrossRef] [Green Version]
  11. Kitti, T.; Seng, R.; Thummeepak, R.; Boonlao, C.; Jindayok, T.; Sutthirat, S. Biofilm formation of methicillin-resistant coagulase-negative Staphylococci isolated from clinical samples in Northern Thailand. J. Glob. Infect. Dis. 2019, 11, 112–117. [Google Scholar]
  12. Hirotaki, S.; Sasaki, T.; Kuwahara-Arai, K.; Hiramatsu, K. Rapid and accurate identification of human-associated Staphylococci by use of multiplex PCR. J. Clin. Microbiol. 2011, 49, 3627–3631. [Google Scholar] [CrossRef] [Green Version]
  13. Shome, B.R.; Das, S.M.; Bhuvana, M.; Krithiga, N.; Velu, D.; Shome, R.; Isloor, S.; Barbuddhe, S.B.; Rahman, H. Multiplex PCR assay for species identification of bovine mastitis pathogens. J. Appl. Microbiol. 2011, 111, 1349–1356. [Google Scholar] [CrossRef]
  14. Chiang, Y.C.; Lu, H.C.; Li, S.C.; Chang, Y.H.; Chen, H.Y.; Lin, C.W.; Tsen, H.Y. Development of PCR primers and a DNA microarray for the simultaneous detection of major Staphylococcus species using groESL gene. Foodborne Pathog. Dis. 2012, 9, 249–257. [Google Scholar] [CrossRef]
  15. Martineau, F.; Picard, F.J.; Menard, C.; Roy, P.H.; Ouellette, M.; Bergeron, M.G. Development of a rapid PCR assay specific for Staphylococcus saprophyticus and application to direct detection from urine samples. J. Clin. Microbiol. 2000, 38, 3280–3284. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Gómez-Sanz, E.; Ceballos, S.; Ruiz-Ripa, L.; Zarazaga, M.; Torres, C. Clonally diverse methicillin and multidrug resistant coagulase negative Staphylococci are ubiquitous and pose transfer ability between pets and their owners. Front. Microbiol. 2019, 10, 485. [Google Scholar] [CrossRef] [Green Version]
  17. Barbier, F.; Ruppe, E.; Hernandez, D.; Lebeaux, D.; Francois, P.; Felix, B.; Desprez, A.; Maiga, A.; Woerther, P.L.; 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, 270–281. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Rolo, J.; de Lencastre, H.; Miragaia, M. Strategies of adaptation of Staphylococcus epidermidis to hospital and community: Amplification and diversification of SCCmec. J. Antimicrob. Chemother. 2012, 676, 1333–1341. [Google Scholar] [CrossRef] [Green Version]
  19. Du, X.; Zhu, Y.; Song, Y.; Li, T.; Luo, T.; Sun, G.; Yang, C.; Cao, C.; Lu, Y.; Li, M. Molecular analysis of Staphylococcus epidermidis strains isolated from community and hospital environments in China. PLoS ONE 2013, 85, e62742. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Abadi, M.I.M.; Moniri, R.; Khorshidi, A.; Piroozmand, A.; Mousavi, S.G.A.; Dastehgoli, K.; Ghazikalayeh, H.M. Molecular characteristics of nasal carriage methicillin-resistant coagulase negative staphylococci in school students. Jundishapur J. Microbiol. 2015, 68, e18591. [Google Scholar] [CrossRef] [Green Version]
  21. Xu, Z.; Shah, H.N.; Misra, R.; Chen, J.; Zhang, W.; Liu, Y.; Cutler, R.R.; Mkrtchyan, H.V. The prevalence, antibiotic resistance and mecA characterization of coagulase negative staphylococci recovered from non-healthcare settings in London, UK. Antimicrob. Resist. Infect. Control 2018, 7, 73. [Google Scholar] [CrossRef] [Green Version]
  22. Jamaluddin, T.Z.; Kuwahara-Arai, K.; Hisata, K.; Terasawa, M.; Cui, L.; Baba, T.; Sotozono, C.; Kinoshita, S.; Ito, T.; Hiramatsu, K. Extreme genetic diversity of methicillin-resistant Staphylococcus epidermidis strains disseminated among healthy Japanese children. J. Clin. Microbiol. 2008, 4611, 3778–3783. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. 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, 561, 315–323. [Google Scholar] [CrossRef] [Green Version]
  24. Rodrigues, A.C.; Belas, A.; Marques, C.; Cruz, L.; Gama, L.T.; Pomba, C. Risk factors for nasal colonization by methicillin-resistant staphylococci in healthy humans in professional daily contact with companion animals in Portugal. Microb. Drug Resist. 2018, 244, 434–446. [Google Scholar] [CrossRef] [PubMed]
  25. 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, 662, 169–173. [Google Scholar] [CrossRef] [PubMed]
  26. Chah, K.F.; Gomez-Sanz, E.; Nwanta, J.A.; Asadu, B.; Agbo, I.C.; Lozano, C.; Zarazaga, M.; Torres, C. Methicillin-resistant coagulase-negative staphylococci from healthy dogs in Nsukka, Nigeria. Braz. J. Microbiol. 2014, 451, 215–220. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Davis, J.A.; Jackson, C.R.; Fedorka-Cray, P.J.; Barrett, J.B.; Brousse, J.H.; Gustafson, J.; Kucher, M. Carriage of methicillin-resistant staphylococci by healthy companion animals in the US. Lett. Appl. Microbiol. 2014, 591, 1–8. [Google Scholar] [CrossRef]
  28. Wedley, A.L.; Dawson, S.; Maddox, T.W.; Coyne, K.P.; Pinchbeck, G.L.; Clegg, P.; Jamrozy, D.; Fielder, M.D.; Donovan, D.; Nuttall, T.; et al. Carriage of Staphylococcus species in the veterinary visiting dog population in mainland UK: Molecular characterization of resistance and virulence. Vet. Microbiol. 2014, 170, 81–88. [Google Scholar] [CrossRef]
  29. Siugzdaite, J.; Gabinaitiene, A. Methicillin-resistant coagulase-negative staphylococci in healthy dogs. Vet. Med. 2017, 62, 479–487. [Google Scholar] [CrossRef] [Green Version]
  30. Schmidt, V.M.; Williams, N.J.; Pinchbeck, G.; Corless, C.E.; Shaw, S.; McEwan, N.; Dawson, S.; Nuttall, T. Antimicrobial resistance and characterization of staphylococci isolated from healthy Labrador retrievers in the United Kingdom. BMC Vet. Res. 2014, 10, 17. [Google Scholar] [CrossRef] [Green Version]
  31. 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, 532, 442–449. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Aslantas, O.; Turkyilmaz, S.; Yilmaz, M.; Yilmaz, E.S. Prevalence of methicillin-resistant staphylococci in dogs. Kafkas Univ. Vet. Fak. Derg. 2013, 119, 37–42. [Google Scholar]
  33. Kern, A.; Perreten, V. Clinical and molecular features of methicillin-resistant, coagulase-negative Staphylococci of pets and horses. J. Antimicrob. Chemother. 2013, 686, 1256–1266. [Google Scholar] [CrossRef] [PubMed]
  34. Park, Y.K.; Paik, Y.H.; Yoon, J.W.; Fox, L.K.; Hwang, S.Y.; Park, Y.H. Dissimilarity of ccrAB gene sequences between methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus among bovine isolates in Korea. J. Vet. Sci. 2013, 143, 299–305. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Weiss, S.; Kadlec, K.; Fessler, A.T.; Schwarz, S. Identification and characterization of methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus pettenkoferi from a small animal clinic. Vet. Microbiol. 2013, 167, 680–685. [Google Scholar] [CrossRef] [PubMed]
  36. McManus, B.A.; Coleman, D.C.; Deasy, E.C.; Brennan, G.I.; O’Connell, B.; Monecke, S.; Ehricht, R.; Leggett, B.; Leonard, N.; Shore, A.C. Comparative genotypes, staphylococcal cassette chromosome mec (SCCmec) genes and antimicrobial resistance amongst Staphylococcus epidermidis and Staphylococcus haemolyticus isolates from infections in humans and companion animals. PLoS ONE 2015, 109, e0138079. [Google Scholar] [CrossRef] [Green Version]
  37. Couto, N.; Monchique, C.; Belas, A.; Marques, C.; Gama, L.T.; Pomba, C. Trends and molecular mechanisms of antimicrobial resistance in clinical staphylococci isolated from companion animals over a 16 year period. J. Antimicrob. Chemother. 2016, 716, 1479–1487. [Google Scholar] [CrossRef]
  38. Han, J.I.; Yang, C.H.; Park, H.M. Prevalence and risk factors of Staphylococcus spp. carriage among dogs and their owners: A cross-sectional study. Vet. J. 2016, 212, 15–21. [Google Scholar] [CrossRef] [PubMed]
  39. Gomez-Sanz, E.; Torres, C.; Ceballos, S.; Lozano, C.; Zarazaga, M. Clonal dynamics of nasal Staphylococcus aureus and Staphylococcus pseudintermedius in dog-owning household members. Detection of MSSA ST398. PLoS ONE 2013, 87, e69337. [Google Scholar] [CrossRef] [Green Version]
  40. Roberts, M.C.; Joshi, P.R.; Greninger, A.L.; Melendez, D.; Paudel, S.; Acharya, M.; Bimali, N.K.; Koju, N.P.; No, D.; Chalise, M.; et al. The human clone ST22 SCCmec IV methicillin-resistant Staphylococcus aureus isolated from swine herds and wild primates in Nepal: Is man the common source? FEMS Microbiol. Ecol. 2018, 94, fiy052. [Google Scholar] [CrossRef] [Green Version]
  41. Gomez-Sanz, E.; Torres, C.; Lozano, C.; Zarazaga, M. High diversity of Staphylococcus aureus and Staphylococcus pseudintermedius lineages and toxigenic traits in healthy pet-owning household members. Underestimating normal household contact? Comp. Immunol. Microbiol. Infect. Dis. 2013, 361, 83–94. [Google Scholar] [CrossRef] [PubMed]
Table
Table 1. Primers used in the species identification of MRCoNS.
Table 1. Primers used in the species identification of MRCoNS.
Primers.Oligonucleotide Sequence (5’-3’ )Ta (°C)Product Size (bp)References
nuc (S. warneri)F-CGTTTGTAGCAAAACAGGGC
R-GCAACGAGTAACCTTGCCAC		60999[12]
rdr (S. epidermidis)F-AAGAGCGTGGAGAAAAGTATCAAG
R-TCGATACCATCAAAAAGTTGG		62130[13]
groESL-F (S. haemolyticus)F-GGTCGCTTAGTCGGAACAAT
R-CACGAGCAATCTCATCACCT		58271[14]
sodA (S. saprophyticus)F-TCAAAAAGTTTTCTAAAAAATTTAC
R-ACGGGCGTCCACAAAATCAATAGGA		55580[15]
Table
Table 2. Primers used in the multiplex SCCmec PCR.
Table 2. Primers used in the multiplex SCCmec PCR.
Primer Sequence (5’-3’)TargetLength (bp)SCCmec Type
F-ATTGCCTTGATAATAGCCYTCT
R-TAAAGGCATCAATGCACAAACACT		ccrA2-B937II, IV
F-CGTCTATTACAAGATGTTA AGGATAAT
R-CCTTTATAGACTGGATTATTCAAAATAT		CcrC518III, V
F-GCCACTCATAACATATGGAA
R-CATCCGAGTGAAACCCAAA		IS1272415I, IV
F-TATACCAAACCCGACAACTAC
R-CGGCTACAGTGATAACATCC		mecA–IS431359V
Table
Table 3. Antibiotic susceptibility pattern of MRCoNS in dog owners and dogs.
Table 3. Antibiotic susceptibility pattern of MRCoNS in dog owners and dogs.
Antibiotics.Susceptibility Patterns (n = 16)Susceptibility Patterns (n = 13)
(Dog owners)(Dogs)
S (%)I (%)R (%)S (%)I (%)R (%)
Erythromycin4 (25) 12 (75)3 (23) 10 (77)
Clindamycin13 (81.2) 3 (18.8)11 (84.6) 2 (15.4)
Co-trimoxazole10 (62.5)1 (6.3)5 (31.2)8 (61.5) 5 (38.5)
Ciprofloxacin13 (81.2) 3 (18.8)10 (76.9) 3 (23.1)
Gentamicin14 (87.5) 2 (12.5)11 (84.6)1 (7.7)1 (7.7)
Chloramphenicol15 (93.7) 1(6.3)12 (92.3) 1 (7.7)
Tetracycline16 (100) 011 (84.6) 2 (15.4)
Linezolid16 (100) 013 (100) 0
Table
Table 4. MIC of oxacillin to MRCoNS isolated from dogs and their owners.
Table 4. MIC of oxacillin to MRCoNS isolated from dogs and their owners.
MRCoNS (Dog)MICs (µg/mL)MRCoNS (Human)MICs (µg/mL)
D14M116
D24M216
D316M332
D416M432
D516M532
D64M68
D78M716
D816M88
D916M98
D1016M104
D1116M1132
D124M1232
D1316M1332
M1416
M154
M1632
Table
Table 5. Inducible and constitutive MLSB pattern in dogs and dog owners.
Table 5. Inducible and constitutive MLSB pattern in dogs and dog owners.
TotaliMLSB PatterncMLSB PatternMSB PatternSusceptible
Number Pattern
Dog owner (16)2 (12.5%)2 (12.5%)8 (50%)4 (25%)
Dog (13)02 (15.4%)8 (61.5%)3 (23.1%)
Table
Table 6. Species and SCCmec types of MRCoNS from dog owners and dogs.
Table 6. Species and SCCmec types of MRCoNS from dog owners and dogs.
SCCmec TypeIsolates from Dog OwnersIsolates from Dogs
Total
n (%)		S. saprophyticusS. haemolyticusS. warneriS. epidermidisTotal
n (%)		S. saprophyticusS. haemolyticusS. warneriS. epidermidis
I0----0----
II1 (6.3)1-- 0----
III2 (12.6)1--11 (7.7)1---
IV7 (43.7)32119 (69.2)3321
V5 (31.3)41--2 (15.4)11--
Non-typable1 (6.3)--1-1 (7.7)--1-
Total169 (56.3%)3 (18.8%)2 (12.5%)2 (12.5%)135 (38.5%)4 (30.8%)3 (23.1%)1 (7.7%)
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Khanal, M.; Joshi, P.R.; Paudel, S.; Acharya, M.; Rijal, K.R.; Ghimire, P.; Banjara, M.R. Methicillin-Resistant Coagulase Negative Staphylococci and Their Antibiotic Susceptibility Pattern from Healthy Dogs and Their Owners from Kathmandu Valley. Trop. Med. Infect. Dis. 2021, 6, 194. https://doi.org/10.3390/tropicalmed6040194

AMA Style

Khanal M, Joshi PR, Paudel S, Acharya M, Rijal KR, Ghimire P, Banjara MR. Methicillin-Resistant Coagulase Negative Staphylococci and Their Antibiotic Susceptibility Pattern from Healthy Dogs and Their Owners from Kathmandu Valley. Tropical Medicine and Infectious Disease. 2021; 6(4):194. https://doi.org/10.3390/tropicalmed6040194

Chicago/Turabian Style

Khanal, Muna, Prabhu Raj Joshi, Saroj Paudel, Mahesh Acharya, Komal Raj Rijal, Prakash Ghimire, and Megha Raj Banjara. 2021. "Methicillin-Resistant Coagulase Negative Staphylococci and Their Antibiotic Susceptibility Pattern from Healthy Dogs and Their Owners from Kathmandu Valley" Tropical Medicine and Infectious Disease 6, no. 4: 194. https://doi.org/10.3390/tropicalmed6040194

APA Style

Khanal, M., Joshi, P. R., Paudel, S., Acharya, M., Rijal, K. R., Ghimire, P., & Banjara, M. R. (2021). Methicillin-Resistant Coagulase Negative Staphylococci and Their Antibiotic Susceptibility Pattern from Healthy Dogs and Their Owners from Kathmandu Valley. Tropical Medicine and Infectious Disease, 6(4), 194. https://doi.org/10.3390/tropicalmed6040194

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