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Article

Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 as a Major MRSA Lineage in Dogs and Cats in Thailand

by
Surawit Chueahiran
1,
Jitrapa Yindee
1,
Pongthai Boonkham
2,
Nipattra Suanpairintr
3 and
Pattrarat Chanchaithong
1,4,*
1
Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
2
Veterinary Diagnostic Laboratory, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
3
Department of Pharmacology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
4
Research Unit in Microbial Food Safety and Antimicrobial Resistance, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand
*
Author to whom correspondence should be addressed.
Antibiotics 2021, 10(3), 243; https://doi.org/10.3390/antibiotics10030243
Submission received: 4 February 2021 / Revised: 23 February 2021 / Accepted: 24 February 2021 / Published: 28 February 2021
(This article belongs to the Section Genetic and Biochemical Studies of Antibiotic Activity and Resistance)
Browse Figure
Review Reports Versions Notes

Abstract

:
The aim of this study was to present molecular and antimicrobial resistance characteristics of methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 isolated from diseased dogs and cats in Thailand. A total of 20 MRSA isolates of 134 Staphylococcus aureus isolated from canine and feline clinical samples during 2017–2020 were CC398, consisting of sequence type (ST) 398 (18 isolates), ST5926 (1 isolate), and ST6563 (1 isolate) by multilocus sequence typing. spa t034 and staphylococcal cassette chromosome mec (SCCmec) V were predominantly associated with ST398. Intraclonal differentiation was present by additional spa (t1255, t4653), non-detectable spa, composite SCCmec with a hybrid of ccrA1B1+ccrC and class A mec complex, and DNA fingerprints by pulsed-field gel electrophoresis. The isolates essentially carried antimicrobial resistance genes, mediating multiple resistance to β-lactams (mecA, blaZ), tetracyclines [tet(M)], aminoglycosides [aac(6′)-Ie-aph(2′)-Ia], and trimethoprim (dfr). Livestock-associated MRSA ST398 resistance genes including lnu(B), lsa(E), spw, fexA, and tet(L) were heterogeneously found and lost in subpopulation, with the absence or presence of additional erm(A), erm(B), and ileS2 genes that corresponded to resistance phenotypes. As only a single CC398 was detected with the presence of intraclonal variation, CC398 seems to be the successful MRSA clone colonizing in small animals as a pet-associated MRSA in Thailand.

1. Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium widespread in human and domestic animals, and is associated with a wide spectrum of infection, from superficial to invasive and systemic infections [1]. Of medical importance, nosocomial infections are caused by a particular subpopulation, known as hospital-associated MRSA (HA-MRSA). Molecular epidemiological tools, such as multilocus sequence typing (MLST), reveal HA-MRSA clonal lineages in terms of clonal complex (CC) and sequence types (ST), including CC8 (ST239), CC22 (ST22), CC30 (ST26) [2]. In addition, CC8 (ST8), CC80 (ST80), CC30 (ST80), CC59 (ST50), and CC93 (ST93) are classified as community-associated MRSA (CA-MRSA) [3]. The nosocomial spread of CA-MRSA in healthcare settings has been reported in many circumstances [4,5,6]. MRSA-colonizing cattle, pigs and poultry are clonal-specific, for CC398 (ST398), CC9 (ST9), CC5 (ST5), and CC49 (ST49), among others, which are all known as livestock-associated MRSA (LA-MRSA). Various MRSA clones have been identified in dogs, cats and horses. Human-related MRSA clones are a major population in small companion animals, and CC398 has been sporadically identified as a subpopulation [7,8,9].
S. aureus may colonize several animal host species, including dogs and cats, but S. pseudintermedius and S. schleiferi subsp. coagulans predominate in canine healthy carriers and in infections. Clonal expansion and multidrug resistance of methicillin-resistant S. pseudintermedius (MRSP) limit effective antimicrobial treatment in small animal practice [10]. Likewise, MRSA is occasionally isolated from canine and feline clinical samples, particularly from skin and soft tissue infections [1,11]. Risk factors contributing to infection in pets include antimicrobial exposures, hospital stays and medical implants [12]. Furthermore, small companion animals can play the role of MRSA reservoirs in the community, and can be a source of infection in humans [13,14]. Interspecies transmission among risk groups including veterinary staff, pet owners and healthy pet carriers was proven using bacterial genetic identities [15]. Recently, increasing reports describing MRSA infections in animals and animal-to-human transmission have raised the alarm of a possible emerging multidrug-resistant pathogen [16,17]. Previous studies illustrate that various human- and livestock-related MRSA clones have been recovered from canine and feline patients and carriers [7,8,9,15,18,19]. Little is known about the clonal spread of MRSA, which is associated with diseases in dogs and cats in Thailand. Genetic features of canine and swine MRSA ST398 from animal carriers were the first-characterized, and have both common and different traits [20,21]. From 2017 to 2020, MRSA were sporadically isolated from canine and feline clinical samples from our routine diagnostic activity, and the majority of the isolates shared some common resistance phenotypes, such as resistance to clindamycin (lincosamides) and susceptibility to erythromycin (macrolides), which are similar to the canine MRSA ST398 isolate in the previous study [20]. MLST analysis illustrated that all clinical MRSA isolates were clonally related to CC398, and other MRSA clones were not detected. This study presents MRSA CC398 isolation from canine and feline clinical samples, along with its intraclonal variation by genetic and antimicrobial resistance features.

2. Results

2.1. Numbers and Origins of MRSA

Of 7499 canine and feline clinical samples, 134 S. aureus was primarily identified during 2017–2020 by Vitek®2 automated identification (ID). Twenty S. aureus isolates exhibited positive MRSA during screening by cefoxitin via Vitek®2 antimicrobial susceptibility testing (AST), and resistance to cefoxitin via disk diffusion method. All cefoxitin-resistant isolates were mecA-positive by PCR. MRSA was recovered from submitted samples each year in 7 of 1772 samples (2017), 5 of 1864 samples (2018), 5 of 2690 samples (2019), and 3 of 1173 samples (January to June 2020). The MRSA-positive samples were submitted from three veterinary hospitals in Bangkok, Thailand. Of 20 MRSA isolates, 15 and 5 isolates were recovered from canine and feline samples, respectively. Lesions or diseases associated with MRSA infection were wounds and abscesses (16), surgical site infections (3), and urinary tract infections (1).

2.2. Genetic Characteristics of Canine and Feline MRSA CC398

From MLST analysis, eighteen MRSA isolates were ST398 (allelic profile 2-35-19-2-20-26-39). Two isolates were ST5926 and a new ST6563, each of which are single locus variants (SLV) of ST398 by tpi gene. The new ST6563 had a new allele variation (allelic number 740) of tpi (A300T). Using an 80% cutoff for similarity, DNA fingerprint patterns by pulsed-field gel electrophoresis (PFGE) of the isolates were clustered into 3 pulsotypes (Figure 1). Fourteen and one MRSA ST398 isolates contained spa t034 and t4652, respectively. spa t1255 was associated with ST5926 and ST6563. Three MRSA ST398 were spa-negative by PCR, and had a composite staphylococcal cassette chromosome mec (SCCmec) which consisted of a class C mec complex and two ccr complexes, including ccrA1B1 and ccrC. This composite SCCmec was also found in one MRSA-ST398-t034 isolate; however, SCCmec V was predominantly associated with 14 ST398 isolates, ST5956 and ST6563.
By broth microdilution, all MRSA were multidrug-resistant (more than 3 antimicrobial classes) and exhibited resistance to, at least, penicillin and cefoxitin (β-lactams), tetracycline (tetracyclines), clindamycin (lincosamides) and ciprofloxacin (fluoroquinolones). No resistance to vancomycin, linezolid, rifampicin and fusidic acid was found in all isolates. Resistance to antimicrobials in high proportions was observed for trimethoprim (19/20), kanamycin (18/20), gentamicin (15/20), and tiamulin (16/20). Distribution of the isolates into categories of susceptible, intermediate or resistant to each antimicrobial agent, along with minimum inhibitory concentrations (MIC), are illustrated in Table 1. The resistance rate of erythromycin, chloramphenicol, and mupirocin was 4/20, 5/20, and 4/20, respectively.

2.3. Antimicrobial Resistance Gene Carriage

Only mecA and tet(M) were detected in all MRSA isolates. blaZ and tet(L) were contained in 18/20 and 16/20 isolates, respectively. Aminoglycoside or aminocyclitol modifying enzyme (AME)-encoding genes were present, including ant(4′)-Ia (17/20), ant(6)-Ia (16/20), bifunctional aac(6′)-Ie-aph(2′)-Ia (18/20), and spw (16/20). Only one isolate did not contain AME-encoding genes. The sixteen spw-positive isolates also contained lnu(B) and lsa(E), and presented a higher MIC for tiamulin (>4 μg/mL). Nineteen of 20 isolates carried either dfrA (16/20) or dfrG (3/20), and one isolate had both dfrA and dfrG. The four dfrG-positive isolates contained erm(B). Both erm(A) and erm(B) were found in the same three isolates, and one isolate carried only erm(B). fexA was detected in five chloramphenicol-resistant isolates, but catpC221 was not found. Four isolates presenting high-level mupirocin resistance contained ileS2. The antimicrobial resistance gene profiles are shown in Table 2.

2.4. Intraclonal Variation and Common Features of Canine and Feline MRSA CC398

Thirteen MRSA isolates were ST398-V-t034 (ST-SCCmec-spa), which was a major characteristic in both pulsotype A and B, and contained common antimicrobial resistance genes, including mecA, ant(6)-Ia, aac(6′)-Ie-aph(2′)-Ia, tet(M) and tet(L), single dfrA, lnu(B), lsa(B), and spw. The ST398-V-t034 pulsotype B were mostly found in 2017, whereas the pulsotype A was found in 2018–2020 (Figure 1). The composite SCCmec was associated with three spa-negative ST398 isolates presenting >90% DNA fingerprint similarity, and one ST398-t034 isolate, which was grouped in pulsotype A and recovered during 2018–2020 (Figure 1). The common features of ST398 containing the composite SCCmec were non ant(6)-Ia, lnu(B), lsa(B) and spw carriage, and a positive result for erm(B) and dfrG detection. The ST5926-V-t1255 and ST6563-V-t1255 isolates present an identical DNA fingerprint pattern (Figure 1). One MRSA-ST398-V-t4562 pulsotype B was isolated in 2017.

3. Discussion

This study presents MRSA CC398 isolated of canine and feline origin which were associated with diseases. With no other CC detected, the evidence shows that CC398 is the major MRSA lineage in cats and dogs in Thailand. MRSA ST398 was first detected in a Thai dog carrier in 2010 [20]. Despite a low prevalence of MRSA in the clinical samples, ST398 and their SLVs detected in this study represent opportunistic infection of this clone in small companion animals. MRSA ST398 was carried by veterinary practitioners, and was also the dominant LA-MRSA clone in pigs and swine workers in Thailand [20,21]. It was the most significant MRSA clone associated with animals and animal-related occupations. CC398 have been heterogeneously isolated together with other sequence types from small animals in other countries, but the majority of the isolates are human-related MRSA clones, such as CC5, CC8, CC22, CC45, and CC239 [8,9,11,15,22]. MRSA CC398 was found in 21.8% and 17.6% of canine and feline MRSA isolates over a five-year period (2010–2015) in France, respectively [8], and was not present in some countries [13,15,19]. In addition, there is no information regarding human-related MRSA clones in companion animals in Thailand. Since its first detection in 2010, we speculate that this clone has persisted and spread, and was occasionally associated with diseases in canine and feline hosts.
ST398-V-t034 pulsotype B was the major population, and was mostly isolated in 2017. ST398-V-t034 is sporadically found in canine carriers in China [22] and in diseased dogs and cats in Austria [9]. The major spa t011 and SCCmec IV are predominant in equine, canine and feline MRSA ST398 in France and Austria [8,9]. t034 is the most prevalent spa associated with CC398 in Asian countries [20,22,23]. In addition to a single-nucleotide polymorphism, SLV ST5926 and ST6563 had a different spa t1255, which represented a minor variation mirroring evolution of the CC398 in cats and dogs. SCCmec IV and V were more common in ST398 isolated from companion animals [8,9], while only SCCmec V is more abundant in LA-MRSA ST398 [21,24,25]. The type V and the composite SCCmec with a hybrid of type 1 ccr and ccrC is also contained in swine MRSA ST398 in Thailand [21]. The Thai swine strains with the composite SCCmec contained spa t034, but the spa could not be detected in the majority of canine and feline ST398-composite SCCmec strains, together with the loss of genes presenting in LA-MRSA. The SCCmec and spa diversifications may be a result of the plasticity of orfX-surrounding regions where SCCmec and spa are located, which promote S. aureus genetic events [26]. However, the presence or absence of spa and the variation of the composite SCCmec need to be deduced by further whole genome analysis. These common and minor different traits may reflect a concomitant CC398 ancestor in a variety of animals, as well as bacterial divergent evolution in different hosts in Thailand.
Antimicrobial resistance genes carried by the present canine and feline MRSA CC398 population were heterogenous. In addition to mecA, multidrug resistance was mediated by common resistance genes in staphylococcal species, such as blaZ, tet(M), ant(4′)-Ia, ant(6)-Ia, and aac(6′)-Ie-aph(2)-Ia [27,28]. tet(M) is frequently distributed in ST398 derived from companion animals, and it can be a differentiation marker from human-related clones [8]. In Thailand, dfrA is remarkably found in ST398-V-t034 from small animals, but LA-MRSA from pigs and MRSP contains dfrG [20,21]. The antimicrobial resistance gene carriages of Thai ST398-V-t034 from swine and from small animals were different. erm genes were not found in ST398-V-t034, in contrast to a wide distribution of erm(A) and erm(C) found in the strains from pigs [21]. However, dfrG was detected in the ST398-composite SCCmec subpopulation with the presence of erm(B) genes, similar to the resistance gene profiles of canine MRSP [27,29]. Taken together, the loss of LA-MRSA-associated resistance genes, including lnu(B), lsa(E), spw, tet(L), and fexA [30,31,32], likely corresponds to antimicrobial use patterns in small-animal practice, which are different than in livestock. In addition, the presence of ileS2 possessing high-level mupirocin resistance may be due to pressure from the introduction of topical mupirocin in small-animal veterinary practice [33]. The heterogenous resistance in the canine and feline MRSA CC398 populations indirectly presented a picture of resistance gene loss and gain in this clone to be fit in canine and feline niches.
The isolates exhibited a resistance to multiple antimicrobial classes that limited the first-line and second-line therapeutic options in small animal practices, such as β-lactams, gentamicin, clindamycin, doxycycline and sulfamethoxazole/trimethoprim. Topical treatment may be an alternative method for wound and skin infection in the most common cases [34,35]. However, systemic treatment and extraordinary use of last-resort antimicrobials is required in the case of invasive infections. Additional resistance can be developed by introduction of new drugs in veterinary practice. Florfenicol and rifampicin treatment were associated with the presence of fexA, cfr, and rpoB mutation in MRSP, respectively [36,37]. Indeed, antimicrobial stewardship programs by effective diagnostic approaches, antimicrobial resistance monitoring program and judicious antimicrobial use are strongly recommended to follow up and diminish ongoing development of resistance.
MRSA was recovered from only 0.26% of clinical samples from dogs and cats. The prevalence of MRSA in companion animals is <2% [7,22]. The proportion of methicillin resistance in S. aureus isolates was 20/134 (14.92%), which was a range similar to a previous study [19], but which cannot be completely compared to other studies, due to the sample sources and population. The majority of the isolates were associated with wound infections and abscess. A wide-scale study in Germany reported that 62.7% of canine wounds were infected by MRSA [11]. Even with low prevalence in companion animals, attention should be continuously paid for S. aureus due to the wide species distribution and their adaptive ability in different hosts, especially MRSA CC398. Furthermore, many studies demonstrated that veterinary staff share common MRSA strains with companion animals and small-animal hospital environments [16,38,39], owing to the proximity and frequent contact. Monitoring at-risk groups, including veterinary personnel and pet owners, healthy pet carriers in the community and environmental contamination should be undertaken in the term of one health approach.
Clinical MRSA isolates in the present study manifested pathogenic potential of CC398 in dogs and cats. S. aureus is more frequently recovered from cat wounds; however, the proportion of MRSA from dog wounds is higher [11]. Circulation of MRSA ST398 in dog kennels can be a source of a variety of infections, including gangrenous mastitis in bitches, and pyoderma and fatal pneumonia in puppies [40]. The evidence from this study supported MRSA ST398 as being not only a recognized worldwide LA-MRSA, but also a pet-associated MRSA. We are unable to speculate on the replacement of previous MRSA clones because prior epidemiological information is lacking. Nevertheless, CC398 has successfully expanded in horses, replacing the previous predominant equine CC8 in Europe [8,9]. Genetic changes supporting clonal proliferation and mechanisms for host adaptation of S. aureus in canine and feline hosts are likely, supported by information obtained from other domestic animals [41,42,43]. Genes encoding survival and virulence factors were not examined in our study, and need to be explored. Little is known about the genetic adaption of S. aureus colonization in dogs and cats. Immune evasion cluster (IEC) delivered by bacteriophages, a factor enhancing MRSA colonization in humans, is inconsistently found in ST398 strains from companion animals, but completely absent in LA-MRSA ST398 [18,41]. Host-specific virulence factors or genome adaptations are not identified in ST22 (EMRSA-15) from humans or companion animals in the UK [44]. High-resolution genome analysis should be further investigated for understanding the complete evolutionary relationship of this clone among domestic animals. Specific markers from deep insights could be informative in term of genomic surveillance for tracing cross-species transmission and for developing preventive measures in the companion animal and livestock sectors.

4. Materials and Methods

4.1. Bacterial Isolates

Staphylococcus aureus isolates were obtained from clinical samples, from diseased cats and dogs submitted to the Veterinary Diagnosis Laboratory of Chulalongkorn University, Bangkok, Thailand, from January 2017 to June 2020. The species identification and antimicrobial susceptibility testing was routinely performed via the Vitek® automated system, with ID-GP and AST-GP81 cards (bioMérieux, France) for clinical purposes. Bacterial strains were stocked in an −80 °C freezer with 20% glycerol in tryptic soy broth. The S. aureus isolates presenting cefoxitin resistance by Vitek® were included for species confirmation, by Microflex Biotyper matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker Dalnotics, Germany) and nuc gene PCR [45], after recovery onto 5% sheep blood agar and DNA extraction using Nucleospin Tissue DNA extraction kits (Machery-Nagel, Germany). The DNA was also used in all PCR-based molecular assays.

4.2. Antimicrobial Susceptibility Testing and Resistance Gene Detection

Cefoxitin disk diffusion test was performed for phenotypic confirmation of MRSA, and mecA was detected by PCR [46]. MIC of antimicrobials was determined by broth microdilution using Sensititre customized EUST 96-well plates (Trek Diagnostic Systems, United Kingdom). Resistance, intermediate and susceptible results were justified by clinical breakpoint from the Clinical and Laboratory Standard Institute, as follows: penicillin, cefoxitin, gentamicin, kanamycin, streptomycin, tetracycline, erythromycin, clindamycin, ciprofloxacin, chloramphenicol, sulfamethoxazole, trimethoprim, vancomycin, rifampicin and linezolid [47,48]. Despite no CLSI-provided breakpoint for mupirocin, fusidic acid, and tiamulin, interpretation from scientific articles was referred to [49,50,51].
Acquired antimicrobial resistance genes encoding mechanisms of resistance to various antimicrobials were detected via PCR, including blaZ (β-lactamase), tet(M) (ribosomal protective proteins), tet(K) and tet(L) (tetracycline efflux proteins), ant(4′)-Ia, ant(6)-Ia and bifunctional aac(6′)-Ie-aph(2′)-Ia (aminoglycoside modifying enzymes), dfrA and dfrG (dihydrofolate reductase inhibitors), erm(A), erm(B) and erm(C) (erythromycin resistance methylase), lnu(B) (lincosamide nucleotydyl transferase), lsa(E) (lincosamide-streptogramin A efflux protein), fexA (florfenicol exporter), catpC221 (chloramphenicol acetyltransferase) and ileS2 (alternative isoleucyl transferase). Specific primers are listed in Supplementary Table S1. All PCR were performed in a total of 25 μL solution, using 5X Firepol Master Mix (Solis Biodyne, Estonia) with 0.2 pmol/μL of each primer. The PCR conditions included an initial denaturation at 95 °C for 3 min, 30 cycles of denaturation at 95 °C for 30 s, annealing at the specific temperature for each primer for 30 s and extension at 72 °C for the specific duration depending on the amplicon size (Supplementary Table S1), followed by a final extension at 72 °C for 5 min.

4.3. DNA Fingerprint Analysis

DNA fingerprints of the MRSA isolates were illustrated by Cfr9I macro-restriction and PFGE [52]. Bacterial cells in agarose plugs were lysed by lysozyme, lysostaphin and detergents. After the plug-washing steps, chromosomal DNA was digested in 50 U of Cfr9I (Thermo Fisher Scientific, CA, USA). Macro-restriction fragments were separated in 1% agarose gel and TE buffer, with a switch time of 5–40 sec and 6 V/cm for 21 h, using CHEF-DRIII PFGE (Bio-Rad, CA, USA). The DNA band patterns were stained with ethidium bromide and documented under a UV illuminator. Relationships among strains were analysed by dendrogram construction using Dice coefficients, using the unweighted matrix pair group method with arithmetic mean (UPGMA) by Bionumeric software version 7.6 (Applied Maths, Belgium) with 1.5% optimization and 1.5% position tolerance. Pulsotypes were classified at >80% band pattern similarity, representing close clonal relatedness.

4.4. spa Typing

Polymorphic X region of spa gene was amplified by 1095F (5′-AGACGATCCTTCGGTGAGC-3′) and 1517R (5′-GCTTTTGCAATGTCATTTACTG-3′) primers [53]. The strains which resulted in negative amplification by 1095F and 1517R were included for spa PCR by additional primers, [spa-1113f (5′-TAA AGA CGA TCC TTC GGT GAG C-3´) and spa-1514r (5′-CAG CAG TAG TGC CGT TTG CTT -3´)] [54]. Amplicons were purified using a Nucleospin DNA purification kit (Machery-Nagel, Germany) for capillary Sanger’s DNA sequencing at a service company (Apical Scientific Co., Ltd., Malaysia). spa sequences were analysed by comparing to spa tandem repeats in the database (http://spaserver.ridom.de/ (accessed on 3 February 2021)).

4.5. SCCmec Identification and MLST

SCCmec types were analysed based on ccr complex type and class of mec gene complex by multiplex PCRs [55] and interpreted according to criteria [56]. Composite SCCmec was classified in cases where more than one ccr complex was detected. MLST of S. aureus was conducted by amplification and nucleotide sequence analysis of arcC, aroE, glpF, gmk, pta, tpi and yqiL [57]. DNA sequences and allelic profiles were electronically submitted to the database (www.pubmlst.org (accessed on 3 February 2021)) for allelic number and ST assignation, respectively.

5. Conclusions

CC398 is the major MRSA lineage in dogs and cats in Thailand. By genetic and antimicrobial resistance features, ST398-V-t034 has proliferated in small animals since its first detection in a dog carrier and veterinarians. Minor differentiation between spa and SCCmec variants and antimicrobial resistance gene carriages described intraclonal variation and its continuous evolution. With only the single clone present in this study, we propose that the MRSA CC398 was a predominant pet-associated MRSA clone of veterinary importance.

Supplementary Materials

The following are available online at https://www.mdpi.com/2079-6382/10/3/243/s1, Table S1: Oligonucleotide primers for resistance gene detection.

Author Contributions

Conceptualization, P.C., S.C., and N.S.; methodology, P.C., S.C., and N.S.; validation, P.C. and N.S.; formal analysis, S.C. and J.Y.; investigation, P.C., S.C., J.Y., and P.B.; writing—original draft preparation, S.C.; writing—reviewing and editing, P.C.; visualization, P.C. and S.C.; supervision, P.C.; project administration, P.C.; funding acquisition, P.C. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Chulalongkorn University-Veterinary Science Research Fund (Grant No. 4/2561) and the Chulalongkorn Academic Advancement into Its 2nd Century Project from Chulalongkorn University.

Institutional Review Board Statement

This study was conducted according to the faculty regulations and approved by the Chulalongkorn University-Faculty of Veterinary Science Biosafety Committee (CU-VET-BC) (Protocol code IBC1831006) on 5 April 2018.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We gratefully acknowledge Akinyemi Fasemore from the University of Wuerzburg, Germany, for assigning a new allelic number and sequence type of Staphylococcus aureus. Surawit Chueahiran received the 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship, Graduate School, Chulalongkorn University.

Conflicts of Interest

The authors declare no conflict of interest.

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Antibiotics 10 00243 g001 550
Figure 1. Genetic relatedness and antimicrobial resistance profile of canine and feline methicillin-resistant Staphylococcus aureus. PEN, penicillin; FOX, cefoxitin; TET, tetracycline; GEN, gentamicin; KAN, kanamycin; STR streptomycin; ERY, erythromycin; CLI, clindamycin; TIA, tiamulin; SMX, sulfamethoxazole; TMP, trimethoprim; CIP, ciprofloxacin; CHL, chloramphenicol; MUP, mupirocin.
Figure 1. Genetic relatedness and antimicrobial resistance profile of canine and feline methicillin-resistant Staphylococcus aureus. PEN, penicillin; FOX, cefoxitin; TET, tetracycline; GEN, gentamicin; KAN, kanamycin; STR streptomycin; ERY, erythromycin; CLI, clindamycin; TIA, tiamulin; SMX, sulfamethoxazole; TMP, trimethoprim; CIP, ciprofloxacin; CHL, chloramphenicol; MUP, mupirocin.
Antibiotics 10 00243 g001
Table
Table 1. Isolates of methicillin-resistant Staphylococcus aureus clonal complex 398 in each minimum inhibitory concentrations of antimicrobials and with antimicrobial resistance genes which corresponded to resistance phenotypes.
Table 1. Isolates of methicillin-resistant Staphylococcus aureus clonal complex 398 in each minimum inhibitory concentrations of antimicrobials and with antimicrobial resistance genes which corresponded to resistance phenotypes.
Antimicrobials *Resistance Break Point
(μg/mL)		Range
Tested
(μg/mL)		Number of Isolates in Each MIC (μg/mL) **Number of Resistant IsolatesResistance
Gene(s) ***		Number of
Isolates with
the Presence of
Resistance Genes ***
≤0.0150.0030.0060.0120.250.51248163264128256512
PEN≥0.250.0012–2 2
(=)		18 (>) 20/20blaZ; mecA18; 20
FOX≥10.5–16 3
(=)		6
(=)		11 (>) 20/20mecA20
TET≥10.5–16 20 (>) 20/20tet(M); tet(L); tet(K)20; 16; 0
GEN≥161–16 2
(≤)		 3
(=)		8
(=)		7
(>)		 15/20aac(6′)-Ie-aph(2′)-Ia18
KAN≥164–64 2 (≤) 9
(=);
9
(>)		 18/20aac(6′)-Ie-aph(2′)-Ia; ant(4′)-Ia18; 17
STR≥164–32 1 (≤)3
(=)		 16 (>) 16/20ant(4′)-Ia; ant(6)-Ia17; 16
ERY≥80.25–8 4
(≤)		10 (=)1
(=)		1
(=)		 4
(>)		 4/20erm(B); erm(A); erm(C)4; 3; 0
CLI≥40.12–4 20 (>) 20/20lnu(B); erm(B); erm(A); erm(C)16; 4; 3; 0
CHL≥324–64 6
(≤)		5
(=)		4
(=)		 5
(=)		 5/20fexA; catpC2215; 0
SMX≥51264–512 6
(≤)		5 (=) 7
(>); 2
(=)		9/20NDND
TMP≥162–32 1
(≤)		 19 (>) 19/20dfrA; dfrG17; 4
MUP≥5120.5–2 & 256 16
(≤)		 4
(>)		 4/20ileS24
VAN≥161–16 19 (≤)2
(=)		 0/20ND0
CIP≥40.25–8 1
(=);
19 (>)		 20/20NDND
RIF≥40.015–0.519 (≤) 1
(=)		 0/20NDND
LZD≥81–8 1
(≤)		5 (=)13 (=)1
(=)		 0/20NDND
FUS≥20.5–4 20 (≤) 0/20NDND
TIA>40.5–4 2
(≤)		2
(=)		 16 (>) 16/20lsa(E)16
* Abbreviation of antimicrobials: PEN, penicillin; FOX, cefoxitin; TET, tetracycline; GEN, gentamicin; KAN, kanamycin; STR, streptomycin; ERY, erythromycin; CLI, clindamycin; CHL, chloramphenicol; SMX, sulfamethoxazole; TMP, trimethoprim; MUP, mupirocin; VAN, vancomycin; CIP, ciprofloxacin; RIF, rifampicin; LZD, linezolid; FUS, fusidic acid; TIA, tiamulin. ** < in parentheses, less than or equal to the MIC; = in parentheses, equal to the MIC; > in parentheses, more than the MIC; Dark gray shade, level of concentration more than interpretive breakpoints referring resistance; light gray shade, level of concentration of intermediate resistance. *** ND, not determined.
Table
Table 2. Characteristics and antimicrobial resistance gene profiles of 20 methicillin-resistant Staphylococcus aureus clonal complex 398 isolates, including sequence type (ST) 398, ST5926 and ST6563, from canine and feline clinical samples, during 2017–2020.
Table 2. Characteristics and antimicrobial resistance gene profiles of 20 methicillin-resistant Staphylococcus aureus clonal complex 398 isolates, including sequence type (ST) 398, ST5926 and ST6563, from canine and feline clinical samples, during 2017–2020.
Sequence TypeSCCmecspa TypePulsotypeNo.Year (No.)Host (No.)Antimicrobial Resistance Genes *
mecAblaZtet(L)tet(M)ant(4′)-Iaant(6)-Iaaac(6′)-Ie-aph(2)-IadfrAdfrGerm(A)erm(B)lnu(B)lsa(E)spwfexAileS2
398Vt034B42017 (3)
2020 (1)		Dog (3)
Cat (1)
398Vt034B22017 (2)Dog (2)
398Vt4652B12017 (1)Dog (1)
398Vt034C12017 (1)Dog (1)
398Vt034A22018 (2)Cat (2)
398Vt034A12018 (1)Dog (1)
398Vt034A12019 (1)Dog (1)
398Vt034A22019 (2)Dog (2)
5926Vt1255A12018 (1)Dog (1)
6563Vt1255A12020 (1)Dog (1)
398Compositet034A12020 (1)Dog (1)
398Composite-A12019 (1)Cat (1)
398Composite-A12018 (1)Cat (1)
398Composite-A12019 (1)Dog (1)
* gray shade, positive.
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Chueahiran, S.; Yindee, J.; Boonkham, P.; Suanpairintr, N.; Chanchaithong, P. Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 as a Major MRSA Lineage in Dogs and Cats in Thailand. Antibiotics 2021, 10, 243. https://doi.org/10.3390/antibiotics10030243

AMA Style

Chueahiran S, Yindee J, Boonkham P, Suanpairintr N, Chanchaithong P. Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 as a Major MRSA Lineage in Dogs and Cats in Thailand. Antibiotics. 2021; 10(3):243. https://doi.org/10.3390/antibiotics10030243

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Chueahiran, Surawit, Jitrapa Yindee, Pongthai Boonkham, Nipattra Suanpairintr, and Pattrarat Chanchaithong. 2021. "Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 as a Major MRSA Lineage in Dogs and Cats in Thailand" Antibiotics 10, no. 3: 243. https://doi.org/10.3390/antibiotics10030243

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