Comparative Susceptibility of Pathogenic Methicillin-Resistant and Methicillin-Susceptible Staphylococcus pseudintermedius to Empirical Cotrimazole for Canine Pyoderma

The prevalence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) that causes pyoderma has been gradually shifting, according to many surveillance studies, with annual changes. The empirical cotrimazole regimen remains interesting, but research on cotrimazole susceptibility to MRSP is limited. The objective of this study was to evaluate the susceptibility of cotrimazole to canine pyoderma MRSP isolates. Sixty isolates of S. pseudintermedius were identified as 16 MRSP and 44 methicillin-susceptible S. pseudintermedius (MSSP) using an oxacillin disk diffusion test and VITEK 2 system with VITEK GP card. Using the VITEK 2 system with a VITEK AST-GP81 card, the susceptibility rates of MRSP (15.00%) and MSSP (35.00%) to cotrimazole was observed. The median MIC of cotrimazole on MSSP (median, ≤10; IQR, 10–320) was lower than that of MRSP (median, ≥320; IQR, 10–320) (p = 0.5889, Mann-Whitney test). Percent attainment of PK/PD targets in MRSP (q 12 h, 43.75; q 8 h, 43.75) were lower than that of MSSP (q 12 h, 52.27; q 8 h, 52.27) (p = 0.7710). These findings show the moderately phenotypic cotrimazole susceptibilities of both MRSP and MSSP. Further study is required to develop clinical trials examining the use of cotrimazole in dogs with pyoderma.


Introduction
Canine pyoderma remains one of the most frequently diagnosed skin infections in dogs [1]. An opportunistic gram-positive Staphylococci is thought to be the primary cause of skin infections in companion animals [2]. Staphylococcus pseudintermedius represents the most prevalent cause of canine pyoderma [3]. When infections are not appropriately treated, S. pseudintermedius might become resistant [4,5]. Consequently, several staphylococci from dermatological canine patients frequently exhibit methicillin resistance [6][7][8]. The majority of methicillin resistant isolates develop multidrug resistance (MDR), which is defined as resistance to at least three antimicrobial classes, and severely restricts the therapeutic options Medicine, Chiang Mai University, Thailand between January to December 2022. The research study was ethically approved by the Animal Care and Use Committee, Faculty of Veterinary Medicine, Chiang Mai University (approval number: S3/2563). The Institute Biosafety Committee, Chiang Mai University, also granted permission to test the pathogens (approval number: CMUIBC A-0763011).

Primary Identification of Coagulase-Positive Staphylococci
All bacterial samples were cultivated using Tryptic soy agar plates (Merck, Darmstadt, Germany) with 5% defibrinated sheep blood, and aerobically incubated at 37 • C for 24 h. The development of presumed staphylococcal colonies was determined by morphological properties including hemolytic characteristics, pigmentation, and typical colony shape. The suspected colonies were sub-cultured onto Mannitol salt agar (Oxoid Ltd., Basingstoke, UK) and incubated aerobically at 37 • C for 24 h. The purified colonies were morphologically identified by gram staining, catalase, oxidase, clumping factor, and tube coagulase tests.

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing (AST) was performed by the Kirby-Bauer disk diffusion method according to The Clinical and Laboratory Standards Institute (CLSI) standards. The coagulase-positive staphylococcal isolates were inoculated into Bacto™ Todd-Hewitt broth (THB) (Becton Dickinson & Co., Sparks, MD, USA) and incubated aerobically at 37 • C for 24 h. Bacterial culture was adjusted to a concentration of 1.5 × 10 8 colony-forming units/mL using a McFarland densitometer (Grant Instruments, Cambridgeshire, UK) and swabbed on BBL™ Mueller-Hinton agar plates (Becton Dickinson & Co., Sparks, MD, USA). All isolates were subjected to the AST by the agar diffusion method with two antimicrobial disks including 1 µg oxacillin (OX) and 23.75 µg sulfamethoxazole-1.25 µg trimethoprim. After 24 h incubation at 30 • C, the size of the bacterial growth inhibition zone dimeters (ZD) was interpreted as sensitive (S) (≥16 mm) and resistant (R), including intermediate resistant (11-15 mm) and resistant (≤10 mm), according to the antimicrobial breakpoints for Staphylococci detailed in the CLSI guidelines.

Confirmation of Staphylococcus pseudintermedius
The presumptive coagulase-positive staphylococcal isolates were then subjected to identification using the VITEK 2 Compact instrument (Biomerieux, Marcy l'Etoile, France) according to the manufacturer's instructions. The disposable VITEK GP cards were used to identify S. pseudintermedius. The GP identity card is a completely closed system, requiring minimal additional reagents. The card was loaded onto a cassette made specifically for the VITEK 2 system, placed inside the apparatus, automatically placed in a vacuum, protected from the elements, and automatically subjected to colorimetric measurement every 15 min for a maximal incubating period of 8 h. The VITEK 2 database version 9.02 was applied. This database enables bacterial identification in a kinetic mode commencing 180 min after the initial beginning of the incubation process.

Determination of Minimum Inhibitory Concentration Values
The presumptive isolates of S. pseudintermedius were also subsequently investigated for antimicrobial susceptibility using the VITEK 2 Compact apparatus. The cefoxitin testing for MRSP confirmation and the MICs of cotrimazole among MRSP and MSSP were achieved by using a single disposable VITEK AST-GP81 card per isolate. The AST-GP81 card was automatically inoculated with a bacterial suspension generated in 0.45% sodium chloride solution at a spectrophotometric turbidity of 0.5. Inoculum preparation and card filling were usually separated by less than 30 min and put into the Vitek-2 apparatus for incubation and reading. The Advanced Expert System was used to provide categorical interpretations of the Vitek-2 AST data.

Target Attainment of Pharmacokinetic and Pharmacodynamics
The pharmacokinetic profile of oral cotrimazole at a dosage of 30 mg/kg for once-daily administration in dogs was utilized [28]. Plasma concentration of cotrimazole above the MIC throughout the whole dosing interval (T 0-24 > MIC) was performed for achieving a specific PK/PD criterion. Successful target attainment in more than half of the total dosing intervals qualifies as acceptable overall for reaching the criterion. Probability of target attainment analysis is used to optimize dosing regimens of cotrimazole for patient dogs infected with MRSP and MSSP.

Statistical Analysis
Descriptive statistics were used to describe data, including frequency, percentage, proportion, range, average with standard deviation, and median with interquartile range to express the basic information on the tested bacteria. The distribution of ZD and MIC values of cotrimazole against bacterial isolates was plotted. The normality of the continuous variable distribution was examined using the Shipiro-Wilk test and normal Q-Q plots. The Mann-Whitney U test was used to assess differences between two independent groups when the dependent variable is ordinal or continuous but not normally distributed. In order to fit the data in the contingency table, the agreement of ZD results of tested isolates against oxacillin and cotrimazole were compared by Cohen's kappa statistic. The oxacillin and cotrimazole susceptibility were also compared using odd ratios (ORs). Cohen's kappa and OR statistics were also used to compare the MIC values of tested isolates against cefoxitin and cotrimazole. The median and 90th percentile were also calculated by regression equations. The association between two continuous variables of antimicrobial ZD and MIC values was assessed using Pearson's correlation coefficients. Percentage probabilities of PK/PD target attainment of cotrimazole in the MRSP and MSSP groups were calculated by time-above-MIC targets. Chi-square tests were used to determine the association between two categorical variables and the difference in proportions. The differences between variables were considered statistically significant when the bicaudal probability was lower than 5% (p < 0.05) due to chance (error type I). Statistical analysis was performed with R statistical software (RStudio, Boston, MA, USA).

Cotrimazole Susceptibility among Coagulase-Positive Staphylococci
Sixty sequential isolates from patient dogs with pyoderma were identified as coagulasepositive staphylococci (CPS) by the conventional method according to CLSI guidelines. Data from the general comparison of the disk diffusion testing results for oxacillin and cotrimazole against all canine pyoderma CPS were demonstrated in Table 1. Among the tested isolates, the percentage of susceptibility to antimicrobials was as follows: 65.00% to oxacillin and 38.33% to cotrimazole [10.00% in oxacillin-resistant isolates (OX-R CPS) and 28.33% in oxacillin-susceptible isolates (OX-S CPS)]. Moreover, the cotrimazole susceptibility was related with a significant increase in chances for OX-S CPS isolates [odd ratio (OR), 1.90; 95% confidence interval (CI), 0.65-6.47] compared to OX-R CPS isolates (p = 0.2538). However, slight agreement between both antimicrobial agents was detected in 32 isolates (53.33% of the observations) with kappa coefficients 0.13 [95%CI, −0.09-0.34; standard error (SE) of kappa 0.11]. By the point of interception, the distribution of cotrimazole ZD on CPS isolates was described in Table 2 and Figure 1a. The ZD range (0-28 mm) and mean ± SD (10.15 ± 11.13) of cotrimazole among total CPS isolates was found. Interestingly, the median ZD of cotrimazole on OX-R CPS [median, 0; interquartile range (IQR), 0-19] was narrower than that of OX-S CPS (median, 15; IQR, 0-24) (p = 0.1631, Mann-Whitney test). Moreover, both OX-R CPS and OX-S CPS groups exhibited susceptibility to cotrimazole; they were 28.57% and 56.41, respectively (Figure 1b).

Cotrimazole Susceptibility among Pathogenic S. pseudintermedius
Sixty presumptive CPS isolates from dogs with pyoderma were confirmed as S. pseudintermedius by the VITEK 2 Compact instrument with VITEK GP cards. For cefoxitin

Cotrimazole Susceptibility among Pathogenic S. pseudintermedius
Sixty presumptive CPS isolates from dogs with pyoderma were confirmed as S. pseudintermedius by the VITEK 2 Compact instrument with VITEK GP cards. For cefoxitin testing, sixteen MRSP isolates were confirmed, whereas the remaining 44 MSSP isolates were identified. The MICs of cotrimazole in MRSP and MSSP were also achieved by using the VITEK AST-GP81 cards. The MICs and ZDs of oxacillin against tested S. pseudintermedius were well-correlated (correlation coefficient −0.9622, p < 0.01). The significant negative association for the MICs and ZDs of cotrimazole on S. pseudintermedius with correlation coefficient −0.9193 was also observed (p < 0.01) (Figure 2). testing, sixteen MRSP isolates were confirmed, whereas the remaining 44 MSSP isolates were identified. The MICs of cotrimazole in MRSP and MSSP were also achieved by using the VITEK AST-GP81 cards. The MICs and ZDs of oxacillin against tested S. pseudintermedius were well-correlated (correlation coefficient −0.9622, p < 0.01). The significant negative association for the MICs and ZDs of cotrimazole on S. pseudintermedius with correlation coefficient −0.9193 was also observed (p < 0.01) (Figure 2). The general information gleaned from the comparison of the MIC values for oxacillin and cotrimazole against sixty canine pyoderma S. pseudintermedius isolates was demonstrated in Table 3. The proportion of antimicrobial susceptibility among the tested isolates was 73.33% to cefoxitin surrogate marker and 50.00% to cotrimazole (15.00% in MRSP and 35.00% in MSSP). Moreover, the cotrimazole susceptibility showed a significant increase in chances for MSSP isolates (OR, 0.71; 95%CI, 0.23-2.40) compared to MRSP isolates (p = 0.5593). Interestingly, the susceptible rate of cotrimazole in the MRSP group was 56.25% (Figure 3b). In addition, the fair agreement between both antimicrobial agents was 28 isolates (46.67% of the observations) with kappa coefficients −0.067 (95%CI, −0.290-0.157; SE of kappa 0.11). The distribution of cotrimazole MIC on S. pseudintermedius isolates was described in Table 4 and Figure 3a. The median MIC of cotrimazole on MSSP (median, ≤10; IQR, 10-320) was lower than that of MRSP (median, ≥320; IQR, 10-320) (p = 0.5889, Mann-Whitney test). Moreover, the MIC90: MIC50 ratio of cotrimazole on MSSP (320/10) was also significantly lower than that of MRSP (320/320).  The general information gleaned from the comparison of the MIC values for oxacillin and cotrimazole against sixty canine pyoderma S. pseudintermedius isolates was demonstrated in Table 3. The proportion of antimicrobial susceptibility among the tested isolates was 73.33% to cefoxitin surrogate marker and 50.00% to cotrimazole (15.00% in MRSP and 35.00% in MSSP). Moreover, the cotrimazole susceptibility showed a significant increase in chances for MSSP isolates (OR, 0.71; 95%CI, 0.23-2.40) compared to MRSP isolates (p = 0.5593). Interestingly, the susceptible rate of cotrimazole in the MRSP group was 56.25% (Figure 3b). In addition, the fair agreement between both antimicrobial agents was 28 isolates (46.67% of the observations) with kappa coefficients −0.067 (95%CI, −0.290-0.157; SE of kappa 0.11). The distribution of cotrimazole MIC on S. pseudintermedius isolates was described in Table 4 and Figure 3a. The median MIC of cotrimazole on MSSP (median, ≤10; IQR, 10-320) was lower than that of MRSP (median, ≥320; IQR, 10-320) (p = 0.5889, Mann-Whitney test). Moreover, the MIC 90 : MIC 50 ratio of cotrimazole on MSSP (320/10) was also significantly lower than that of MRSP (320/320).

PK-PD Target Achieving of Cotrimazole on Pathogenic S. pseudintermedius
Cotrimazole achievement rates relating to S. pseudintermedius were evaluated based on the time the free drug stayed over the plasma MIC between treatment intervals ( Table  5). The PK parameters of cotrimazole in dogs orally administered (

PK-PD Target Achieving of Cotrimazole on Pathogenic S. pseudintermedius
Cotrimazole achievement rates relating to S. pseudintermedius were evaluated based on the time the free drug stayed over the plasma MIC between treatment intervals ( Table 5)

Discussion
The genus Staphylococcus is common in epidermal and nasal flora and leads to opportunity-based infections in both humans and animals. When compared to coagulasenegative staphylococci, CPS may result in more severe infections [29,30]. A great many prior investigations have demonstrated that S. pseudintermedius is a prevalent cause of canine pyoderma [29,31,32]. In a recent investigation, all examined isolates were conventionally identified as CPS. All CPS-tested isolates were indeed subsequently confirmed as S. pseudintermedius using the VITEK 2 Compact instrument and VITEK GP cards. This actively demonstrates that all pathogenic bacteria from clinical specimens of dogs with pyoderma were consistent with the previous studies. The range of concentrations deter-Life 2023, 13, 1210 8 of 12 mined by the Vitek-2 AST-GP card is in accordance with veterinary breakpoints established according to the current CLSI guidelines for the oxacillin utilized in this investigation [33].
The particular reason for using the VITEK 2 GP test technique was validated in accordance with the Association of Official Analytical Collaboration (AOAC) recommendations for the identification of Staphylococcus species usingthe Performance Tested Method SM (PTM) and Official Methods of Analysis SM (OMA). Furthermore, a previous research investigation indicated that the Vitek 2 Compact instrument accurately identifies 123 (93.2%; n = 132) different Staphylococcus strains from the database species. In addition, the research points out the remarkable accomplishment of the colorimetric Vitek 2 GP card, which is capable of being utilized in medical, veterinary, agricultural, and food laboratories [34]. As a consequence of this, previous research has recommended that the VITEK 2 GP test card method be used as the Official First Action for the detection of certain Gram-positive bacteria [35]. Moreover, the accuracy of VITEK-2 for phenotypic identification of Staphylococci is higher than that of the MicroScan and Crystal GP systems [36]. Through the use of molecular techniques, genotypic bacterial species might be reliably detected. Unfortunately, 16s rDNA sequencing did not confirm all bacterial isolates in this study.
The susceptibility of all CPS Isolates to cotrimazole was determined by AST using an OX disk. In accordance with EUCAST recommendations, oxacillin has been used as a surrogate for methicillin-resistant AST. The oxacillin disk (1 g) accurately predicted mecA-mediated methicillin resistance in S. pseudintermedius. [37,38]. Likewise, this study revealed that one-third of tested CPS (21 isolates) were OX-R CPS. Furthermore, 16 MRSP isolates were identified by the cefoxitin testing on OX-R CPS isolates using VITEK AST-GP81 cards. Previous investigations have demonstrated the correlation of cefoxitin and oxacillin ZD and MIC values with mecA/mecC PCR and the diagnostic test accuracy of disk diffusion relative to broth microdilution for canine clinical S. pseudintermedius [39]. Moreover, the Vitek 2 automated system also provides a highly reliable approach for detecting Staphylococcal mecA and mecC positive isolates [40][41][42]. This actively demonstrates that the five remaining isolates that showed oxacillin resistance but not the cefoxitin resistance phenotype, were classified as MSSP. However, 1.1% of methicillin-resistant Staphylococci probably remain sensitive to cefoxitin, according to a prior study [43].
In this study, the cotrimazole susceptibility among the tested S. pseudintermedius was 50.00% with the 50th percentile of the MIC less than 10 mg/L. The finding is consistent with previous research on the susceptibility of S. pseudintermedius isolated from infected dogs to cotrimazole [44][45][46]. Moreover, many previous studies demonstrated that the median MIC was less than 10 mg/L [47][48][49]. Surprisingly, this research also demonstrated that the cotrimazole sensitivity of the OX-R CPS and MRSP groups was 28.57% and 56.25%, respectively. These findings differed from the previous study in that MRSP was completely resistant to cotrimazole [50]. However, the MIC 50 /MIC 90 ratio of cotrimazole on MSSP was much lower than that of MRSP. In addition, cotrimazole demonstrated a low MIC range for MRSP, and both MIC 90 and MIC 50 of MRSP were above the break point. These results suggest that MRSP developed resistance to cotrimazole at a higher rate than MSSP. We are aware of some limitations in our study. MIC values were determined using automated testing procedures rather than broth microdilution. The methicillin-resistance of bacterial strains is caused by the mec gene, which encodes a second penicillin binding protein with a low affinity for all β-lactam antibiotics [51]. Consequently, a group of antimetabolite sulfonamide has been less affected by mec gene mutation. However, Staphylococcus isolates may mutate a DfrB gene causing bacteria resistance if they are exposed to cotrimazole for a duration longer than two weeks [52].
Disk diffusion and broth microdilution methods have been approved as AST reference procedures by the CLSI and the EUCAST. The automated approaches are preferred to manual labor methods. The Vitek-2 system has been attached to the Advanced Expert System, which was developed to evaluate Vitek-2 results on the basis of specific bacterial species. AST reliability of Vitek-2 has been validated for numerous bacterial species of Staphylococci and Enterococci [53]. However, when testing antimicrobial agents against particular Life 2023, 13, 1210 9 of 12 staphylococci, Vitek-2 might provide the results that are not accurate [54]. However, this present study revealed a strong correlation between ZD and MIC values of both oxacillin and cotrimazole against S. pseudintermedius; hence, ZD might be used to predict MIC values. These findings are in agreement with previously published research that reported almost perfect agreement between disk diffusion to Vitek-2 of oxacillin and cotrimazole against S. pseudintermedius isolates collected in clinical microbiology laboratories in Italy during 2018 to 2020 [55].
For surface and superficial conditions, topical therapy should be the only on-animal antimicrobial treatment administered. However, systemic treatment is suggested for deep pyoderma, multifocal or severe superficial infections in dogs that are not susceptible to topical therapy. This recommendation, which was reached through consensus among the Antimicrobial Guidelines Working Group of the International Society for Companion Animal Infectious Disease (ISCAID), focuses on the treatment of superficial bacterial folliculitis in particular and classifies antimicrobial agents into first-and second-tier categories [56]. In addition, the MIC value is necessary for antimicrobial treatment regimens that predict outcomes. Interestingly, this PK/PD target of cotrimazole might be reached with approximately 50% of both MRSP and MSSP. However, the pharmacodynamic parameter for optimizing the dose of cotrimazole is limited [57]. Cotrimazole has time-dependent bacteriostatic activity and the potential for concentration-dependent bactericidal activity against particular species [58]. The pharmacokinetic of cotrimazole in dogs has been also infrequently documented. As a result, achievement rates of cotrimazole to S. pseudintermedius were estimated based on the duration that the free drug remained over the plasma MIC between dosing intervals. The effectiveness of systemic antimicrobial medication for both MRSP and MSSP infections is primarily influenced by bacterial susceptibility. Moreover, it also depends on other criteria of rational drug use, including drug administration, correct dosing, and dog's owner compliance [59]. Systemic therapy combined with topical antibiotic treatment is advised wherever possible to minimize contamination of the environmental microorganism and the likelihood of spreading the infection to other hosts, as well as to potentially decrease the total duration of systemic prescription.
Multi-center surveillance studies showed progressive shifts with annual trends of antimicrobial susceptibility [60]. The development of evidence-based research in veterinary medicine is essential since occurrences of pyoderma-causing MRSP have been reported [61,62]. Empirical antimicrobial agent selection is no longer reliable for pyoderma therapy in locations with high MRSP prevalence. The empirical amoxicillin-clavulanate and first-generation cephalosporins have a poor effectiveness for superficial bacterial folliculitis in dogs. Therefore, the treatment of canine pyoderma is becoming more challenging due to bacteria like MRSP. The outcomes showed that the remaining empirical cotrimazole regimen is an attractive alternative for the MRSP canine pyoderma. Future susceptibility tests should focus on S. pseudintermedius isolated from different geographic regions, time dynamics, and with an increased variety of antimicrobials susceptibility patterns.

Conclusions
The results highlighted the first study on cotrimazole susceptibility and PK/PD target attainment in pathogenic S. pseudintermedius isolated from dogs with pyoderma. Through this research, different levels of phenotypic cotrimazole susceptibility of MRSP and MSSP was observed. Interestingly, when given cotrimazole at at least a 12-h dosing interval, the attainment rate of pathogenic MRSP was more than 50%. Further studies are necessary to expand the clinical trial of cotrimazole in dogs with pyoderma.