Evolution of the Prevalence of Antibiotic Resistance to Staphylococcus spp. Isolated from Horses in Florida over a 10-Year Period

Simple Summary The purpose of this study was to determine how common antibiotic-resistant infections are in horses, particularly Staphylococcus species. These are bacteria that are normally found on the skin of horses. Overgrowth of these bacteria can lead to infection. In recent years, the emergence of resistance to antibiotics to treat these infections has been shown with these bacteria in humans and dogs. Determining how widespread Staphylococcal resistant bacteria are in horses helps to educate the veterinary profession on potential changes to horse resistance. This can help guide appropriate antibiotic usage as well as prove the need for innovative treatment options for both veterinary and human medicine. This study found increasing resistance in a class of antibiotics in the population observed at our institution. In addition, the species of Staphylococcal bacteria affects the resistance. Larger studies with more horses are needed to evaluate the clinical usefulness of these results. Abstract Previous studies documented antibiotic resistance in horses but did not focus on skin specifically. We investigated antibiotic resistance and correlations between resistance patterns in skin infections. Records from 2009 to 2019 were searched for Staphylococcal infection and susceptibility results. Seventy-seven cases were included. Organisms identified were S. aureus (48/77), S. pseudintermedius (7/77), non-hemolytic Staphylococcus (8/77), beta-hemolytic Staphylococcus (6/77), and other species (8/77). Samples included pyoderma (36/77), wounds (10/77), abscesses (15/77), incision sites (5/77), nose (8/77), and foot (3/77). A trend analysis using non-parametric Spearman’s test showed significant upward trend of resistance (p < 0.05) for 3/15 antibiotics (ampicillin, cefazolin, penicillin). Susceptibility was significantly different by Staphylococcal species for 8/15 antibiotics. Gentamicin showed significant susceptibility differences based on source (all abscesses were susceptible to gentamicin). Steel-Dwass test showed statistically significant (p = 0.003) difference between incision sites and abscesses. A non-parametric Kendall’s T-test found significantly negative correlation between cefazolin and amikacin sensitivity (p = 0.0108) and multiple positive correlations of resistance (p < 0.05). This study confirms increasing resistance in dermatologic samples. It is unlikely that the sample source affects resistance, but Staphylococcus species may affect it. Study limitations include lack of information about previous antibiotic use and small sample size.


Introduction
Cutaneous bacterial infections represent a frequent, widespread challenge in equine medicine with allergic horses being at increased risk for recurrent Staphylococcal infections. Empiric and prophylactic use of antibiotics is widely practiced to treat these infections [1], and their overuse is threatening to diminish their value as therapeutic agents. With difficulty determining and/or controlling the underlying causes of the infections, antibiotics are frequently the mainstay of treatment.
Of the 77 samples, the majority came from pyoderma lesions (36/77; 46.8%). The other locations of sample selection included superficial wounds (10/77; 13%), abscesses (15/77; 19.5%), surgical incision sites (5/77; 6.5%), the nose (8/77; 10.4%), and the foot (3/77; 3.9%). Using the Kruskal-Wallis rank sum test, sites of sample collection were compared to each antibiotic susceptibility. Gentamicin was the only antibiotic that showed a significant difference in susceptibility based on the sample collection site. Therefore, data points for gentamicin were analyzed using the Steel-Dwass method to determine which sample type contained Staphylococcus that were more resistant or susceptible. It was found that all 15 Staphylococcus spp. isolated from abscesses were susceptible to gentamicin. However, 70% of Staphylococcus spp. isolated from surgical incision sites, 33.3% isolated from the foot, 30.5% isolated from pyoderma lesions, 30% isolated from superficial wounds, Vet. Sci. 2023, 10, 71 4 of 11 and 12.5% isolated from the nose were resistant to gentamicin. Statistical significance was achieved (p = 0.003) in the Steel-Dwass test when comparing surgical incision site to abscesses ( Table 1). The comparison of pyoderma lesions to abscesses was not statistically significant (p = 0.1225). To investigate an association between year of sample isolation and resistance patterns, a trend analysis was completed using a nonparametric Spearman's test. The results indicated that 3/15 antibiotics tested over the 10-year period showed a significant (p = 0.05) upward trend of resistance (Table 2).
Using the Kruskal-Wallis rank sum test, Staphylococcal isolate groups were compared to each antibiotic susceptibility. Results of 8/15 antibiotics tested showed a significant difference in susceptibility based on the isolate group. These eight antibiotics were then analyzed with the Steel-Dwass test to determine which organisms demonstrated greater resistance and which organisms followed similar resistance patterns for each antibiotic. Ampicillin, azithromycin, cefazolin, clarithromycin, erythromycin, imipenem, oxacillin and penicillin all had at least one Staphylococcal isolate more likely to be resistant than the others. The MG consisting of S. hyicus, S. xylosus, S. epidermidis, S. schleiferi, and isolates reported as Staphylococcus spp. showed statistical significance that they were more likely to be resistant to ampicillin, azithromycin, cefazolin, erythromycin, imipenem, oxacillin, and penicillin than S. aureus. The S. pseudintermedius group showed equal likelihood to be resistant to ampicillin and penicillin as S. aureus. The non-hemolytic Staphylococcus group was more likely to be resistant to azithromycin, clarithromycin, erythromycin, and oxacillin than the S. aureus group. The MG was more likely to be resistant to cefazolin than the beta-hemolytic Staphylococcus group. There were also numerous organism groups that showed very similar resistance prevalence to one another for each antibiotic. These similarities can be seen in Table 3 along with the statistics for the aforementioned relationships. The organism Vet. Sci. 2023, 10, 71 5 of 11 groups with a p-value close to one demonstrate a similar likelihood to be resistant to that particular antibiotic. Number and percentage of susceptible Staphylococcal species for each sample collection site examined during the time period 1 January 2009-1 October 2019 at the author's institution. Gentamicin was the only antibiotic showing statistical significance based on sample collection site. This occurred between abscesses and surgical incision sites and the percent susceptible for each site are highlighted in yellow. TMS, trimethoprim/sulfonamide.
To investigate an association between year of sample isolation and resistance patterns, a trend analysis was completed using a nonparametric Spearman's test. The results indicated that 3/15 antibiotics tested over the 10-year period showed a significant (p = 0.05) upward trend of resistance (Table 2). To investigate an association between year of sample isolation and resistance patterns, a trend analysis was completed using a nonparametric Spearman's test. The results indicated that 3/15 antibiotics tested over the 10-year period showed a significant (p = 0.05) upward trend of resistance (Table 2). To investigate an association between year of sample isolation and resistance patterns, a trend analysis was completed using a nonparametric Spearman's test. The results indicated that 3/15 antibiotics tested over the 10-year period showed a significant (p = 0.05) upward trend of resistance (Table 2). To investigate an association between year of sample isolation and resistance patterns, a trend analysis was completed using a nonparametric Spearman's test. The results indicated that 3/15 antibiotics tested over the 10-year period showed a significant (p = 0.05) upward trend of resistance (Table 2). To investigate an association between year of sample isolation and resistance patterns, a trend analysis was completed using a nonparametric Spearman's test. The results indicated that 3/15 antibiotics tested over the 10-year period showed a significant (p = 0.05) upward trend of resistance (Table 2). To investigate an association between year of sample isolation and resistance patterns, a trend analysis was completed using a nonparametric Spearman's test. The results indicated that 3/15 antibiotics tested over the 10-year period showed a significant (p = 0.05) upward trend of resistance (Table 2). Test results for association between date of sample collection and resistance for each antibiotic. Antibiotics highlighted in yellow show an upward trend over the last 10 years. The p values highlighted in red illustrate the significant trends. The difference plots show a measure of strength and direction of association using the grey bars in the far-right column.
Using the Kruskal-Wallis rank sum test, Staphylococcal isolate groups were compared to each antibiotic susceptibility. Results of 8/15 antibiotics tested showed a significant difference in susceptibility based on the isolate group. These eight antibiotics were then analyzed with the Steel-Dwass test to determine which organisms demonstrated greater resistance and which organisms followed similar resistance patterns for each antibiotic. Ampicillin, azithromycin, cefazolin, clarithromycin, erythromycin, imipenem, oxacillin and penicillin all had at least one Staphylococcal isolate more likely to be resistant than the others. The MG consisting of S. hyicus, S. xylosus, S. epidermidis, S. schleiferi, and isolates reported as Staphylococcus spp. showed statistical significance that they were more likely to be resistant to ampicillin, azithromycin, cefazolin, erythromycin, imipenem, oxacillin, and penicillin than S. aureus. The S. pseudintermedius group showed equal likelihood to be resistant to ampicillin and penicillin as S. aureus. The non-hemolytic Staphylococcus group was more likely to be resistant to azithromycin, clarithromycin, erythromycin, and oxacillin than the S. aureus group. The MG was more likely to be resistant to cefazolin than the beta-hemolytic Staphylococcus group. There were also numerous organism groups that showed very similar resistance prevalence to one another for each antibiotic. These similarities can be seen in Table 3 along with the statistics for the aforementioned relationships. The organism groups with a p-value close to one demonstrate a similar likelihood to be resistant to that particular antibiotic. Test results for association between date of sample collection and resistance for each antibiotic. Antibiotics highlighted in yellow show an upward trend over the last 10 years. The p values highlighted in red illustrate the significant trends. The difference plots show a measure of strength and direction of association using the grey bars in the far-right column.
Using the Kruskal-Wallis rank sum test, Staphylococcal isolate groups were compared to each antibiotic susceptibility. Results of 8/15 antibiotics tested showed a significant difference in susceptibility based on the isolate group. These eight antibiotics were then analyzed with the Steel-Dwass test to determine which organisms demonstrated greater resistance and which organisms followed similar resistance patterns for each antibiotic. Ampicillin, azithromycin, cefazolin, clarithromycin, erythromycin, imipenem, oxacillin and penicillin all had at least one Staphylococcal isolate more likely to be resistant than the others. The MG consisting of S. hyicus, S. xylosus, S. epidermidis, S. schleiferi, and isolates reported as Staphylococcus spp. showed statistical significance that they were more likely to be resistant to ampicillin, azithromycin, cefazolin, erythromycin, imipenem, oxacillin, and penicillin than S. aureus. The S. pseudintermedius group showed equal likelihood to be resistant to ampicillin and penicillin as S. aureus. The non-hemolytic Staphylococcus group was more likely to be resistant to azithromycin, clarithromycin, erythromycin, and oxacillin than the S. aureus group. The MG was more likely to be resistant to cefazolin than the beta-hemolytic Staphylococcus group. There were also numerous organism groups that showed very similar resistance prevalence to one another for each antibiotic. These similarities can be seen in Table 3 along with the statistics for the aforementioned relationships. The organism groups with a p-value close to one demonstrate a similar likelihood to be resistant to that particular antibiotic. Test results for association between date of sample collection and resistance for each antibiotic. Antibiotics highlighted in yellow show an upward trend over the last 10 years. The p values highlighted in red illustrate the significant trends. The difference plots show a measure of strength and direction of association using the grey bars in the far-right column.
Using the Kruskal-Wallis rank sum test, Staphylococcal isolate groups were compared to each antibiotic susceptibility. Results of 8/15 antibiotics tested showed a significant difference in susceptibility based on the isolate group. These eight antibiotics were then analyzed with the Steel-Dwass test to determine which organisms demonstrated greater resistance and which organisms followed similar resistance patterns for each antibiotic. Ampicillin, azithromycin, cefazolin, clarithromycin, erythromycin, imipenem, oxacillin and penicillin all had at least one Staphylococcal isolate more likely to be resistant than the others. The MG consisting of S. hyicus, S. xylosus, S. epidermidis, S. schleiferi, and isolates reported as Staphylococcus spp. showed statistical significance that they were more likely to be resistant to ampicillin, azithromycin, cefazolin, erythromycin, imipenem, oxacillin, and penicillin than S. aureus. The S. pseudintermedius group showed equal likelihood to be resistant to ampicillin and penicillin as S. aureus. The non-hemolytic Staphylococcus group was more likely to be resistant to azithromycin, clarithromycin, erythromycin, and oxacillin than the S. aureus group. The MG was more likely to be resistant to cefazolin than the beta-hemolytic Staphylococcus group. There were also numerous organism groups that showed very similar resistance prevalence to one another for each antibiotic. These similarities can be seen in Table 3 along with the statistics for the aforementioned relationships. The organism groups with a p-value close to one demonstrate a similar likelihood to be resistant to that particular antibiotic. Test results for association between date of sample collection and resistance for each antibiotic. Antibiotics highlighted in yellow show an upward trend over the last 10 years. The p values highlighted in red illustrate the significant trends. The difference plots show a measure of strength and direction of association using the grey bars in the far-right column.
Using the Kruskal-Wallis rank sum test, Staphylococcal isolate groups were compared to each antibiotic susceptibility. Results of 8/15 antibiotics tested showed a significant difference in susceptibility based on the isolate group. These eight antibiotics were then analyzed with the Steel-Dwass test to determine which organisms demonstrated greater resistance and which organisms followed similar resistance patterns for each antibiotic. Ampicillin, azithromycin, cefazolin, clarithromycin, erythromycin, imipenem, oxacillin and penicillin all had at least one Staphylococcal isolate more likely to be resistant than the others. The MG consisting of S. hyicus, S. xylosus, S. epidermidis, S. schleiferi, and isolates reported as Staphylococcus spp. showed statistical significance that they were more likely to be resistant to ampicillin, azithromycin, cefazolin, erythromycin, imipenem, oxacillin, and penicillin than S. aureus. The S. pseudintermedius group showed equal likelihood to be resistant to ampicillin and penicillin as S. aureus. The non-hemolytic Staphylococcus group was more likely to be resistant to azithromycin, clarithromycin, erythromycin, and oxacillin than the S. aureus group. The MG was more likely to be resistant to cefazolin than the beta-hemolytic Staphylococcus group. There were also numerous organism groups that showed very similar resistance prevalence to one another for each antibiotic. These similarities can be seen in Table 3 along with the statistics for the aforementioned relationships. The organism groups with a p-value close to one demonstrate a similar likelihood to be resistant to that particular antibiotic. Test results for association between date of sample collection and resistance for each antibiotic. Antibiotics highlighted in yellow show an upward trend over the last 10 years. The p values highlighted in red illustrate the significant trends. The difference plots show a measure of strength and direction of association using the grey bars in the far-right column.
Using the Kruskal-Wallis rank sum test, Staphylococcal isolate groups were compared to each antibiotic susceptibility. Results of 8/15 antibiotics tested showed a significant difference in susceptibility based on the isolate group. These eight antibiotics were then analyzed with the Steel-Dwass test to determine which organisms demonstrated greater resistance and which organisms followed similar resistance patterns for each antibiotic. Ampicillin, azithromycin, cefazolin, clarithromycin, erythromycin, imipenem, oxacillin and penicillin all had at least one Staphylococcal isolate more likely to be resistant than the others. The MG consisting of S. hyicus, S. xylosus, S. epidermidis, S. schleiferi, and isolates reported as Staphylococcus spp. showed statistical significance that they were more likely to be resistant to ampicillin, azithromycin, cefazolin, erythromycin, imipenem, oxacillin, and penicillin than S. aureus. The S. pseudintermedius group showed equal likelihood to be resistant to ampicillin and penicillin as S. aureus. The non-hemolytic Staphylococcus group was more likely to be resistant to azithromycin, clarithromycin, erythromycin, and oxacillin than the S. aureus group. The MG was more likely to be resistant to cefazolin than the beta-hemolytic Staphylococcus group. There were also numerous organism groups that showed very similar resistance prevalence to one another for each antibiotic. These similarities can be seen in Table 3 along with the statistics for the aforementioned relationships. The organism groups with a p-value close to one demonstrate a similar likelihood to be resistant to that particular antibiotic. Test results for association between date of sample collection and resistance for each antibiotic. Antibiotics highlighted in yellow show an upward trend over the last 10 years. The p values highlighted in red illustrate the significant trends. The difference plots show a measure of strength and direction of association using the grey bars in the far-right column.
Using the Kruskal-Wallis rank sum test, Staphylococcal isolate groups were compared to each antibiotic susceptibility. Results of 8/15 antibiotics tested showed a significant difference in susceptibility based on the isolate group. These eight antibiotics were then analyzed with the Steel-Dwass test to determine which organisms demonstrated greater resistance and which organisms followed similar resistance patterns for each antibiotic. Ampicillin, azithromycin, cefazolin, clarithromycin, erythromycin, imipenem, oxacillin and penicillin all had at least one Staphylococcal isolate more likely to be resistant than the others. The MG consisting of S. hyicus, S. xylosus, S. epidermidis, S. schleiferi, and isolates reported as Staphylococcus spp. showed statistical significance that they were more likely to be resistant to ampicillin, azithromycin, cefazolin, erythromycin, imipenem, oxacillin, and penicillin than S. aureus. The S. pseudintermedius group showed equal likelihood to be resistant to ampicillin and penicillin as S. aureus. The non-hemolytic Staphylococcus group was more likely to be resistant to azithromycin, clarithromycin, erythromycin, and oxacillin than the S. aureus group. The MG was more likely to be resistant to cefazolin than the beta-hemolytic Staphylococcus group. There were also numerous organism groups that showed very similar resistance prevalence to one another for each antibiotic. These similarities can be seen in Table 3 along with the statistics for the aforementioned relationships. The organism groups with a p-value close to one demonstrate a similar likelihood to be resistant to that particular antibiotic. Test results for association between date of sample collection and resistance for each antibiotic. Antibiotics highlighted in yellow show an upward trend over the last 10 years. The p values highlighted in red illustrate the significant trends. The difference plots show a measure of strength and direction of association using the grey bars in the far-right column.
Using the Kruskal-Wallis rank sum test, Staphylococcal isolate groups were compared to each antibiotic susceptibility. Results of 8/15 antibiotics tested showed a significant difference in susceptibility based on the isolate group. These eight antibiotics were then analyzed with the Steel-Dwass test to determine which organisms demonstrated greater resistance and which organisms followed similar resistance patterns for each antibiotic. Ampicillin, azithromycin, cefazolin, clarithromycin, erythromycin, imipenem, oxacillin and penicillin all had at least one Staphylococcal isolate more likely to be resistant than the others. The MG consisting of S. hyicus, S. xylosus, S. epidermidis, S. schleiferi, and isolates reported as Staphylococcus spp. showed statistical significance that they were more likely to be resistant to ampicillin, azithromycin, cefazolin, erythromycin, imipenem, oxacillin, and penicillin than S. aureus. The S. pseudintermedius group showed equal likelihood to be resistant to ampicillin and penicillin as S. aureus. The non-hemolytic Staphylococcus group was more likely to be resistant to azithromycin, clarithromycin, erythromycin, and oxacillin than the S. aureus group. The MG was more likely to be resistant to cefazolin than the beta-hemolytic Staphylococcus group. There were also numerous organism groups that showed very similar resistance prevalence to one another for each antibiotic. These similarities can be seen in Table 3 along with the statistics for the aforementioned relationships. The organism groups with a p-value close to one demonstrate a similar likelihood to be resistant to that particular antibiotic. Test results for association between date of sample collection and resistance for each antibiotic. Antibiotics highlighted in yellow show an upward trend over the last 10 years. The p values highlighted in red illustrate the significant trends. The difference plots show a measure of strength and direction of association using the grey bars in the far-right column.
Using the Kruskal-Wallis rank sum test, Staphylococcal isolate groups were compared to each antibiotic susceptibility. Results of 8/15 antibiotics tested showed a significant difference in susceptibility based on the isolate group. These eight antibiotics were then analyzed with the Steel-Dwass test to determine which organisms demonstrated greater resistance and which organisms followed similar resistance patterns for each antibiotic. Ampicillin, azithromycin, cefazolin, clarithromycin, erythromycin, imipenem, oxacillin and penicillin all had at least one Staphylococcal isolate more likely to be resistant than the others. The MG consisting of S. hyicus, S. xylosus, S. epidermidis, S. schleiferi, and isolates reported as Staphylococcus spp. showed statistical significance that they were more likely to be resistant to ampicillin, azithromycin, cefazolin, erythromycin, imipenem, oxacillin, and penicillin than S. aureus. The S. pseudintermedius group showed equal likelihood to be resistant to ampicillin and penicillin as S. aureus. The non-hemolytic Staphylococcus group was more likely to be resistant to azithromycin, clarithromycin, erythromycin, and oxacillin than the S. aureus group. The MG was more likely to be resistant to cefazolin than the beta-hemolytic Staphylococcus group. There were also numerous organism groups that showed very similar resistance prevalence to one another for each antibiotic. These similarities can be seen in Table 3 along with the statistics for the aforementioned relationships. The organism groups with a p-value close to one demonstrate a similar likelihood to be resistant to that particular antibiotic. Test results for association between date of sample collection and resistance for each antibiotic. Antibiotics highlighted in yellow show an upward trend over the last 10 years. The p values highlighted in red illustrate the significant trends. The difference plots show a measure of strength and direction of association using the grey bars in the far-right column.
Using the Kruskal-Wallis rank sum test, Staphylococcal isolate groups were compared to each antibiotic susceptibility. Results of 8/15 antibiotics tested showed a significant difference in susceptibility based on the isolate group. These eight antibiotics were then analyzed with the Steel-Dwass test to determine which organisms demonstrated greater resistance and which organisms followed similar resistance patterns for each antibiotic. Ampicillin, azithromycin, cefazolin, clarithromycin, erythromycin, imipenem, oxacillin and penicillin all had at least one Staphylococcal isolate more likely to be resistant than the others. The MG consisting of S. hyicus, S. xylosus, S. epidermidis, S. schleiferi, and isolates reported as Staphylococcus spp. showed statistical significance that they were more likely to be resistant to ampicillin, azithromycin, cefazolin, erythromycin, imipenem, oxacillin, and penicillin than S. aureus. The S. pseudintermedius group showed equal likelihood to be resistant to ampicillin and penicillin as S. aureus. The non-hemolytic Staphylococcus group was more likely to be resistant to azithromycin, clarithromycin, erythromycin, and oxacillin than the S. aureus group. The MG was more likely to be resistant to cefazolin than the beta-hemolytic Staphylococcus group. There were also numerous organism groups that showed very similar resistance prevalence to one another for each antibiotic. These similarities can be seen in Table 3 along with the statistics for the aforementioned relationships. The organism groups with a p-value close to one demonstrate a similar likelihood to be resistant to that particular antibiotic.
Test results for association between date of sample collection and resistance for each antibiotic. Antibiotics highlighted in yellow show an upward trend over the last 10 years. The p values highlighted in red illustrate the significant trends. The difference plots show a measure of strength and direction of association using the grey bars in the far-right column.           Additionally, a nonparametric Kendall's τ test was used to analyze the correlations between each antibiotic. Many statistically significant positive correlations were found between antibiotics. Only one significant negative correlation was found between cefazolin and amikacin. Strong positive correlations of susceptibility were found between multiple antibiotics and can be seen in the supporting information (Supplemental Table  S1). Finally, the overall prevalence of resistance or intermediate susceptibility for seven antibiotics was determined for each of the organism groups in the 10-year time period (Table 4 and Table 5). Additionally, a nonparametric Kendall's τ test was used to analyze the correlations between each antibiotic. Many statistically significant positive correlations were found between antibiotics. Only one significant negative correlation was found between cefazolin and amikacin. Strong positive correlations of susceptibility were found between multiple antibiotics and can be seen in the supporting information (Supplemental Table  S1). Finally, the overall prevalence of resistance or intermediate susceptibility for seven antibiotics was determined for each of the organism groups in the 10-year time period (Table 4 and Table 5). Additionally, a nonparametric Kendall's τ test was used to analyze the correlations between each antibiotic. Many statistically significant positive correlations were found between antibiotics. Only one significant negative correlation was found between cefazolin and amikacin. Strong positive correlations of susceptibility were found between multiple antibiotics and can be seen in the supporting information (Supplemental Table  S1). Finally, the overall prevalence of resistance or intermediate susceptibility for seven antibiotics was determined for each of the organism groups in the 10-year time period (Table 4 and Table 5). Additionally, a nonparametric Kendall's τ test was used to analyze the correlations between each antibiotic. Many statistically significant positive correlations were found between antibiotics. Only one significant negative correlation was found between cefazolin and amikacin. Strong positive correlations of susceptibility were found between multiple antibiotics and can be seen in the supporting information (Supplemental Table  S1). Finally, the overall prevalence of resistance or intermediate susceptibility for seven antibiotics was determined for each of the organism groups in the 10-year time period (Table 4 and Table 5). Additionally, a nonparametric Kendall's τ test was used to analyze the correlations between each antibiotic. Many statistically significant positive correlations were found between antibiotics. Only one significant negative correlation was found between cefazolin and amikacin. Strong positive correlations of susceptibility were found between multiple antibiotics and can be seen in the supporting information (Supplemental Table S1). Finally, the overall prevalence of resistance or intermediate susceptibility for seven antibiotics was determined for each of the organism groups in the 10-year time period (Tables 4 and 5).

Discussion
The results of this study suggest that resistance for certain antibiotics is present and patterns are being seen across antibiotic classes. Ampicillin, cefazolin, and penicillin showed Vet. Sci. 2023, 10, 71 8 of 11 a statistically significant upward trend of resistance over the 10-year period examined. Since these antibiotics are not commonly used by dermatologists, this could be due to their increased use for infections not related to the skin. Penicillin is frequently used for surgical prophylaxis in horses undergoing colic surgery [1] and is used as a first line choice for treatment of streptococcal infections, the cause of equine strangles and upper and lower respiratory infections in horses [27]. Ampicillin is used in horses for streptococcal lower airway infections in addition to being a treatment for neonatal sepsis, while cefazolin is a treatment choice for culture-confirmed susceptible Staphylococcus spp. [27]. All of these uses could be leading to the increased resistance seen in this study. It is important to note that other horses in the same pasture or boarding facility may have been treated previously and created a selective pressure on the bacteria in the area [4].
Blood supply of skin is comparatively poor to other organs and the upper end of dose ranges are used to treat skin infections. To be effective, antibiotics must reach sufficient concentrations at the site of infection. Tissue distribution is an important factor for antibiotic efficacy and due to the challenges of achieving these concentrations in the epidermis, systemic treatments for superficial pyoderma involve larger doses and longer durations compared to treatments for other infections. These factors may play a role in why dermatology cases tend to develop more resistance than other patients. Some antibiotics, such as rifampin, macrolides, and fluoroquinolones, are lipophilic, thus accumulate in larger amounts in the skin [28]. This paper focused specifically on how antibiotic sensitivity in vitro has changed over time and when translating these results to clinical practice it is important to put them into context of the characteristics of the specific antibiotic.
Trimethoprim/sulfonamide also showed an increasing trend of resistance that did not reach statistical significance, but may be of clinical relevance as this is often a first choice for systemic treatment of horses with skin infections due to the cost and spectrum of activity of this class of antibiotic [29,30]. In a Canadian study, trimethoprim/sulfonamide's increased resistance had been shown previously in coagulase-positive staphylococci [6]. However, another study in France showed that TMS resistance was stationary over a four-year time period [31]. Therefore, further investigation is warranted. It is possible that TMS would have reached statistical significance in this study if a longer period of time was analyzed or if the study started before 2009 due to the fact that the population of bacteria could have already matured or developed resistance prior to the start of the study. Alternatively, the bacterial population in different geographical locations potentially have different resistance patterns in part due to the distinct ways medicine is practiced or the different antibiotics used in various locations leading to unique pressure on the bacteria.
The low numbers of oxacillin resistance of all Staphylococcal species in this study is in contrast to what is seen in small animal medicine in which an increase has been seen in oxacillin resistance in dogs in recent years [32][33][34]. However, the low prevalence of oxacillin resistance seen in this study is similar to that seen in livestock [35]. It is hypothesized that there is less methicillin/oxacillin resistance in livestock and horses when compared to small animals due to the increased regulations on antibiotic use and decreased available antibiotics for use in the large animal species. A study in the Netherlands showed decreased antibiotic use was associated with declining methicillin resistance [36].
It is possible that some of the resistance documented in penicillin, ampicillin, and cefazolin is due to increased numbers of staphylococci producing beta lactamase and not due to an acquired mecA gene mutation as is required for methicillin resistance. The semi-synthetic penicillins can circumvent beta lactamase activity [37]. This could explain why some of the beta-lactam antibiotics have increased resistance. In addition, the use of fluoroquinolones is likely higher in dogs than horses due to the cost. A previous study in Germany reported the use of fluoroquinolones to have been less than 1% of antibiotics administered or dispensed to horses [38]. Fluoroquinolones have been shown to increase the risk of antibiotic resistance in humans and dogs [39][40][41].
Moreover, gentamicin was the only antibiotic that showed a statistical difference in susceptibility based on the sample collection type. The difference was found between the surgical incision sites and the abscesses while all other sample types showed no statistical difference. It was hypothesized that there would be no difference in susceptibility based on sample type and this difference could potentially be due to small sample sizes. There were only five samples in the surgical incision group and 15 in the abscess group. However, all of the abscesses were susceptible to gentamicin while only one of the surgical incision sites was susceptible. Further investigation with larger sample sizes would be warranted to confirm sample type affects susceptibility.
There were many significant positive correlations found between the antibiotics that were analyzed and one significant negative correlation (Supplemental Table S1). Many of the positive correlations were between antibiotics in the same drug class which was expected due to their similar mechanisms of action. In addition, many other positive correlations were found between antibiotics in the same tier, such as rifampin and chloramphenicol. This was also to be anticipated due to the similar amount of use these antibiotics get causing similarities in selective pressure. The single negative correlation was between cefazolin and amikacin and is likely not of clinical relevance.
Lastly, the Staphylococcal species often did affect the antibiotic susceptibility as expected. Historically, coagulase negative staphylococci have been more resistant to antibiotics than coagulase positive staphylococci [42]. This aligns with the current study where the "other" group, which consisted of S. hyicus, S. xylosus, S. epidermidis, and S. schleiferi, showed a statistical significance of being more resistant to beta-lactam antibiotics and macrolides when compared to S. aureus. In addition, S. aureus and S. pseudintermedius, both coagulase positive staphylococci, showed equal likelihood to be resistant to ampicillin and penicillin. From a clinical point of view, this data is important as most pyodermas that dermatologists treat are coagulase positive staphylococci.
As previously mentioned, one of the main limitations of this study is a small sample size. The numbers in this study are likely an underestimation of the population of horses treated at the author's academic institution. This is due to the retrospective nature of the study and the difficulty associated with searching medical record systems. Moreover, the previous antibiotic history was unknown or not reported in a majority of the cases. Of the 77 cases included, 49 had an unknown antibiotic history and 24 had at least one antibiotic used prior to being cultured. We suspect that there is a selection bias in this study due to the fact that culture and sensitivity panels are often submitted after a patient is not responding to an empiric treatment or after previously being treated with antibiotics. Therefore, higher resistance was expected. In addition, this can be assumed due to the tertiary referral nature of the institution being studied. Many of the patients at the institution have had chronic infections that could not be cleared by their primary veterinarian or the first line antibiotic was not working. To end, this study is important to help show the progress of bacterial resistance and guide field veterinarians on their empiric choices. Continual monitoring of resistance patterns is essential for better antibiotic use and maximizes the chance of successful therapy.

Conclusions
This study suggests that resistance of beta-lactam antibiotics for Staphylococcus is on the rise at the author's tertiary referral institution, the specific Staphylococcal spp. isolated affects resistance and antibiotics in similar classes are likely to follow similar resistance patterns. Further larger-sample studies are needed to assess the clinical usefulness of the aforementioned results.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/vetsci10020071/s1, Table S1: Test results reporting the correlations between each antibiotic. Numbers listed in orange (p < 0.01) or red (p < 0.05) reached statistical significance. Many statistically significant positive correlations were found between antibiotics. Only one significant negative correlation was found between cefazolin and amikacin.