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Genotypic profile of Staphylococcus spp., Enterococcus spp., and E. coli colonizing dogs, surgeons, and environment during the intraoperative period: a cross-sectional study in a veterinary teaching hospital in Brazil

BMC Veterinary Research volume 21, Article number: 147 (2025) Cite this article

Abstract

Aims

This prospective cross-sectional study aimed to determine the occurrence of resistance genes and genetic diversity in Staphylococcus spp., Enterococcus spp., and Escherichia coli isolated from dogs' superficial surgical site (SS), surgeons' hands, and the operating room (OR) during the intraoperative period.

Methods

Thirty dogs undergoing clean/clean-contaminated (G1, n = 20) and contaminated surgeries (G2, n = 10), along with eight surgeons, were included in the study. Specimens were collected using sterile swabs, transported in 0.1% peptone salt solution, and spread onto blood agar. Environmental samples were collected through passive exposure using BHI agar plates. Seventy-five isolates were selected and classified using MALDI-TOF MS. Resistance genes were screened via PCR: tet(M), ermA, aacA-aphD, blaZ, mecA, blaTEM-1, blaSHV, blaSHV-1, blaCTX-M-1, 3 e 15, blaCTX-M-2, blaCMY-2, mcr1, mcr2, mcr3, mcr4, and ndm. Genetic diversity was assessed through PFGE analysis using SmaI and XbaI restriction enzymes, with clustering performed by the UPGMA method. The chi-square test compared the frequency of resistance gene detected.

Results

Staphylococcus pseudintermedius (83.33%), Enterococcus spp. (52.63%), and E. coli (62.50%) were more frequently isolated from dogs' skin, while coagulase-negative staphylococci (CoNS; 62.50%) were more frequent in the OR. Resistance genes detected in Staphylococcus spp. included blaZ (79.17%), mecA (43.75%), tet(M) (41.67%), and aacA-aphD (25%). Among Enterococcus spp., tet(M) (78.95%) and blaZ (10.53%) were identified. S. pseudintermedius harbored tet(M) and aacA-aphD genes more frequently than CoNS. No E. coli isolates tested positive for the investigated genes. Twenty-four PFGE banding patterns were observed in CoNS (24/24), 15 in S. pseudintermedius (15/24), 4 in E. coli (4/8), and 7 in Enterococcus spp. (7/19). Genetically related S. pseudintermedius and E. coli were obtained from SS and OR in G2. Seven indistinguishable Enterococcus spp. were identified across different procedures and patients.

Conclusion

Our study revealed high rates of methicillin-resistant Staphylococcus spp. and tetracycline-resistant Enterococcus spp. colonizing the environment in a veterinary teaching hospital in Brazil. PFGE analysis indicated a high diversity of CoNS and Enterococcus spp. Genetically related strains in S. pseudintermedius, Enterococcus spp., and E. coli emphasize the importance of effective infection control policies to minimize the spread of resistant bacteria.

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Background

Infections caused by multidrug-resistant (MDR) organism currently represent one of the most significant global challenges within the context of One Health, owing to their capacity to increase morbidity, mortality, and healthcare-associated costs [1]. Infections caused by bacteria classified as MDR are challenging because these bacteria exhibit resistance to at least one drug from three or more antimicrobial classes, severely limiting treatment options [2].

The hospital environment is reported as the main risk factor for acquiring resistant pathogens during hospitalization, both in human and veterinary settings. Community reservoirs of MDR bacteria could contribute to the heightened prevalence of these strains in the Intensive Care Unit (ICU), thereby compromising patient treatment and outcomes [3, 4].

Methicillin-resistant Staphylococcus spp. (MRS), Vancomycin-resistant Enterococcus spp. (VRE), and Extended-Spectrum β-Lactamase (ESBL)-producing Enterobacterales, such as Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis, are among the most common pathogens causing infections in critically ill veterinary patients [4, 5].

Although epidemiological data in Brazil's veterinary context is lacking, several studies have already reported a high prevalence of these pathogens colonizing patients or causing infections, especially MRS and ESBL-producing organisms [6,7,8,9,10,11,12,13]. Additionally, worldwide studies are also documenting the colonization of patients, health-care professionals, and the environment within veterinary hospital settings by MDR bacteria [6, 11, 14].

This carriage could contribute to the development of surgical site infections (SSI), as well as to the dissemination of these strains among humans and dogs [4, 15]. Some studies have reported genetically related strains isolated from humans, veterinary patients, and the environment [16, 17]. In this way, molecular epidemiology appears as an important tool to better screen and elucidate the reservoirs and transmission dynamics of MDR bacteria in veterinary settings, aiming to develop strategies to overcome these challenges.

With this proposal, this cross-sectional epidemiological study aimed to determine the occurrence of resistance genes and the banding patterns in pulsed-field gel electrophoresis (PFGE) of Staphylococcus spp., Enterococcus spp., and E. coli isolated from dog’s superficial surgical site (SS), surgeons’ hands, and the operation room (OR) during the intraoperative period of clean/clean-contaminated, and contaminated surgeries in a veterinary teaching hospital located in the southeast of Brazil.

Results

Genotypic resistance

Among the bacterial species analyzed, within each species, S. pseudintermedius (83.33% [20/24]), E. coli (62.50% [5/8]), and Enterococcus spp. (52.63% [10/19]) were more frequently isolated from dogs’ skin, while coagulase-negative staphylococci (CoNS; 62.50% [15/24]), were more frequent in the OR environment. Figure 1 A summarizes the bacteria isolated by the source of collection.

Fig. 1
figure 1

Percentage distribution of overall bacterial species (A) and within Staphylococcus spp. (B) isolated per source of collection during the intraoperative period. Abbreviations: coagulase-negative staphylococci (CoNS); dogs’ superficial surgical site (SS); surgical site infection (SSI)

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Regarding the staphylococcal species (Fig. 1B), the most frequent was S. pseudintermedius (50% [24/48]), followed by S. haemolyticus (19% [9/48]), and S. epidermidis (15% [7/48]). Among enterococcal species, Enterococcus faecium (89.47% [17/19]) was the most frequent, followed by E. faecalis (10.53% [2/19]).

When comparing the occurrence between the groups, S. pseudintermedius were more frequent in G2 (70% [21/30]) than in G1 (16.67% [3/18]), whereas CoNS were more frequent in G1 (83.33% [15/18]) than in G2 (30% [9/30]). Additionally, Enterococcus spp. were more frequent in G1 (68.42% [13/19]), whereas E. coli isolates were only identified in G2 (Fig. 2). Two out of 10 dogs in G2 (20%) developed SSI within 30 days after the intervention. These infections were caused by mixed species involving S. pseudintermedius, Proteus mirabilis, and Pseudomonas aeruginosa (P2), as well S. pseudintermedius, and Enterobacter spp. (P10). No patients in G1 presented with SSI within 30 days after the procedure.

Fig. 2
figure 2

Percentage distribution of overall bacterial species isolated during the intraoperative period, per type of surgery, in clean/contaminated (G1) and contaminated surgery (G2). *The outer ring differentiates between the two groups: clean/contaminated surgeries (G1, blue) and contaminated surgeries (G2, orange), while the inner ring represents the bacterial species isolated in G1 (right) and G2 (left). Abbreviations: clean/contaminated surgery (G1); contaminated surgery (G2); coagulase-negative staphylococci (CoNS)

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In terms of the presence of resistance genes, blaZ was the most frequently detected overall. No statistically significant difference was found in the frequency of resistance genes (FR) between isolates obtained from different sources (p > 0.05), as shown in Fig. 3A and Table 1. Among Enterococcus spp., tet(M) (78.95% [15/19]), was the most frequently detected, followed by blaZ (10.53% [2/19]). None of the isolates tested positive for ermA. No screened resistance genes were found in the E. coli isolates.

Fig. 3
figure 3

Frequency of resistance genes detected in Staphylococcus sp., and Enterococcus sp. per collection source (A), and in S. pseudintermedius and coagulase-negative staphylococci (B) isolated from different sources during the intraoperative period in clean/clean-contaminated and contaminated surgery. *A statistical difference was found when comparing the rates of positive resistance genes among staphylococcal isolates using the chi-square test, with a significance threshold set at 0.05 and analysis conducted with R 4.3.3 software. Abbreviations: coagulase-negative staphylococci (CoNS); dogs’ superficial surgical site (SS); surgical site infection (SSI)

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Table 1 Frequency of Staphylococcus spp. isolates positive for selected resistance genes by source of collection
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Among the staphylococcal isolates (Fig. 3B; Table 2), blaZ had the highest occurrence (79.17% [38/48]), followed by mecA (43.75% [21/48]), tet(M) (41.67% [20/48]), and aacA-aphD (25% [12/48]). CoNS (50% [12/24]) exhibited a higher frequency of mecA-positive isolates compared to S. pseudintermedius (37.50% [9/24]), although no statistical difference was observed between the rates (p = 0.5606). However, for the tet(M) and aacA-aphD genes, S. pseudintermedius showed a higher proportion (p = 0.0404 and p = 0.0196, respectively) of positive isolates (58.33% [14/24] and 41.67% [10/24], respectively) compared to CoNS (25% [6/24] and 8.33% [2/24], respectively). The phenotypic results for these isolates, obtained in a previous study [11], are summarized in Additional File 1.

Table 2 Frequency of isolates positive for selected resistance genes in Staphylococcus pseudintermedius and coagulase-negative staphylococci species
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Four out of 10 procedures (40%) whithin G2 and 7 out of 20 (35%) within G1 had at least one MRS isolated from any source.

Genetic diversity

Twenty-four PFGE banding patterns were observed in CoNS. S. haemolyticus, and S. epidermidis presented a Diversity Ratio (DR) of 100%. Fifteen PFGE banding patterns were observed in S. pseudintermedius (DR = 58.33% [14/24]), 4 in E. coli (DR = 50% [4/8]), and 7 in Enterococcus faecium and E. faecalis (DR = 36.84% [7/19]).

Regarding the staphylococcal species, CoNS isolates exhibited greater diversity compared to S. pseudintermedius, a coagulase-positive staphylococci (Fig. 4). Genetically related S. pseudintermedius isolated from different sources were obtained during two procedures (P2 and P10) in G2 (Fig. 4A). In contrast, none of the S. haemolyticus, and S. epidermidis isolates exhibited a high degree of similarity (≥ 90%) among the three distinct sources (Fig. 4B; 4C). Three isolates were obtained from SS (keys: 85, 87, and 90), 1 from OR (key: 82), and 1 causing SSI (key: 91) in P2. In P10, 1 isolate obtained from SS (key: 130) were genetically related to 1 isolate causing SSI (key: 137).

Fig. 4
figure 4

Dendrogram produced by comparing banding patterns on PFGE of Staphylococcus pseudintermedius (A), S. haemolyticus (B), S. epidermidis (C), and other coagulase-negative staphylococci (D) species isolated from dogs’ superficial surgical site (red); surgeons’ hands (green), and operation room (blue). The dendrogram was generated using the UPGMA clustering method in BioNumerics 7.1 software. The Dice coefficient and optimization was set at 1%. PFGE similarity cutoff of 90% (dashed line) was applied. Red squares highlight the genetically related isolates. *Note: Species and genes names are not italicized due to software limitations (BioNumerics 7.1). Abbreviations: G1 = clean/clean contaminated surgery; G2: contaminated surgery; SS: dogs’ superficial surgical site; SSI: surgical site infection; Env: environment; Surg: surgeon

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Four episode of genetically related Enterococcus spp. were identified across different procedures, and distinct patients. Isolates 42 and 61 (from P15 and P19); 102, 104, and 108 (from P3 and P4); and 9, 10, and 15 (from P5 and P6) were obtained from SS and exhibited high similarity (Fig. 5A). Another episode was found among eight isolates obtained from OR environment (11, 16, 34, and 66), SS (60, and 65), and surgeons’ hands (98 and 99).

Fig. 5
figure 5

Dendrogram produced by comparing banding patterns on PFGE of Enterococcus spp. (A) and E. coli (B) isolated from dogs’ superficial surgical site (red); surgeons’ hands (green), and operation room (blue). The dendrogram was generated using the UPGMA clustering method in BioNumerics 7.1 software. The Dice coefficient and optimization was set at 1%. PFGE similarity cutoff of 90% (dashed line) was applied. Red squares highlight the genetically related isolates. *Note: Species and genes names are not italicized due to software limitations (BioNumerics 7.1). Abbreviations: G1 = clean/clean contaminated surgery; G2: contaminated surgery; SS: dogs’ superficial surgical site; SSI: surgical site infection; Env: environment; Surg: surgeon

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Despite the limited number of E. coli isolates obtained in this study, one episode of genetically related isolates was found (Fig. 5B). In P10, three isolates obtained from the environment (keys: 121, 122, and 123) were similar to one obtained from SS (key: 147).

Discussion

This cross-sectional epidemiological study reveals the presence of significant resistance genes identified in Staphylococcus spp., Enterococcus spp., and E. coli, along with their genotypic patterns.

S. pseudintermedius, S. epidermidis, and S. haemolyticus are common pathogens that colonize the skin and mucous membranes of dogs and humans, except for S. pseudintermedius, which is typically found in dogs but rarely in humans. They are oportunistic pathogens and a common cause of healthcare-associated infections, including SSIs [18, 19]. The colonization by MDR Staphylococcus spp. appears as one of the main risk factors for acquiring serious infections [5].

Only one (12.5% [1/8]) S. pseudintermedius isolate was obtained from surgeon’s hands in this study; however it was not genetically related to other isolates found in the environment or dog’s skin, suggesting human colonization by this strain. The close contact between humans and pets nowadays is increasing the prevalence of this species, particularly among veterinary workers [14, 16, 17, 20, 21].

Additionally, genetically related strains obtained from different sources were found among S. pseudintermedius isolated in two procedures in G2 (20% [2/10]), while in G1, there was no evidence of shared strains during a same procedure. This is likely due to contaminated surgeries presenting a higher microbial load in the surgical site, despite of not showing an infection. This high microbial content could also increase the possibility of fomites and environment contamination during the procedure, as well as increasing the SSI rates [22]. Thus, two out of 10 dogs in G2 (20%) developed SSI within 30 days after the intervention, caused by a mixed infection involving S. pseudintermedius. These isolates are similar to those obtained from the patients' skin during the intraoperative period (Fig. 4A).

Previous studies conducted in veterinary settings have already reported genetic relatedness in bacterial strains among dogs, personnel, and the hospital enviroment, identifying this as a risk factor for acquiring healthcare-associated infections [14, 23]. Interestingly, a high occurence of staphylococcal isolates in the present study harbored blaZ (79.17%), mecA (43.75%), tet(M) (41.67%), and aacA-aphD (25%), genes conferring resistance to penicillins, beta-lactams, tetracycline, and aminoglycosides drugs, respectively. These results, corroborate previous findings regarding phenotypic resistance published by the author’s group. High rates of antimicrobial resistance to penicillin (80–100%), oxacillin (50%), cefoxitin (53.33–75.86%), tetracycline (65–73.33%), gentamicin (34.48–66.67%), and erythromycin (73.33–75.86%) in disk diffusion tests were observed [12]. However, despite the high rates of phenotypic erythromycin resistance found, no isolates were positive for the ermA gene in the current study. This finding highlights the need for further investigation into the resistance mechanism of these isolates [24]. Similar resistance rates were recently reported by Teixeira et al. (2023) [13], regarding aminoglycosides (84%), penicillin (76%), tetracycline (53.3%), and oxacillin resistance (24%) located in the southeast of Brazil.

Staphylococcus spp. strains harboring the mecA gene are generally multidrug-resistant, as this gene confers resistance to all drugs of the β-lactam class, with the exception of fifth-generation cephalosporins, and often correlates with resistance to other antimicrobial classes as well [25]. This behavior severely restricts treatment options and compromises patient outcomes. Additionally, tetracycline (tet), gentamicin (aacA-aphD) and erythromycin (ermA) could be important alternative choices to treat MRS infections, avoiding the use of antibiotics with high toxicity, such as rifampicin or chloramphenicol, or those antimicrobials considered critically important in human medicine, such as vancomycin, linezolid, and fluoroquinolones [26]. Hence, the high rates of tet(M) and aacA-aphD, in addition to blaZ and mecA, detected in our study are concerning.

These rates of MRS contrast with the low frequency (< 10%) of MRS colonization reported in companion animal practice, veterinary healthcare professionals, and veterinary settings in some countries, such as Austria [27], Bangladesh [28], Germany [29], Tanzania [30], and United States [23, 31]. However, they corroborate with the moderate or high occurrence (> 10%) observed in Brazil [6, 7, 10, 11, 13], Iran [32], Italy [33], Nigeria [34], Poland [35], Portugal [36], South Africa [37], and Switzerland [38] with occurrence rates ranging from 10 to 85.9%.

While the importance of S. epidermidis as a pathogen in human medicine is well-established, its significance in veterinary medicine remains unclear and potentially underestimated. However, there is increasing recognition of CoNS, mainly S. epidermidis and S. haemolyticus, as potential pathogens causing nosocomial infections in veterinary settings [7, 12, 38]. The present study showed a higher frequency of methicillin-resistance in CoNS (50%) isolates than in S. pseudintermedius (37.50%), a coagulase-positive staphylococci. Although no statistical significance was found, this finding corroborates with the previous phenotypic results published by the author’s group [11], and with the findings reported by Adiguzel et al. (2022) [39].

The high frequency of staphylococcal isolates harboring mecA gene in the present study, as well as those frequently reported worldwide over the last two decades, underscore the exponential emergence of MRS globally. This trend is notable even in countries that traditionally report a low prevalence of these strains, highlighting the imperative for effective surveillance efforts aimed at devising strategies to mitigate this threat [40]. Therefore, meticulous attention to infection control policies is imperative to address the spread of these resistant strains and mitigate their impact on animal health.

Enterococcus spp. are opportunistic pathogens capable of surviving for months in the environment under adverse conditions. They can cause nosocomial infections, especially in immunocompromised patients [41]. While less prevalent compared to Staphylococcus spp. in causing infections in dogs, the incidence of Enterococcus spp. infections is increasing, with multidrug-resistant strains posing a significant concern in both human and canine critical care settings [42, 43]. Despite their lower prevalence, infections caused by enterococcal species are challenging to treat due to their diverse intrinsic antimicrobial resistance mechanisms and high level of acquired resistance, severely limiting treatment options [41, 44].

In the present study, Enterococcus faecium (89.47%) and E. faecalis (10.53%) were more frequently obtained from SS (52.63%) and from the OR environment (31.58% [6/19]). These isolates exhibited a different diversity and similarity pattern (Fig. 5A) compared to S. pseudintermedius and CoNS, showing a lower FR than the other species. Interestingly, eight indistinguishable isolates by PFGE banding patterns were observed in five procedures in G1 (isolates key: 11, 16, 34, 60, and 65) and two procedures in G2 (isolates key: 66, 98, and 99). All but one of these isolates harbored the tet(M) gene. This observation likely indicates the persistence of a specific strain in the surgical environment and underscores the need for more appropriate cleaning and disinfection procedures tailored to this species. Chung et al. (2014) [45] previously reported a potential clonal cluster of antibiotic-resistant Enterococcus spp. isolated from both dogs and healthcare professionals in a veterinary hospital in Korea. Additionally, Ghosh et al. (2011) [46] documented a low FR of E. faecium in the feces of dogs in an ICU, along with strains genetically related among dogs, humans, and hospital outbreaks.

A high rate of enterococcal isolates harbored the tetracycline resistance gene (78.95%), which correlates with the phenotypic resistance reported previously (78.95%) by the author’s group [11] and with findings reported by other authors [47,48,49,50,50]. However, the detection frequency of blaZ (10.53%), and ermA (0%) contrast with the phenotypic results for penicillin (68.42%), and erythromycin (84.24%) resistance for these specific isolates [11], and with those reported by other authors [47]. Several studies have reported an absence of a strong association between phenotypic and genotypic resistance to erythromycin. This highlights the urgent need for comprehensive molecular investigations of these species [49].

Finally, E. coli was isolated from two patients undergoing two different procedures. In one of these procedures, isolates were obtained from both the surgical site (isolate key: 147) and the environment (isolate key: 121, 122, and 123), exhibiting high similarity in PFGE banding patterns. Furthermore, all isolates tested negative for the investigated resistance genes, despite being phenotypically classified as MDR (Additional File 1). As the most often identified Gram-negative bacterium from animals, E. coli is responsible for a wide range of illnesses, including sepsis syndromes, gastrointestinal disorders, enterotoxemy, and SSI. Contaminated surgeries have a higher microbial load in the tissue and/or chronic inflammation, making these sites more susceptible to contamination by environmental bacteria [4, 22]. Thus, it was expected that this bacterial species would be isolated only in G2.

The limited number of E. coli isolates in this study poses challenges for conducting a reliable analysis of genotypic antimicrobial resistance frequency. Additionally, the small sample size of procedures, dogs, and surgeons included in this study is another limitation, along with the restricted diversity of resistance genes screened. Despite these limitations, our study provides essential insights for epidemiological surveillance and suggests avenues for further cross-sectional and longitudinal research.

Conclusion

Our cross-sectional epidemiological study revealed high rates of MRS and tetracycline-resistant Enterococcus spp. colonizing the environment in a veterinary teaching hospital in the southeast of Brazil. PFGE analysis indicated a high diversity of CoNS among dogs, veterinarians, and the operating room. Genetically related strains were found in S. pseudintermedius, Enterococcus spp., and E. coli isolates, emphasizing the importance of effective infection control policies to minimize the spread of multidrug-resistant bacteria. Hospital environments and dogs can serve as sources of multidrug-resistant bacterial strains. Enhancing awareness and monitoring of these organisms in veterinary environments can aid in the prevention and control of infections. Continuous passive and active surveillance are crucial for achieving this goal.

Methods

Study design and population

A prospective cross-sectional epidemiological study was conducted at a veterinary teaching hospital in São Paulo state, Brazil. Seventy-five isolates were obtained from the superficial surgical site (SS) of 30 dogs that had not received antimicrobial therapy in the 72 h prior to collection, as well as from the operating room environment, and 8 veterinary surgeons in 30 different procedures. These dogs underwent either clean/clean-contaminated (G1 [n = 20]) or contaminated (G2 [n = 10]) surgeries.

The isolates were obtained from a previously published study conducted at the same veterinary teaching hospital during the year 2019 [11]. The owners of the dogs and the surgeons selected for this study provided informed consent for their participation.

Sample collection

In a previous study [11], specimens were collected using a dry, sterile cotton-tipped swab and transported in a sterile tube containing 0.1% peptone salt solution. For surgeons samples, swabs were rubbed in a circular motion from the wrists to the fingertips (both dorsal and palmar surfaces), with this procedure repeated three times for each finger. For dog’s skin samples, the swab was also rubbed over the superficial surgical site (SS). The swabs were then spread onto 5% bovine blood agar (Oxoid, United Kingdom).

Environmental samples were collected via passive exposure using a Petri dish with Brain Heart Infusion agar (BHI; Oxoid, United Kingdom), which was left open on a 1-m-high support positioned near the surgery table during the procedure. All samples were incubated at 37 °C for 24 h under aerobic conditions. All morphologically distinct colonies from each sample were selected for further analysis.

Bacterial species identification

Each obtained colony was preserved in BHI broth with a 30% glycerol solution (1:1) at −80 ºC and subsequently inoculated into BHI broth prior to each analysis.

Forty-eight Staphylococcus sp., 19 Enterococcus sp., and 8 Escherichia coli isolates, identified by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS), were selected for this study. Sample preparation, data acquisition, and analysis were conducted as previously described by Sauer et al. (2008) [51] and Freiwald & Sauer (2009) [52]. The MALDI Biotyper 3.1 software from Bruker Daltonics was utilized to identify bacterial isolates based on their mass spectra obtained by an Autoflex III Smartbeam mass spectrometer. Standard Bruker interpretative criteria were applied; scores ≥ 2.0 were considered indicative of high reliability, while scores between 1.7 and 1.99 were considered indicative of moderate reliability. Isolates with a score < 1.7 were excluded from this study.

Resistance genes detection

DNA extraction

The DNA extraction of Staphylococcus spp. and Enterococcus spp. isolates was carried out following the method proposed by Bag et al. (2016) [53] and Moraes et al. (2022) [54] with some modifications. Briefly, aliquots of 1.5 mL of bacterial culture from each isolate were centrifuged at 8000 rpm for 5 min at 12 °C to obtain a sufficient cell pellet. The culture medium was discarded, and the bacterial pellet was resuspended in 700 µL of DNA extraction buffer [Tris–HCl 160 mM pH 8.0; EDTA 60 mM pH 8.0; NaCl 20 mM; SDS 0.5% (w/v)]. Cell lysis occurred at 65 °C for 40 min. Subsequently, 300 µL of 5 M potassium acetate solution was added to the solution, which, after homogenization, was kept on ice bathing for 30 min. Purification was performed using 600 µL of chloroform:isoamyl alcohol 24:1 (v/v) under centrifugation at 12,000 rpm at 10 °C for 10 min. The clear supernatant was transferred to new tubes to which 1000 µL of chilled absolute ethanol was added. The solution was homogenized and kept in a freezer at −20 °C for 12 h for DNA precipitation. Subsequently, the tubes were centrifuged at 12,000 rpm at 10 °C for 17 min to obtain the DNA pellet. The supernatant was discarded, and the pellet was washed with 700 µL of 70% (v/v) ethanol, dried at 55 ºC, and resuspended in 30–50 µL of TE buffer 10:1 (Tris–HCl 10 mM pH 8.0; EDTA 1 mM pH 8.0).

The DNA of Gram-negative bacteria was obtained using the boiling extraction method, as described by Keskimaki et al. (2001) [55].

PCR amplification

Resistance genes were detected through PCR amplification of selected genes. PCR conditions for detection of tested genes were carried out following previously described protocols provided in the Addtional File 2.

The genes tet(M) [56], aacA-aphD [56], ermA [56], and blaZ [57] were screened in Staphylococcus spp. and Enterococcus spp. isolates, while mecA [58] was exclusively targeted in Staphylococcus spp. For E. coli, the following genes were investigated: blaTEM-1 [59], blaSHV [60], blaSHV-1 [59], blaCTX-M-1, 3 e 15 [61], blaCTX-M-2 [61], blaCMY-2 [62], mcr1 [63], mcr2 [63], mcr3 [63], mcr4 [63], and ndm [64].

The PCR protocol followed the method described by CHINA et al. (1996) [65] with slight modifications. Briefly, a DNA aliquot (2 µL) was added to a mixture containing 0.4 µL of dNTP (2 mM), 2 µL of 10X buffer solution (200 mM Tris–HCl; 500 mM KCl; 20 mM MgCl2 [pH 8.5]), 0.8 µL of MgCl2 (25 mM), 0.2 µL of Taq DNA polymerase, and 1 µL of each primer (10 pmol), forward and reverse. The final volume of 20 µL was achieved with sterile MilliQ water. An aliquot of 5 µL of Gel Loading Dye Blue (0.25% bromophenol blue in 50% glycerol) was added to the PCR product. A molecular marker (100 pb or 1000 pb) was applied to a 1.5% agarose gel with SYBR® Safe DNA Gel Stain 10X (Thermo Fisher Scientific, Brazil). The eletrophoresis was runned in a Tris–Borate, EDTA buffer (120 V).

Bacterial genotyping

Strain genotyping was conducted using PFGE. Genomic DNA from all Staphylococcus spp. and Enterococcus spp. isolates was digested with SmaI, and from E. coli and Salmonella spp. with XbaI. Subsequently, they were separated by PFGE using the standardized Centers for Disease Control (CDC) protocol [66]. The pulse switch times for E. coli were 2.2 s initial time, 54.2 s final time, with a gradient of 6 V cm−1 and an angle of 120°, at 14 °C for 21 h; and for Staphylococcus spp. and Enterococcus spp. were 5.0 s initial time, 40.0 s final time, with a gradient of 6 V cm−1 and an angle of 120°, at 14 °C for 19 h. The universal size marker Salmonella serotype Braenderup H9812 were used on every gel [67].

Data analysis

Data generated were subjected to descriptive statistics using WPS Office spreadsheet© version 5.7.1 (Kingsoft Office Corporation, China) and expressed in percentages.

The frequency of resistance genes (FR) was calculated by WPS Office spreadsheet according to the formula:

$$FR(\%)=(\frac{\text{number of positive isolates for a specific gene}}{\text{total number of isolates tested for a specific gene}})\text{ x }100$$

The chi-square test was applied using R 4.3.3 software© (R Foundation for Statistical Computing, Austria) to compare bacterial resistance rates between isolates obtained from different sources (within the same species) or among different species. A significance threshold of 0.05 was applied.

DNA fingerprints were analyzed using BioNumerics 7.1 software (Applied Maths NV, bioMérieux, Belgium), and the similarity of PFGE fragments obtained was compared using a Dice coefficient with a tolerance and optimization of 1%. The dendrogram was generated using the UPGMA clustering method. A PFGE similarity cutoff of 90% was applied to consider isolates as genetically related.

Diversity ratio (DR) was calculated by WPS Office spreadsheet according to the formula:

$$DR(\%)=(\frac{\text{number of PFGE banding patterns}}{\text{total number of isolates}})\text{ x }100$$

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

BHI:

Brain heart infusion

CoNS:

Coagulase-negative staphylococci

DR:

Diversity ratio

ESBL:

Extended-spectrum β-lactamase

FR:

Frequency of resistance gene

G1:

Clean/clean-contaminated surgery

G2:

Contaminated surgery

ICU:

Intensive care unit

MALDI-TOF MS:

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry

MDR:

Multidrug-resistant

MRS:

Methicillin-resistant Staphylococcus spp.

OR:

Operation room

PFGE:

Pulsed-field gel electrophoresis

SS:

Superficial surgical site

VRE:

Vancomycin-resistant Enterococcus spp

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Acknowledgements

We express our gratitude to Prof. Dr. Eliana Gertrudes de Macedo Lemos from the Department of Agricultural and Environmental Biotechnology at the School of Agricultural Sciences and Veterinary Medicine (UNESP) for providing access to Bionumerics 7.1® software. We also acknowledge São Paulo State University (UNESP) for its support of our study.

Funding

This study was financed, in part, by São Paulo Research Foundation (FAPESP). Process number#2019/20585-0 and #2023/06904-0. And by the National Council for Scientific and Technological Development (CNPq), process number #307791/2021-1. The funding body played no role in the design of the study, data collection, analysis, interpretation, or in writing the manuscript.

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Authors and Affiliations

  1. Department of Clinic and Veterinary Surgery, School of Agricultural and Veterinarian Sciences, São Paulo State University (UNESP), Jaboticabal, São Paulo, 14884-900, Brazil

    Mareliza Possa de Menezes, Mariana Bugov & Paola Castro Moraes

  2. Department of Pathology, Reproduction, and One Health, School of Agricultural and Veterinarian Sciences, São Paulo State University (UNESP), Jaboticabal, São Paulo, 14884-900, Brazil

    Marita Vedovelli Cardozo & Natália Pereira

  3. Embrapa Beef Cattle, Campo Grande, Mato Grosso do Sul, Brazil, 79106-550

    Newton Valerio Verbisck

  4. Research and Development Center in Animal Health, General Bacteriology Laboratory, Biological Institute, São Paulo, São Paulo, 04016-035, Brazil

    Vanessa Castro & Alessandra Figueiredo de Castro Nassar

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  1. Mareliza Possa de Menezes
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Contributions

MVC, PCM, and MPM participated in the conceptualization, study design, and manuscript writing. MPM collected, processed, analyzed datas, and prepared Figs. 1–4. MPM, NP, and MB performed PCR and PFGE analysis. MPM, NVV, VC, and AFCN performed MALDI-TOF analysis. MPM, MVC, and NVV interpreted data. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Paola Castro Moraes.

Ethics declarations

Ethics approval and consent to participate

This study followed the recommendations of the Brazilian National Council for the Control of Animal Experimentation (CONCEA) and National Health Council (CNS), and was approved by the Institutional Ethics Committee in the Use of Animals (protocol number 5436/20), the Research Ethics Committee (CAAE: 07111419.4.0000.9029, and 39163720.5.0000.9029), and the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) (protocol nº A621D91). Dogs’ owners and surgeons were informed of the methods and purpose of the study and provided written informed consent.

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Not applicable.

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The authors declare no competing interests.

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Supplementary Information

12917_2025_4611_MOESM1_ESM.docx

Additional file 1: Summary of phenotypic and genotypic resistance in strains isolated from dogs’ surgical site, surgeons’ hands, and operation room during the intraoperative period in clean/clean-contaminated and contaminated surgery. Addtional Table 1. Summary of phenotypic and genotypic resistance in strains isolated from dogs’ surgical site, surgeons’ hands, and operation room during the intraoperative period in clean/clean-contaminated surgery. Addtional Table 2. Summary of phenotypic and genotypic resistance in strains isolated from dogs’ surgical site, surgeons’ hands, and operation room during the intraoperative period in contaminated surgery

12917_2025_4611_MOESM2_ESM.docx

Additional file 2: Resistance genes amplified by PCR in Escherichia coli, Staphylococcus spp., and Enterococcus isolated from dogs’ surgical site, surgeons’ hands, and operation room in a Veterinary Teaching Hospital in Brazil. Addtional Table 1. Resistance genes amplified by PCR in Escherichia coli isolated from dogs’ surgical site, surgeons’ hands, and operation room in a Veterinary Teaching Hospital in Brazil. Addtional Table 2. Resistance genes amplified by PCR in Staphylococcus spp., and Enterococcus spp. isolated from dogs’ surgical site, surgeons’ hands, and operation room in a Veterinary Teaching Hospital in Brazil.

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Possa de Menezes, M., Vedovelli Cardozo, M., Pereira, N. et al. Genotypic profile of Staphylococcus spp., Enterococcus spp., and E. coli colonizing dogs, surgeons, and environment during the intraoperative period: a cross-sectional study in a veterinary teaching hospital in Brazil. BMC Vet Res 21, 147 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04611-4

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BMC Veterinary Research

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