Abstract: Objective: The intent of this study was to characterize the potential bacterial contamination before and after sterilization in a limited resource setting and re-assess good surgical practices. This included the monitoring of antimicrobial susceptibility of recovered isolates, which have bearing on optimal antimicrobial use. Materials and Methods: Samples were collected from three surgical sites on the patient (two before cutting and one after), surgeon’s hand and pooled sample from surgical instruments in sterilization process. Antimicrobial susceptibility was performed on recovered bacteria. Descriptive analyses were done to compare between isolate recovery between sites and susceptibility findings. Results: Dogs enrolled in the study ranged in age from 0.5-6 years (mean 2.4). Bacterial isolates were recovered from 28 (93%) dogs; 17 (57%) surgeon’s hands and 8 (27%) pooled surgical equipment swabs. Eighty two unique bacterial isolates were recovered from 150 collected samples. Of the 82 isolates, 18 were identified as Staphylococcus sp. and 64 were identified as Bacillus sp. and Staphylococcus sp., were generally susceptible to amoxicillin/clavulanic acid, cephalexin, chloramphenicol and gentamycin. Thirteen different antimicrobial resistance patterns were observed for Staphylococcus sp. Four isolates were pan-sensitive. About 10 of 18 isolates were resistant to three of more classes of antibiotics. Conclusion: Reproductive surgical sterilization is an important tool in efforts to control pet overpopulation, even in limited resource settings. With ongoing concerns about emerging antibiotic resistance, implementation of optimum aseptic technique is needed. Findings from this study support standard and appropriate surgical protocols in dog control sterilization in limited resource settings which supports optimal antibiotic use policies.
INTRODUCTION
Stray and feral dogs are linked to serious human health (i.e., rabies), animal health and welfare problems1. In many countries, dog population control is a complex issue which is influenced by socio-economic, political and religious factors. The World Organization for Animal Health (OIE) recognizes the importance of controlling dog populations without causing unnecessary animal suffering1. It also recognizes the important role of veterinary care in controlling animal populations, preventing zoonotic diseases and ensuring animal welfare.
In 2014 in the Chiang Mai municipal area, there are an estimated 6624 owned dogs and 612 free roaming or temple dogs. In the downtown area, these free roaming or discarded dogs scrounge for food near tourist areas, food markets and city dumps2. City officials receive numerous complaints regarding these free or stray dogs in the city.
As a means to control the stray dog populations, the ompanion Animal Control Project of Chiang Mai Municipality has developed a program that includes public education, dog registration, unwanted dog adoptions and dog sterilization. According to the guidelines of International Companion Animal Management Coalition3, sterilization practices needs to be done by a veterinarian under aseptic conditions with appropriate anesthesia and pain management. Appropriate aseptic technique includes surgical prep of the patient, sterilization of surgical instruments and maintenance of asepsis during surgery. Previous studies have found that, in surgeon practice if using a hand rub (Hand rubbing) alcohol with ethanol after washing with mild soap can reduce the amount of bacteria as well and found fewer bacteria at the end of surgery than hand scrubbing4,5. This includes monitoring for surgical wound infections post-operatively6 that may have some problem from surgical7. Inflammation post surgery infection that is found in a third of the problems encountered by surgical sterilization8. The primary problem from contaminated is a wound infection9 Staphylococcus intermedius is a common problem of skin infection in dogs10,11. Steptococcus spp. and Staphylococcus spp., are the microbe that can be found to infect on the skin and the subcutaneous layer and become to be an abscess. Other infections that can cause dermatitis is Blastomyces dermatitidis and Sporothix schenckii12. Moreover, lock jaw and pyoderma are caused by skin infection, lock jaw is caused by Costridium tetani and pyoderma can caused by many bacterial include Corynebacterium spp., Staphylococcus aureus and Staphylococcus pyogeneus13.
The intent of this study was to characterize the potential bacterial contamination before and after sterilization in a limited resource setting and re-assess good surgical practices. This included the monitoring of antimicrobial susceptibility of recovered isolates, which have bearing on optimal antimicrobial use. The goal was to provide the est practices in a limited funded dog control program ( ompanion Animal Control Project of Chiang Mai Municipality). This information will useful for other local municipalities and veterinarians involved in dog population control programs.
MATERIALS AND METHODS
Enrolled animals: This was a voluntary program supported by Chiang Mai University College of Veterinary Medicine and the ompanion Animal Control Project of Chiang Mai Municipality between January, 2015 and April, 2015. Thirty healthy female owned dogs, between the ages of 1-10 years were enrolled (The protocol of this study was improved by Animal Ethic Committee, Faculty of Veterinary Medicine, Chiang Mai University to use animal enrolled in study (No. S30/2557)). These dogs varied by breed and there was no history of prior antibiotic use and all of the data were managed with commercial available software program (Excel 2013, Microsoft Corp, Redmond, Wash). Surgical procedures were done by Veterinary Services of the Chiang Mai City Municipality.
Sample collection: Sample collection occurred from February through April, 2015. There were 4 surgeons performing ovariohysterectomies at the sterilization room of the Veterinary section, Chiang Mai Municipality. Four sets of surgical instruments were sanitized in alkyl dimethyl ammonium chloride (Umonium38, Huckert International, Nivelles, Belgium) for at least 15 min. Cotton swabs were used to collect samples from three surgical sites (two before cutting and one after), surgeon’s hand and pooled sample from surgical instruments. After surgical prep with betadine scrub and an alcohol wipe, samples were obtained from the (1) Area surrounding the incision (ASR) within the prepped area, (2) The incisional site before surgery (INB) and (3) The incision site after closure and suturing (INA). A swab sample from the surgeon hand was collected after scrubbing (SHD). A pooled swab sample was collected from the following instruments including the rat toothed forceps, tissue scissors, Allis tissue forceps and the needle holder (INS).
Bacteriological methods: The samples were immediately inoculated on sheep blood agar and Mac-Conkey agar. Agar plates were incubated at 37°C for 24-48 h at the Central Laboratory of Faculty of Veterinary Medicine, Chiang Mai University. If multiple colonies were present up to three colonies were characterized. This was more common from the dog skin collection sites. Bacterial identification was performed by examining colony type and morphology, catalase test and Gram stain. Biochemical tests were used to confirm Bacillus sp. and Staphylococcus sp.14.
Antimicrobial susceptibility: Antimicrobial susceptibility was performed by disc diffusion according to Clinical and Laboratory Standards Institute15 guidelines in Mueller-Hinton agar (Oxoid). The following 12 antimicrobials were used: Ampicillin (AMP: 10 μg), amoxicillin (AML: 10 μg), amoxicillin and clavulanic acid (AMC: 30 μg, amoxicillin: 20 μg and clavulanic acid: 10 μg), cefotaxim (CTX: 30 μg), cephalexin (CEP: 30 μg), (chloramphenicol ｩ: 30 μg), ciprofloxacin (CIP: 5 μg), doxycycline (DO: 30 μg), enrofloxacin (ENR: 5 μg), gentamycin (CN: 10 μg), penicillin (P: 10 U), streptomycin (STP: 25 μg), sulfametoxazole and trimethoprim (SXT: 23.5 and 1.25 μg).
Analysis: Descriptive summary statistics were provided to characterize the frequency of isolation by site and describe the resistance profiles of Staphylococcus and Bacillus isolates. When multiple isolates were available by site with similar susceptibility profiles, only one was used in the summary analysis. The chi-square test was used when appropriate to compare bacterial isolates among before operation procedure and after operation procedure. Analyses were performed with free available statistical software (R program, R version 3.3.1, The R Foundation of Statistical Computing).
RESULTS
Age was available on 27 of 30 dogs. Dogs ranged in age from 0.5-6 years (mean 2.4). Bacterial isolates were recovered from 28 (93%) dogs; 17 (57%) surgeon hands and 8 (27%) pooled surgical equipment swabs. Multiple different isolates were recovered from the area around the surgery site. For the analysis only unique isolate patterns were included based on the susceptibility profile. Eighty two unique bacterial isolates were recovered from 150 collected samples (Table 1). Of the 82 isolates, 18 were identified as Staphylococcus sp. and 64 were identified as Bacillus sp. In three surgeries, Staphylococcus sp., was recovered from multiple samples (i.e., surgeon hands and the skin during the same surgery). In 24 surgeries, Bacillus sp., was recovered from one or more sites. Total of 7 isolates (n = 7) were recovered from the disinfected surgical equipment. A comparison of frequency of bacterial recovery from the surgical incision site before and after the surgical procedure was similar (p = 0.8).
The susceptibility of Staphylococcus sp. and Bacillus sp., isolates to individual antimicrobials is shown in Table 2 and 3. Staphylococcus sp., were generally susceptible (e.g., <85%) to amoxicillin/clavulanic acid, cephalexin, chloramphenicol and gentamycin. Staphylococcus isolates were resistant (e.g., <50%) to ampicillin, penicillin, streptomycin and sulfametoxazole/trimethoprim. Bacillus sp., isolates were susceptible (e.g., <85%) to most antibiotics except penicillin (72% susceptible).
Resistance profiles from the Staphylococcus isolates are summarized in Table 4. Thirteen different antimicrobial resistance patterns were observed for Staphylococcus sp. Four isolates were pan-sensitive. Ten of 18 isolates were resistant to three of more classes of antibiotics. Seventeen different antimicrobial resistance patterns were observed for Bacillus sp., 35 (55%) were susceptible to all antibiotics in the panel. Eleven isolates only had reduced susceptibility to penicillin.
| Table 1: | Recovery of Staphylococcus spp. and Bacillus spp. by site |
| Table 2: | Antibiotic Staphylococcus sp., susceptibility of recovered isolates |
| *Susceptibility done on 17 isolates | |
| Table 3: | Antibiotic Bacillus sp., susceptibility of recovered isolates |
| Table 4: | Antimicrobial resistant patterns of Staphylococcus spp., isolates (n = 14)* |
| *Four isolates were pan-sensitive | |
Five Bacillus sp., isolates were resistance to 3 or more different antimicrobial classes.
DISCUSSION
This study demonstrated that even in settings with limited resources such as city animal control programs, aseptic practices can be followed limiting the potential contamination of surgical sites16,17. These surgeries were done in a single room, where preparation including hair clipping, skin disinfection, surgery and surgical recovery. To the researchers knowledge, none of the dogs developed post-surgical infections. Surgeons need to pay attention and follow standard procedures for surgical preparation including use of a surgical hand scrub, wearing sterile gloves and practicing sterile technique to reduce bacterial contamination. Other aspects of operating room etiquette include limiting access of personnel to the surgical room during procedures and maintaining flow from lean to irty areas.
This study also provides insight into the degree of bacterial resistance observed among commensal bacteria in a Thailand dog population control program. The identification of Staphylococcus with reduced susceptibility to a number of different classes of antibiotics is evidence that multi-drug resistant infections are a present day reality18. Both Staphylococcus and Bacillus were readily recovered from the area around the incisional site irrespective of previous disinfection. Staphylococcus sp., is a normal commensal of the canine skin19,20 and in some studies 46% of skin samples readily recovered Staphylococcus9,21. Also, in a study evaluating three different disinfectant protocols, researchers recovered Staphylococcus intermedius in 9.2% and Staphylococcus sp., in 8.4% of samples collected after antisepsis17. This is not surprising; the surgery site is not a sterile environment. This fact re-emphasizes the value of aseptic technique for routine procedures. Some of the recovered bacteria were multi-drug resistant, especially the Staphylococcus isolates22,23. This may reflect the emergence of multi-drug resistant Staphylococcus pseudintermedius24,25 and healthy dogs can be a reservoir of antimicrobial-resistant bacteria which may be transferred to surgeon and their owners26.
To prevent nosocomial and zoonotic transmission, incorporation of infection prevention practices is important for all veterinary settings just like in human health-care setting. These efforts include the comprehensive promotion of infection prevention practices in companion animal settings including animal shelters27. The thoughtful and appropriate use of antibiotics as part of antibiotic stewardship programs should be incorporated in these plans28. Generally, in less than ideal settings antibiotics are often prescribed before and after the surgical procedure. The use of antibiotics is generally contra-indicated for the healthy patient undergoing routine, aseptic procedures. Findings from the study have implications for standard and appropriate surgical protocols in dog control sterilization in such settings and the optimal use of antibiotics to prevent veterinary-setting acquired infections. As a result of this study, the Veterinary Services of the City of Chiang Mai Municipality changed their policy of providing antibiotics post surgically.
In pet population control efforts globally, reproductive surgical sterilization is an important tool in broad efforts to control pet over-population29. Adherence to aseptic technique is possible in limited resource settings. With current concerns about the transfer and transmissibility of antibiotic resistant elements, it is important to utilize antibiotics optimally and review practices even in sub-optimal conditions.
CONCLUSION
Reproductive surgical sterilization is an important tool in efforts to control pet overpopulation and also to eliminate rabies virus, even in limited resource settings. With ongoing global concerns about emerging antibiotic resistance, implementation of optimum aseptic technique is needed. Findings from this study support standard and appropriate surgical protocols in dog control sterilization in limited resource settings that can uses where similar places and which supports optimal antibiotic use policies.
ACKNOWLEDGMENTS
We appreciate the support from the ompanion Animal Control Project of Chiang Mai Municipality and also the Central Laboratory of Faculty of Veterinary Medicine, Chiang Mai University for the laboratory helpful.