Presurgical Antiseptic Efficacy of Chlorhexidine Diacetate and Providone-Iodine in the Canine Preputial Cavity
Antiseptic flushing of the canine prepuce and its exclusion from the surgical field are recommended before abdominal surgery to reduce the risk of bacterial contamination. The authors cultured the preputial cavity of 60 dogs prior to and following flushing with 0.05% chlorhexidine diacetate, 1% povidone-iodine, or 0.9% saline control. Bacterial growth was evaluated using a semiquantitative method, and bacterial organisms were subsequently identified. There were no significant differences between povidone-iodine and the saline control in any of the variables assessed. Chlorhexidine resulted in a significant decrease in the proportion of positive postflush cultures compared with povidone-iodine. Although not significant, the difference in adverse reactions between povidone-iodine (25%) and chlorhexidine diacetate (5%) suggests clinical relevance. Based on the results of this study, a 2 min flush with 0.05% chlorhexidine diacetate is recommended for presurgical preparation of the preputial cavity.
Introduction
Antiseptic flushing of the canine prepuce and its exclusion from the surgical field are recommended before abdominal surgery to reduce the risk of bacterial contamination.1 Preputial antiseptic flushing is also the recommended treatment of purulent discharge associated with balanoposthitis.2 Previous studies have evaluated the ability of various antiseptics to reduce the bacterial population on the skin of dogs; however, none have evaluated the effect of antiseptics within the preputial cavity.3–8
In a study of preputial antisepsis in uncircumcised children, it was determined that povidone-iodine could not completely eliminate bacterial contamination. Twenty-six percent of these children had persistent positive cultures and were considered at risk for developing an infection postoperatively if their prepuce was used in reconstructive surgery.9
Bacteria commonly isolated from within the prepuce of dogs are similar to cutaneous organisms. These include potential pathogens such as Pasteurella multocida, β-hemolytic streptococci, Escherichia coli, Staphylococcus aureus, and coagulase-positive staphylococci.10–13 This mixed population supports the recommendation for effective presurgical antisepsis while accounting for the sensitivity of regional mucus membranes when the penis and prepuce are included in the surgical field of procedures such as laparotomy.1,2,14
Postoperative wound infection remains a major concern in human and veterinary medicine.15–20 The incidence of postoperative infections is related in part to the degree of contamination within the surgical field.15–17,20,21 Reducing contamination may be especially important in males, who are at an increased risk for postoperative infections in both humans and animals.21,22 Because the prepuce and penis may be included and manipulated within the surgical field during procedures such as cystotomy and urethrostomy, the authors wanted to examine the impact of antiseptic flushing on residual bacterial contamination within the canine preputial cavity. The authors hypothesized that there would be no significant difference in bacterial reduction or residual bacterial growth between 0.05% chlorhexidine diacetate (CD) and 1% povidone-iodine solution (PI).
Materials and Methods
Patient Selection
Sixty male dogs presenting for various surgical procedures were sampled. Informed consent was obtained from all owners prior to the sampling procedure. Dogs were excluded if they had a recent history (<2 wk) of urinary tract disease, had evidence of active pyoderma, or were currently receiving antibiotics for any reason. Twenty dogs were assigned to one of three groups (i.e., CD, PI, or 0.9% saline control [SC]) through block randomization, with 10 dogs per block.
Patient Sampling
For the purpose of this study, the preputial cavity was defined as the space from the mucocutaneous junction of the preputial orifice to the fornix, inclusive of all penile mucosa extending to the urethral orifice. Antiseptic solutions were prepared daily by dilution in sterile buffered 0.9% saline. Unused antiseptic was discarded at the end of the day. The CD solution was prepared by adding 25 mL of 2% chlorhexidine diacetate solutiona to 975 mL of 0.9% saline. To make the PI solution, 100 mL of 10% povidone-iodine solutionb was added to 900 mL of 0.9% saline.
After induction of anesthesia, all dogs were placed in dorsal recumbency and 5 cm of peripreputial hair was clipped. The preputial orifice and surrounding skin were scrubbed with a gloved hand using 4% chlorhexidine gluconate for 2 min then rinsed with a sterile saline-soaked sponge to reduce the risk of sample contamination. Care was taken to ensure the antiseptic was not introduced into the preputial cavity. A commercial swabc containing transport media was premoistened with one drop of sterile saline. The preputial cavity was cultured by advancing the swab to the level of the fornix and rotating it around the bulbus glandis for 5 sec to ensure adequate sampling. The antiseptic solution (or saline for control group) and saline rinse were poured into individually sterilized bowls. Due to the colors of the different antiseptics, the primary investigator was not blinded to the solution being tested. The preputial cavity was flushed using a sterile 12 mL curved tip syringe. The syringe was introduced into the preputial orifice and the prepuce was filled until it was mildly distended. This amount varied based on individual patient size and anatomy. The prepuce was manually agitated, drained, and the procedure was repeated. All dogs were flushed six times over 2 min. After antiseptic flushing, the preputial cavity of all dogs was flushed twice with 0.9% saline to remove residual antiseptic before collecting the postflushing swab for culture using the same technique as described for the antiseptic.
All samples were collected by the primary investigator. Sterile gloves were worn for all sample collections. Five minutes after sample collection, the preputial and penile mucosa were examined for evidence of tissue reactions such as erythema, wheals, and “weeping” of serum.3,4 Adverse reactions were subjectively graded as none, mild (erythema of mucosal surface), moderate (erythema and/or wheals), or severe (erythema, wheals, and weeping of serum).
Sample Handling and Preparation
All swabs were refrigerated at 4°C until they were transferred to a reference diagnostic laboratoryd. All samples were plated within 24 hr of collection. All isolations and culture evaluations were performed by the same microbiologist who was blinded to the type of flush solution tested. Swabs were plated on 5% bovine blood agar, phenylethyl alcohol agar, and MacConkey agar sequentially, and streaked for isolation. Blood and phenylethyl agar plates were incubated at 37°C in 5% CO2. MacConkey agar plates were incubated at 37°C in ambient air. Cultures were examined after 24 hr and 48 hr of incubation. Semiquantitative scoring of total bacterial growth was performed using a quadrant streak method.23,24 Growth was recorded as none (0), very light for ≤10 colonies in the first quadrant only (1), light for >10 colonies in the first quadrant only (2), moderate for growth spreading into the second quadrant (3), and heavy for growth that spread into the third or fourth quadrant (4). The highest level of growth on any of the three plates was recorded as the bacterial growth score (BGS). Bacterial species and their prevalence were noted prior to subculturing for identification purposes. Bacillus spp., Corynebacterium spp., and nonhemolytic streptococci were identified to the genus level only. Potential pathogens such as coagulase-positive staphylococci, Pseudomonas spp., and coliforms were identified to the species level using conventional biochemical testing. Due to recent debate in the veterinary literature, the Staphylococcus intermedius group (SIG), which includes S. delphini, S. intermedius, and S. pseudintermedius, were not differentiated and were classified as SIG for the purpose of this study.
Statistical Analysis
Data were analyzed using statistical softwaree and a public domain statistical calculator. Reduction in BGS between sampling times was compared among all groups and between groups using the Wilcoxon rank-sum test. The proportion of positive preflush samples among groups was compared using χ2 analysis. Proportion of samples with positive postflush cultures, high residual bacterial growth (defined as postflush BGS >1), and adverse reactions were compared between groups within sampling times using the Fisher's exact test. For pairwise comparisons between groups, the Bonferroni adjusted significance level was set at PB<0.017 (=0.05/3), otherwise the significance level was set at P<0.05. Samples with negative preflush cultures (BGS=0) were excluded from analysis of proportion of positive postflush cultures and proportion with high residual BGS.
Results
Sixty male dogs (11 castrated and 49 intact) were included in the study. Median age was 1.25 yr (range, 0.5–14 yr). The most common breeds were Chihuahua (n=7), mixed-breeds (n=6), dachshund (n=6), boxer (n=5), pug (n=4), Yorkshire terrier (n=4), American pit bull terrier (n=4), and Labrador retriever (n=3).
Data regarding the number of positive cultures, changes in BGS, and adverse reactions have been summarized in Table 1. Sixty-five percent of preflush samples exhibited bacterial growth. This difference was not significant among groups (P=0.80). The number of positive pre- and postflush cultures were 14/20 and 11/20 for SC, 13/20 and 1/20 for CD, and 12/20 and 7/20 for PI, respectively. There was a significant reduction in the postflush BGS in all three groups (P=0.01). All negative preflush samples remained negative postflushing, and no samples increased in BGS postflushing.
Excludes samples with no growth (BGS=0) preflush
Data are presented as number (percent) unless otherwise indicated. BGS, bacterial growth score; CD, 0.05% chlorhexidine diacetate; PI, 1% povidone-iodine solution; SC, 0.9% saline control.
The bacterial species identified prior to and following preputial flushing have been summarized in Table 2. The bacterial species identified following preputial flushing with PI included coagulase-negative staphylococci, SIG, β-hemolytic streptococci, E. coli, Klebsiella spp., and Enterobacter spp. The single dog with a positive culture after flushing with CD grew a SIG.
Data are presented as number (percent). CD, 0.05% chlorhexidine diacetate; PI, 1% povidone-iodine solution; SC, 0.9% saline control; SIG, Staphylococcus intermedius group.
Comparison of CD, PI, and SC
Compared with the SC, CD resulted in a significant reduction in postflush BGS (PB=0.008), a significant reduction in the proportion of samples with high residual bacterial growth (PB=0.016), and a significant reduction in the proportion of positive postflush cultures (PB<0.001). When PI was compared with the SC, there was no significant difference in the reduction of postflush BGS (PB=0.088), proportion with high residual bacterial growth (PB=0.701), or proportion of positive postflush cultures (PB=0.401). Finally, when CD was compared with PI, there was a significant reduction in positive postflush cultures (PB=0.011) with CD; however, there was no significant reduction of postflush BGS (PB=0.110) or proportion of samples with high residual bacterial growth (PB=0.039).
Adverse Reactions
There were no adverse reactions noted in SC. One dog in CD (1/20) and five dogs in PI (5/20) exhibited mild adverse reactions consisting of diffuse erythema of the penile and preputial mucosa. There was no significant difference in the incidence of adverse reactions between groups CD and SC (PB=0.999), PI and SC (PB=0.047), or CD and PI (PB=0.181).
Discussion
The results of this study support the authors’ hypothesis in part as there was no significant difference in bacterial reduction between CD and PI, but there was a significant decrease in the proportion of samples with positive postflush cultures with CD. CD significantly reduced bacterial growth compared with the SC. When PI was compared with the SC, there were no significant differences in any of the variables assessed. Although not significant, the difference in adverse reactions between PI (25%) and CD (5%) may influence decision making in the clinical setting.
Chlorhexidine and povidone-iodine are the most commonly used antiseptics in human and veterinary surgery.3–8,25–28 Chlorhexidine is rapidly bactericidal with a broad spectrum of activity, low toxicity, and is minimally affected by the presence of organic debris.14,25 The scrub formulation should only be used on intact skin, whereas the solution is reserved for wounds and mucus membrane application.14 The concentration of CD used in this study was chosen based on its bactericidal efficacy in experimentally induced wounds with minimal tissue reaction.29 This concentration has also been shown to be lethal to 100% of S. intermedius within 60 sec in vitro.30 In the current study, chlorhexidine was safe and highly efficacious, with only one dog having very light residual bacterial growth after antiseptic flushing. This suggested that when prepared with chlorhexidine using the methods described in this study, the prepuce would not contribute to contamination of the surgical field.
Potential pathogens identified in the PI postflush group included β-hemolytic streptococci, SIG, E. coli, Enterobacter spp., and Klebsiella spp. When identified intraoperatively, certain bacteria have been associated with a higher chance of subsequent wound infection.31 These include Klebsiella spp., Enterobacter spp., and E. coli, all of which were found in the postflush PI group and could contribute to postoperative infections.31 The surviving bacterial species, combined with the high proportion of dogs with persistent bacterial contamination after preparation with PI, make the use of PI suboptimal compared with CD in this study.
The concentration of PI used in this study was chosen based on its bactericidal activity against S. aureus. This dilute concentration has shown efficacy against S. aureus in vitro in as little as 15 sec, and Mycobacterium chelonei in as little as 2 min.32 Concentrations <1% resulted in significantly greater survival of S. aureus in one study.33 The efficacy of antiseptic flushing with PI may have been improved by further dilution or longer contact time. Povidone-iodine's antimicrobial activity is directly related to the level of free iodine in the solution.32 The concentration of free iodine follows a bell-shaped curve with dilute solutions having increased bactericidal activity compared with the 10% stock solution.25,32 Dilute solutions, however, are less effective than the stock solution in the presence of organic material.34 The presence of smegma within the preputial cavity may have provided organic debris competing for free iodine, which decreased its antiseptic efficacy. In humans, cleansing of the prepuce with surgical soap before antiseptic preparation resulted in lower bacterial counts than treatment with povidone-iodine alone, but failed to completely eradicate all bacterial contamination.9
The authors do not expect preoperative antisepsis to result in complete sterility of the operative site and recommend selecting the antiseptic that results in the greatest bacterial reduction with the lowest risk of adverse reactions. In this study, CD resulted in a significant decrease in positive postflush cultures compared with PI. Compared with SC, PI did not significantly reduce bacterial growth, and resulted in 33% of postflush cultures having unacceptably high residual bacterial growth. This was consistent with a report on the use of povidone-iodine used as a wound antiseptic in which neither povidone-iodine nor saline had significant bactericidal activity.35
In experimental and clinical trials of skin preparation in dogs, no significant difference was identified between chlorhexidine gluconate and povidone-iodine scrubs with regard to the percentage of negative cultures and percentage with high residual bacterial growth.3,4 There was a significant difference in the incidence of skin reactions, with approximately 50% of patients treated with povidone-iodine scrub developing acute contact dermatitis.3,4 In the current study, the effect of CD within the preputial cavity compared favorably to those of other skin preparation techniques, with 92% of patients having negative cultures after antiseptic flushing and none having high residual bacterial growth. The effect of PI did not compare as well, with 58% of patients having positive postflushing cultures and 33% having high residual bacterial growth. It is important to note that the authors chose >10 colony-forming units/plate to represent high residual bacterial growth. This number was chosen arbitrarily, and further research is required to determine an acceptable level of contamination for clean surgical procedures.
Previous studies evaluating the bacterial flora within the canine prepuce reported bacterial growth in 86–90% of dogs sampled.10–12 The lower percentage of dogs in this study with positive cultures prior to flushing was unexpected. This may be explained by the different sampling methods and the ability to reduce contamination from the external preputial environment, peripreputial skin, and hair in the current study. Alternatively, the age, breeding behavior, and regional differences of animals in the current study may have influenced the presence or absence of quantifiable bacterial growth within the preputial cavity.
The bacterial species that were identified prior to flushing were similar to those previously reported, which included SIG, β-hemolytic streptococci, E. coli, coagulase-negative staphylococci, Bacillus spp., and Enterobacter spp. Only one study has reported S. intermedius, with a prevalence of 9.5% within the prepuce.10 Another study identified S. intermedius in 10% and coagulase-negative staphylococci in 33% of urogenital samples from dogs.36 In the study reported here, the authors identified SIG in 23% and coagulase-negative staphylococci in 27% of the preflush samples. This is important because S. pseudintermedius and other coagulase-positive staphylococci are considered the pathogens responsible for most canine cutaneous infections.15,36 Staphylococcus intermedius is one of the most commonly isolated bacteria from surgical sites immediately postoperatively, with approximately 14–16% of samples being positive for contamination.5,37 In the current study, SIG was identified in 5% and 10% of samples following flushing with CD and PI, respectively.
Study Limitations
Quantitative plate counting is considered the gold standard for bacterial quantification; however, semiquantitative bacterial culture techniques have been validated in studies on IV catheter contamination and surgical site contamination.24,38–40 When evaluating the presence of bacterial growth, quantitative culture by immersion of catheter tips in saline followed by sonication or vortexing was not significantly different than the semiquantitative method of roll plating.38,39 The simplicity of the semiquantitative technique makes it the procedure of choice for routine work in the microbiology laboratory.39 As the goals of this study were to evaluate the presence or absence of bacterial growth after antiseptic flushing and to determine whether residual bacterial growth was excessive (defined as >10 colony-forming units/plate), the semiquantitative method provided a simple, reliable method to achieve these goals. A single swab was used to inoculate plates from least inhibitory to most selective. This allowed the final two plates to help identify and separate species whereas the initial plate was most representative of the total bacterial count. The use of swabs to evaluate bacterial growth after antisepsis has been validated in various animal and human models.8,9,24,41
No antiseptic inactivator was used in this study. The effect of inactivators may be negated during periods of storage or transport, making their use of questionable value when samples are not immediately inoculated.42 In the current study, the swabs were plated between 18 hr and 20 hr after collection, and the authors elected to flush the preputial cavity with saline to dilute any residual antiseptic to levels that were not bactericidal. During the study design, it was determined that the mean residual volume of antiseptic remaining in the preputial cavity was 2.5%. Two saline rinses resulted in final dilutions of 1.6×104 for PI and 6.4×104 for CD, which were well below the 2×102 for PI and 4×103 for CD that render them ineffective (thus negating the need for neutralization).33 Saline rinsing following antiseptic flushing is not routinely performed in the clinical setting. Although bacterial growth would no longer be affected beyond the inherent residual effect of the antiseptic, saline rinsing and antiseptic dilution may have reduced the true incidence and severity of adverse reactions.
The authors found that the volume of flush used varied based on the patient size and individual anatomy, and that patients of the same breed and size exhibited wide variations in their preputial capacity. Because all procedures were performed by the same investigator, the subjectivity of flushing was consistent.
Only the immediate effect of both antiseptics within the preputial cavity was studied. The duration of residual activity is unknown; however, chlorhexidine and povidone-iodine have been shown to be equally effective in dogs for up to 1 hr when applied to the skin of the trunk and for 24 hr when applied to the paws under a bandage.3,4,6 Potential modifications to the authors’ protocol that may have increased antiseptic efficacy include increased contact time, increased number of flushes, or preflushing to reduce the initial bacterial and biologic load. Future areas of research could include a blinded comparison of postoperative infection rates following laparotomies when the prepuce is incorporated in the surgical field.
The authors chose to only evaluate aerobic bacterial growth due to the difficulty in culturing anaerobic organisms and their unknown clinical significance. No attempt was made to compare culture findings based on the age or sexual status (intact versus neutered) of dogs in this study. It is unknown if age or the presence of male sex hormones influences bacterial population characteristics within the canine prepuce.
Conclusion
There was no significant difference between CD and PI in reduction of bacterial growth within the canine preputial cavity; however, CD resulted in a significant decrease in the proportion of positive postflush cultures. There was no significant difference in bacterial reduction between PI and the SC. The effect of residual bacterial contamination within the preputial cavity remains uncertain. These results suggest that when prepared with CD, the preputial cavity does not contribute to contamination of the surgical field. Based on the results of this study, a 2 min flush with 0.05% chlorhexidine diacetate is recommended for presurgical preparation of the preputial cavity.
Contributor Notes
S. Neihaus’ present affiliation is Chicago Veterinary Emergency and Specialty Center, Chicago, IL.
