Surgical site infections are potential complications of surgical procedures and can result in increased patient morbidity, additional treatment costs, and frustration of clients and veterinarians.1–3 Methicillin-resistant Staphylococcus pseudintermedius has rapidly emerged as a leading cause of SSI in dogs.1,3–6 Treatment of MRSP can be challenging because multidrug resistance is common and, possibly, because MRSP has the ability to adhere to materials and form biofilms.5,7,8
Foreign material, such as suture material introduced into a patient, poses an ideal substrate for bacterial adherence. Some bacteria can quickly adhere to surfaces and become reversibly or irreversibly attached through various mechanisms. Attached bacteria may be difficult to eliminate because they can be protected within biofilm; therefore, they can avoid impacts of the immune system and antimicrobial treatment.9,10 Avoiding contamination of a surgical site is the ultimate preventive measure but is not always possible. Therefore, measures to reduce the implications of bacterial contamination are needed.
One such measure involves the suture because different materials and surface coatings can impact the ability of bacteria to attach and survive. Many currently available sutures are coated with triclosan, a broad-spectrum biocide that has efficacy against gram-positive and -negative bacteria, including methicillin-resistant Staphylococcus aureus and MRSP.11–13 Although there has been extensive clinical research on the use of TC suture in human medicine, to the authors' knowledge, there remains little information on its use in veterinary medicine. A study14 was conducted to evaluate TC suture and wound closure with TC-polydioxanone or uncoated polydioxanone after tibial plateau leveling in dogs.14 The authors of that study14 found that SSI rates did not differ between these 2 suture groups.
In addition to the impact of coatings, various suture materials may have differences in propensities for bacterial adherence and colonization. Sutures are categorized into structural classes that differ in surface area available for bacterial attachment and consist of various materials that may also impact adherence. Structurally, suture is categorized as monofilament, multifilament, and barbed monofilament. Multifilament suture has a greater surface area than does monofilament suture, and bacterial adherence is greater for multifilament suture than for monofilament suture.15–18 Barbed suture is the newest suture class. It is a knotless wound closure device that has gained popularity in human medicine because of the ease of use (especially for minimally invasive surgery such as laparoscopy), reduction in surgical time, and cosmetic appeal.19,20 Investigators of several studies21–24 have described the use of barbed sutures in veterinary surgery. Similar to multifilament suture, barbed suture has a larger surface area, compared with that of its nonbarbed monofilament counterpart; therefore, bacterial adherence might be expected to be greater than that for monofilament suture.
Despite the increasing concern of SSI attributable to MRSP in veterinary medicine, little is known about the potential role of suture selection on adherence of MRSP and subsequent patient risk for development of SSI. The objective of the study reported here was to compare adherence of MRSP with monofilament, multifilament, barbed, and TC suture materials commonly used in small animal surgery.
Materials and Methods
Sample
Ten epidemiologically unrelated MRSP isolates with strong biofilm-forming ability were evaluated. Five types of suture were used to test bacterial adherence.
MRSP isolates
Ten epidemiologically unrelated MRSP isolates from dogs with clinical infections were used. The isolates previously had been categorized as strong biofilm producers.8 Isolates had been identified as MRSP on the basis of results of species-specific PCR assay and detection of mecA with a PCR assay or penicillin-binding protein 2a with a latex agglutination test.25 Isolates were stored at −80°C in protein-free, serumfree cryopreservation medium.a
Suture material
The 5 suture materials used in the study were monofilament polyglecaprone 25,b monofilament polydioxanone,c TC–monofilament polydioxanone,d braided polyglactin 910,e and barbed monofilament polydioxanone.f All suture material was 3-0 in size. Suture was cut aseptically into 2.5-cm segments. Two suture segments were used for each experiment (total, 5 cm/experiment). Portions of the barbed suture that lacked barbs were excluded from testing.
Adherence of MSRP to suture materials
Isolates were subcultured onto blood agar plates by incubation overnight at 35°C. Resulting colonies were inoculated into 5 mL of tryptone soy broth plus 1% glucose. Bacterial suspensions were created at a 0.5-McFarland standard (approx 108 CFUs/mL)26,27 by use of a calibrated spectrophotometer. Suspensions were mixed in a vortex device to break up cell clusters.26 Suture segments were then exposed to bacterial suspensions for 2 minutes, as described elsewhere.12 After exposure was completed, suture was gently rinsed 3 times with PBS solution and aseptically transferred to sterile tubes containing 5 mL of tryptone soy broth plus 1% glucose. Tubes were incubated for 24 hours at 35°C in an oscillating water bath.g Testing of all MRSP isolate–suture combinations was performed in triplicate with 2 suture segments/tube. Segments of polyglecaprone 25 were added to 5 mL of tryptone soy broth plus 1% glucose without bacteria to serve as a negative control sample.
After incubation was complete, suture segments were rinsed 3 times with PBS solution to remove nonadherent bacteria and then transferred into sterile test tubes that each contained 10 mL of PBS solution.26 Suture segments were subjected to sonicationh for 5 minutes to remove adherent bacteria, which was followed by mixing in a vortex device for 1 minute to ensure uniform suspension.27,28 Resulting suspensions were used to create serial dilutions of up to 10−4 CFUs/mL. An aliquot (100 μL) of each dilution was inoculated onto blood agar and incubated overnight at 35°C. The following day, the number of CFUs was manually counted and recorded. Counts for plates containing between 20 and 200 distinct colonies were used to calculate the mean number of CFUs per suture segment for each MRSP isolate–suture combination. No growth at the lowest dilution was recorded as 0.
Box-and-whisker plots of the mean log10 CFUs of MRSP that adhered to each of 5 suture materials. Each box represents the middle 2 quartiles (25% to 75%), the horizontal line in each box represents the median, and the whiskers represent the range (0% to 100%). Each black circle represents the mean log10 CFUs for triplicate assay of each suture strand for each of the 10 MRSP isolates.
Citation: American Journal of Veterinary Research 77, 2; 10.2460/ajvr.77.2.194
Scanning electron microscopy
To confirm the presence of adherent bacteria and associated biofilm on suture after incubation, rinsing with PBS solution, and sonication, scanning electron microscopy of 1 segment for each of the 5 suture types was performed. Suture was inoculated with MRSP and incubated as described previously. After incubation was completed, suture segments were rinsed 3 times with PBS solution and then immediately fixed in 2% glutaraldehyde. Fixed samples were stored at 4°C until the time of further processing. In addition, suture segments were rinsed 3 times in PBS solution, subjected to sonication, and mixed in a vortex device as described previously; segments were then immediately fixed in 2% glutaraldehyde and stored at 4°C until time of further processing. The day before image acquisition, suture segments were washed 3 times (10 min/wash) with Sorensen phosphate buffer (0.07M; pH, 6.8). Segments then were fixed by incubation with 1% osmium tetroxide for 1 hour at 22°C, washed 3 times (15 min/wash) in Sorensen phosphate buffer, dehydrated by immersion in a series of ethanol solutions (50%, 70%, 80%, and 90%) and 3 immersions in 100% ethanol (15 min/immersion), critical point dried, mounted onto metal specimen stubs, and sputter coated with 20 nm of gold-palladium. The following day, scanning electron microscropyi was performed on suture segments. Suture was subjectively evaluated for adherent cells and extracellular matrix.
Statistical analysis
Mean values were calculated for the triplicate of each MRSP isolate–suture combination. Normality was assessed by use of the Shapiro-Wilk goodness-of-fit test. A Steel-Dwass test was used to compare median log10 CFUs among suture types. Values were considered significant at P < 0.05.
Results
There was significantly (P = 0.001) less adherence of MRSP to TC–monofilament polydioxanone than to polyglecaprone 25, braided polyglactin 910, barbed monofilament polydioxanone, or monofilament polydioxanone (Figure 1). There was significantly (P = 0.03) less adherence of MRSP to polyglecaprone 25 than to braided polyglactin 910. There was no significant difference in adherence of MRSP between barbed monofilament polydioxanone and polyglecaprone 25 (P = 0.07), braided polyglactin 910 (P = 1.00), or monofilament polydioxanone (P = 0.10).
Evaluation of SEM images revealed evidence of adherent bacteria on all suture types, except TC–monofilament polydioxanone (Figure 2).
Scanning electron microscopic images of barbed monofilament polydioxanone after incubation with MRSP (A) and after subsequent sonication to remove adherent bacteria (B). Sputter coated with gold-palladium; bar = 300 and 135 μm for panels A and B, respectively.
Citation: American Journal of Veterinary Research 77, 2; 10.2460/ajvr.77.2.194
Discussion
Bacterial adherence to suture has received considerable attention in human medicine; however, this topic has received little attention in veterinary medicine. In the study reported here, there were significant differences in MRSP adherence to various suture types; this finding could have clinical relevance. Adherence of MRSP to TC–monofilament polydioxanone was significantly less, compared with adherence of MRSP to uncoated suture. Other in vitro studies,12,13,29–31 which involved the use of a variety of bacteria, also found significant antibacterial effects of TC suture. Furthermore, TC suture has been extensively evaluated in humans, with clinical trials finding that the use of TC suture is associated with lower SSI rates.32,33 Despite this fact, to the authors' knowledge, only 1 clinical trial has been conducted to evaluate the use of TC suture in veterinary medicine.14 Authors of that study14 did not find a reduction in SSI rate with the use of TC suture in dogs that underwent tibial plateau leveling osteotomy. Further evaluation of TC suture in veterinary patients is required to determine whether the in vitro effects found in the present study will correspond to a clinical benefit.
Similar to results for other biocides, resistance to triclosan can occur. However, triclosan resistance in MSRP has not been reported, and the concentration of triclosan used to coat the suture (0.236%) is > 4,000 times as high as the minimum inhibitory concentration recorded for 25 canine MRSP isolates in 1 study.11 Rarity (or absence) of triclosan resistance in MRSP, extremely high concentrations of triclosan achieved at suture sites, and negligible exposure of patients' endogenous microbiota as a result of use of coated suture suggest that the emergence of bacterial resistance is of limited concern.
Triclosan is thought to behave in a manner similar to that of antiseptics, exerting its bactericidal effects through several mechanisms.11,13,16 In contrast, antibacterials have only 1 mechanism by which they exert their effects, and bacterial resistance is more easily achieved. Finally, triclosan elutes from the suture, which provides an antibacterial environment at the suture site and in surrounding tissues.12,13 As a result of elution that protects incision sites, multiple mechanisms of bacterial killing, and high concentrations relative to the minimum inhibitory concentration that is safe for patients, triclosan appears to be an excellent agent to combat MRSP colonization and prevent SSI.
In accordance with results of other studies,15–17 we found in the study reported here that bacterial adherence to braided polyglactin 910 was greater than bacterial adherence to TC–monofilament polydioxanone and polyglecaprone 25. This was not surprising, considering a multifilament suture has an increased surface area (compared with that of a monofilament suture), which allows trapping of bacteria. Investigators of 1 study30 found that TC–polyglactin 910 reduced bacterial adherence such that it was comparable to bacterial adherence for monofilament suture. This finding, combined with the fact that TC–monofilament polydioxanone had the lowest MRSP adherence of any suture material tested in the present study, may indicate that TC suture is preferable for use in patients at increased risk of SSI, especially if polyglactin 910 is selected for use by a surgeon. Although polyglactin 910 offers distinct advantages (including easier handling, increased tensile strength, and better knot security) over polyglecaprone 25, monofilament polydioxanone, and barbed suture, the potential implications of SSI and the increasingly common problem of SSI attributable to MRSP indicate that the potential beneficial effects of TC suture or suture with lower MRSP adherence should not be overlooked.
The use of barbed suture is gaining popularity in veterinary medicine.21–23 The use of barbed suture may be most advantageous when performing minimally invasive surgery (eg, laparoscopy) because tying knots inside a body cavity can be challenging and may result in prolonged surgical time, a factor that has a clearly established association with SSI in human and veterinary medicine. Another important consideration with barbed suture is the concern that the increased surface area, relative to that of monofilament suture, would allow for greater bacterial adherence. The study reported here is one of only a few studies in human and veterinary medicine that have been conducted to evaluate bacterial adherence to barbed suture; to our knowledge, the present study is the only one in which MRSP adherence to barbed suture has been assessed. Results for the present study are in agreement with those of another study29 in which investigators found that barbed monofilament suture had bacterial adherence similar to that for monofilament suture. Further in vivo evaluation of barbed suture is required to determine the risk of SSI, compared with the risk of SSI after use of monofilament suture; however, analysis of the in vitro results reported here suggested that there was no additional bacterial adherence to barbed suture, compared with the adherence to monofilament suture.
The study reported here had limitations. Caution must be used when extrapolating results from an in vitro study to a clinical situation because a surgically closed wound likely represents a considerably different environment for bacterial adherence to suture material. Additionally, we chose strong biofilm-forming isolates of MRSP and a standardized bacterial amount (0.5 MacFarland standard), which may not be representative of the type of bacteria and bacterial load found in surgically closed wounds.
For the study reported here, we found that adherence of MRSP was lowest to TC–monofilament suture (of all the absorbable sutures tested); thus, TC–monofilament suture should be considered for use in surgical patients at high risk of SSI. Adherence of MRSP to barbed suture was comparable to adherence of MSRP to uncoated monofilament suture. This suggested that barbed suture may be used in procedures that benefit from its distinct advantages, without concern of increased bacterial adherence and subsequent SSI development. In accordance with results of other studies of other bacteria, adherence of MRSP to braided polyglactin 910 was greater than adherence to polyglecaprone 25 or TC–monofilament polydioxanone. Thus, TC–polyglactin 910 may be considered for use if multifilament suture is selected and may provide the benefit of a reduced potential for development of SSI.
Acknowledgments
Supported by Johnson & Johnson Inc. Ms. Morrison was supported by a Zoetis Summer Student Fellowship.
Presented in part at the 3rd International Society for Companion Animal Infectious Disease Symposium, Niagara-on-the-Lake, ON, Canada, October 2014, and the 12th Veterinary Endoscopic Society Conference, Santa Barbara, Calif, April 2015.
ABBREVIATIONS
| MRSP | Methicillin-resistant Staphylococcus pseudintermedius |
| SSI | Surgical site infection |
| TC | Triclosan-coated |
Footnotes
Cryostor, Innovatek Medical Inc, Mississauga, ON, Canada.
Monocryl, provided by Ethicon Inc, Somerville, NJ.
PDS II, provided by Ethicon Inc, Somerville, NJ.
PDS Plus Antibacterial, provided by Ethicon Inc, Somerville, NJ.
Vicryl, provided by Ethicon Inc, Somerville, NJ.
Stratafix Spiral PDO, provided by Ethicon Inc, Somerville, NJ.
SHEL LAB shaking water bath, 17 L, Sheldon Manufacturing Inc, Cornelius, Ore.
Ultrasonic cleaner model 2510, Branson Ultrasonic Corp, Danbury, Conn.
Hitachi S-570, Hitachi America Ltd, Washington, DC.
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