Lavage with at least 2.5 mL/cm sterile saline reduces bacterial load in a subcutaneous tissue model

Cody B. Westmoreland Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Search for other papers by Cody B. Westmoreland in
Current site
Google Scholar
PubMed Close
 BS
,
Sara D. Lawhon Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Search for other papers by Sara D. Lawhon in
Current site
Google Scholar
PubMed Close
 DVM, PhD, DACVM
,
Jing Wu Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Search for other papers by Jing Wu in
Current site
Google Scholar
PubMed Close
 MD, PhD
,
Nicholas D. Jeffery Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Search for other papers by Nicholas D. Jeffery in
Current site
Google Scholar
PubMed Close
 BVSc, PhD, DECVN
, and
Vanna M. Dickerson Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX

Search for other papers by Vanna M. Dickerson in
Current site
Google Scholar
PubMed Close
 DVM, MS, DACVS https://orcid.org/0009-0008-0096-9942
Open access

Abstract

Objective

To quantify the volume of lavage required to decrease the bacterial load below a standard of 105 CFUs/mL on a subcutaneous tissue model.

Methods

This was a benchtop experimental study conducted between May 1, 2023, through July 31, 2023 that included 20 sterile silicone skin models with a 10-cm incision. The silicone skin model was inoculated with a 1,000-fold dilution of approximately 1.5 X 108 CFUs/mL of isolated Staphylococcus pseudintermedius or Escherichia coli. Bacterial quantification samples were taken preinoculation, 20 minutes postinoculation, and after incremental saline lavage to total a volume of 2.5, 5, and 10 mL/cm incisional length bacterial quantification.

Results

For S pseudintermedius and E coli, a reduction of bacterial colonies below 105 CFUs/mL was noted in all lavage volumes, with an initial 3-log decrease from postinoculation to the 2.5 mL/cm lavage for both S pseudintermedius and E coli. Microbial growth demonstrated a 1-log reduction with increased lavage volumes from 2.5 mL/cm to 10 mL/cm for S pseudintermedius. For E coli, microbial growth demonstrated a 1-log reduction with the second lavage (total of 5 mL/cm) and a half-log reduction with the third lavage (total of 10 mL/cm).

Conclusions

Surgeons should consider a minimum of 2.5 mL/cm lavage when aiming to decontaminate SC tissues in linear surgical wounds. Further work in clinical cases is required to evaluate differences in live tissue compared to this model.

Clinical Relevance

This work provides surgeons with a reference point for deciding what volume of lavage to use for purposes of decontaminating an SC space.

Abstract

Objective

To quantify the volume of lavage required to decrease the bacterial load below a standard of 105 CFUs/mL on a subcutaneous tissue model.

Methods

This was a benchtop experimental study conducted between May 1, 2023, through July 31, 2023 that included 20 sterile silicone skin models with a 10-cm incision. The silicone skin model was inoculated with a 1,000-fold dilution of approximately 1.5 X 108 CFUs/mL of isolated Staphylococcus pseudintermedius or Escherichia coli. Bacterial quantification samples were taken preinoculation, 20 minutes postinoculation, and after incremental saline lavage to total a volume of 2.5, 5, and 10 mL/cm incisional length bacterial quantification.

Results

For S pseudintermedius and E coli, a reduction of bacterial colonies below 105 CFUs/mL was noted in all lavage volumes, with an initial 3-log decrease from postinoculation to the 2.5 mL/cm lavage for both S pseudintermedius and E coli. Microbial growth demonstrated a 1-log reduction with increased lavage volumes from 2.5 mL/cm to 10 mL/cm for S pseudintermedius. For E coli, microbial growth demonstrated a 1-log reduction with the second lavage (total of 5 mL/cm) and a half-log reduction with the third lavage (total of 10 mL/cm).

Conclusions

Surgeons should consider a minimum of 2.5 mL/cm lavage when aiming to decontaminate SC tissues in linear surgical wounds. Further work in clinical cases is required to evaluate differences in live tissue compared to this model.

Clinical Relevance

This work provides surgeons with a reference point for deciding what volume of lavage to use for purposes of decontaminating an SC space.

Keywords: infection; surgery; small animal; contamination; lavage

Surgical site infections (SSIs) are a recurrent challenge in postoperative management. Infections can result in incisional dehiscence, illness, increased cost to the client, and, in some cases, may lead to death or euthanasia. Surgical site infection is of particular concern in patients undergoing gastrointestinal surgery as contact with gastrointestinal contents (either directly or indirectly via soiled gloves or instruments) may lead to contamination of the SC tissues. Gastrointestinal surgeries are considered clean-contaminated procedures, which have historically been associated with a 5.9% SSI rate.1 However, more recent literature focusing on SSI following gastrointestinal surgery specifically reports a higher infection incidence of 7% to 10%.2,3 Escherichia coli was the most common aerobic bacterium isolated following gastrointestinal procedures in these studies, and multidrug-resistant infections are common.2,3 Staphylococcus pseudintermedius is another bacterium commonly implicated in SSIs.4

Many clinical strategies may be employed to reduce the risk of SSI, including aseptic preparation of the skin and the use of intraoperative prophylactic antibiotics.5 In cases of abdominal surgery, peritoneal lavage promotes the removal of bacteria that may have translocated into the peritoneal cavity or that are present due to contamination following gastrointestinal surgery or perforation. It has been documented that in cases of septic peritonitis, 200 to 300 mL/kg of lavage volume provides clinically relevant decreases in multidrug-resistant bacterial isolates.6 While human literature supports that wound lavage is superior to no lavage at preventing SSI, a volume for wound lavage has not been clearly defined.7

The SC tissue remains a potential location of contamination during gastrointestinal surgery. Bacterial burden of 105 CFUs/g of tissue has previously been shown to be associated with a higher risk of developing a clinical infection; wound irrigation reduces that bacterial load.8 However, to our knowledge, there is no validated standard volume of SC lavage to reduce bacterial growth below that level.8 The purpose of this study was to establish a volume of lavage capable of reducing bacterial growth below 105 CFUs/mL in an SC tissue model.

Methods

Setup

An artificial human silicone model (model No. SP01; Shawn Science) mimicking the tissue layers encountered during routine abdominal surgery, including skin, SC tissue, and muscle, was used for each experiment. A 10-cm longitudinal incision was made in the center of each model with a #10 surgical blade through the skin and SC model layers, up to but not including the muscle model layer.

A fresh 24-hour culture of either E coli (ATCC 25922) or a clinical isolate of S pseudintermedius originally derived from a dog and confirmed as S pseudintermedius by matrix-assisted laser desorption/ionization time-of-flight (Biotyper; Bruker Daltonics) and 16S ribosomal DNA sequencing was transferred into a sterile vial containing 3 mL of 0.9% NaCl. The bacterial concentration was standardized to a McFarland 0.48 to 0.52 (approx 1.5 X 108 CFUs/mL) using a calibrated nephelometer. The bacterial suspension was diluted in a 10-fold series using 1 mL to 9 mL of 1X PBS (catalog No. 10010023; Life Technologies Corp) or Dulbecco PBS (DPBS; catalog No. 21–0310CV; Coming) solution based on laboratory availability until a dilution power of 10−3 from the original standard was achieved (approx 1.5 X 105 CFUs/mL). The 10−3 dilution was used to inoculate the model. Three additional 10-fold dilutions were made using 100 µL to 900 µL of 1X PBS or DPBS solution to make a dilution factor of 10−4, 10−5, and 10−6. One hundred µL of the 10−4, 10−5, and 10−6 dilutions was individually plated to trypticase soy agar supplemented with 5% sheep blood plates and incubated at 37 °C overnight in an air atmosphere for preinoculation bacterial quantification.

Inoculation

Before each lavage trial started, the model was placed on a standard surgical plastic tray in a horizontal position, wrapped in a single layer of muslin, and plasma gas was sterilized for 1 hour. The surgical wrap was opened, sterile gloves were donned via standard open-glove technique, the plasma sterilization indicator (Comply 1248; 3M Healthcare) was verified to have passed, and the tray with the model was removed and placed onto an absorbent pad within a biosafety cabinet (Figure 1). A sterile swab was drawn over the 10-cm incision using a back-and-forth motion to obtain a preinoculation sample for culture (negative control). The model was then inoculated via pipetting 3 mL of the 10−3 dilution suspension into the incisional area and allowed to remain for 20 minutes at room temperature. This inoculation period was chosen as an estimate for the amount of time it may take a novice surgeon to perform an intestinal procedure from opening to closing the gastrointestinal tract, during which time the SC tissue could hypothetically be exposed to bacteria from the intestine. After the inoculation period, the remaining solution was removed from the model using a sterile syringe and placed into the postinoculation test tube. A 1-L 0.9% sterile NaCl IV bag (Vetivex; Dechra) was used as the source for the lavage fluid and was decontaminated with alcohol wipes between each use and discarded after 1 week of use.6 For lavage 1, sterile saline (25 mL volume, equal to 2.5 mL/cm of incision) was withdrawn aseptically from the IV bag using a sterile multiple-access port and a 60-mL syringe. The lavage was applied to the model by hand approximately 2 cm from the incision at an angle of 45° and a rate of 3 to 4 mL/s.9,10 The remaining volume of lavage within the incision was removed using a 3-mL syringe (without needle) and transferred into a small test tube. Following the removal of the remaining saline, the skin model incision site was swabbed using aseptic technique for a postlavage culture. Lavage 2 was immediately performed with an additional 25 mL and sampled as for lavage 1. Finally, lavage 3 was performed with an additional 50 mL of sterile lavage and sampled. Ultimately, lavage 1 equated to a total volume of 25 mL (2.5 mL/cm), lavage 2 equated to a total lavage volume of 50 mL (5 mL/cm of incision), and lavage 3 equated to a total volume of 100 mL (10 mL/cm of incision), respectively.

Figure 1
Figure 1

Sterilized silicone models of the SC tissue were inoculated with Staphylococcus pseudintermedius or Escherichia coli. After 20 minutes of incubation, a culture was taken to quantify initial bacterial counts. The models were subsequently lavaged with sterile saline to total volumes of 2.5, 5, and 10 mL/cm and cultured after each volume.

Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.01.0030

Quantification

A 10-fold dilution series was performed by adding 100 µL of each sample to 900 µL of 1X PBS or DPBS solution to achieve a dilution factor up to 10−3. Each dilution was performed in triplicate. A standard trypticase soy agar plate with 5% sheep blood was used for bacterial quantification. Ten µL of each sample and dilution power were pipetted onto a plate in 5 equidistant spots, being careful to avoid touching each other or the edge of the plate. Additionally, the preinoculation tubes were plated at 10−4 to 10−6 dilution power for comparative quantification. The preinoculation and postlavage cultures taken were streaked for isolation onto a plate in a system of 4 quadrants for semiquantitative analysis. Growth in the third to fourth quadrants indicates a bacterial count above 105 CFUs/g of tissue, so this scale was used to verify that remaining bacteria counts were below 105 CFUs/mL.11 All plates were incubated at 37 °C in a 5% carbon dioxide atmosphere overnight. After 24 hours, each plate was removed and manually analyzed for total CFUs formed. The number of CFUs were converted into CFUs/mL by multiplying the number of CFUs by the following formula to account for the 50 µL pipetted on the plate:
CFUs/mL=CFU X dilution factor X 10 X 2.

Statistical analysis

Numerical data were assembled in Excel (Microsoft Corp), and additional columns were included to denote counts less than 1 X 105 CFUs/mL as “successes.” Two analytic approaches were used. First, multilevel linear regression was used to examine the effect on the bacterial population of each incremental wash step. Second, multilevel logistic regression was used to determine the proportion of “successful lavages” associated with each increment of wash. In this context, a “success” was defined as a bacterial population of < 1 X 105 CFUs/mL. Stata (version 18; StataCorp) was used for all summary statistics and analyses; P < .05 was used to reject null hypotheses.

Results

A lack of growth from the preinoculation culture (CFUs = 0) along with a passing plasma sterilization indicator confirmed that all models were sterile prior to inoculation. All S pseudintermedius and E coli models had > 105 CFUs/mL bacterial counts 20 minutes postinoculation (Table 1). Histograms showed that bacterial counts (in CFUs/mL) were not normally distributed and were converted for analysis to log10CFUs/mL. When considering binary analysis with < 105 CFUs/mL bacterial load as a success, all models had < 105 CFUs/mL of both S pseudintermedius and E coli after 2.5 mL/cm of lavage; all lavage volumes were considered a “success.”

Table 1

Median and IQR (CFUs/mL) of Staphylococcus pseudintermedius and Escherichia coli after inoculation, 2.5 mL/cm saline lavage (lavage 1), 5 mL/cm saline lavage (lavage 2), and 10 mL/cm saline lavage (lavage 3).
Treatment S pseudintermedius (CFUs/mL) median (IQR) E coli (CFUs/mL) median (IQR)
Baseline (prelavage) 7.8 X 105 (5.4 X 105 to 9.6 X 105) 9.7 X 105 (7.8 X 105 to 1.1 X 106)
Lavage 1 (2.5 mL/cm) 4.9 X 103 (2.3 X 103 to 9.5 X 103) 4.0 X 103 (1.2 X 103 to 7.1 X 103)
Lavage 2 (5 mL/cm) 1.0 X 103 (6.0 X 102 to 1.8 X 103) 4.0 X 102 (0 to 1.0 X 103)
Lavage 3 (10 mL/cm) 4.0 X 102 (1.3 X 102 to 6.0 X 102) 2.0 X 102 (0 to 4.0 X 102)

Analysis using multilevel logistic regression indicated that lavage significantly reduced bacterial load (β = 3.96; 95% CI, 2.00 to 5.92; P < .001; Figure 2; Table 1). An approximately 3-log decrease from the postinoculation baseline to the first lavage of 2.5 mL/cm was noted with both S pseudintermedius and E coli, followed by an approximately 1-log reduction between each successive volume (2.5 mL/cm to 5 mL/cm and 5 mL/cm to 10 mL/cm) for S pseudintermedius. For E coli, an approximately 1-log reduction was seen between 2.5 mL/cm to 5 mL/cm lavage volumes, and a half-log reduction was seen between 5 mL/cm and 10 mL/cm lavage volumes. A 90% and 99.9% reduction in bacterial counts from lavage 1 (2.5 mL/cm) to lavage 3 (10 mL/cm) was noted with S pseudintermedius and E coli, respectively.

Figure 2
Figure 2

Log (CFUs/mL) of S pseudintermedius and E coli prior to lavage (0) and after 2.5 mL/cm (1), 5 mL/cm (2), and 10 mL/cm (3) of saline lavage. Mean ± SD of the log(CFUs/mL) is graphically represented.

Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.01.0030

Discussion

When starting with a load of approximately 1.5 X 108 CFUs/mL of a single isolate of S pseudintermedius or E coli, applying 2.5 mL/cm of lavage to a silicone model resulted in successful reduction of bacterial load below 105 CFUs/mL. Additionally, there was an exponential reduction of bacteria between lavages 1 (2.5 mL/cm) and 3 (10 mL/cm), suggesting that the use of more than 2.5 mL/cm may be prudent in clinical cases to reliably ensure adequate reduction of bacterial load within SC tissues, particularly traumatic wounds with inconsistent size and depth proportions.

Wound irrigation has been studied extensively in human medicine, with evidence-based recommendations for various types of wounds, both surgical and traumatic.12,13 While recommendations exist regarding desired lavage pressure or solution, guidance on the ideal lavage volume for wounds is lacking, even in human literature.12,13 One frequently cited human study14 evaluated lacerations irrigated with 250 mL saline/5 cm of wound (50 mL/cm) delivered in a syringe to 220 mL saline/5 cm of wound (44 mL/cm) delivered via pressurized canister, with a primary goal of comparing the time to complete lavage and overall wound complication rates. Reasoning behind the choice of these lavage volumes was not defined. The initial lavage volume tested in this study (2.5 mL/cm) was chosen by considering that if 200 to 300 mL/kg is adequate for an entire peritoneal cavity, a closed incision would require a much lower volume; the midrange volume of 250 mL/kg was divided by 100 to reach 2.5 mL/cm as a starting lavage volume for this study.

There are several challenges associated with using a silicone skin model to represent not only the dermis but also the SC tissues. Bacterial adherence to silicone surfaces does occur and is a concern for silicone-based medical implants; however, the authors are not aware of a study specifically comparing bacterial adherence on silicone to that in natural wounds.15 We expect patient tissues to show increased bacterial adhesion such that a volume greater than 2.5 mL/cm may be more ideal in reducing the starting bacterial concentrations below 105 CFUs/g of tissue, particularly for nonlinear wounds with varying depths and degrees of contamination. Given the further reduction in bacterial counts seen in this model with 5 mL/cm and 10 mL/cm of lavage, 10 mL/cm should be considered in clinical patients.

Our model represents a linear surgical wound that was contaminated with a singular isolate of S pseudintermedius or E coli. In the practice of wound management or abdominal closure following contaminated surgeries, such consistency does not persist. The usage of a single wound type and single bacterial isolates was chosen to investigate trends in reduction; however, it is important to note the challenge with extrapolating such a trend to other strains of bacteria, which may adhere more tightly or have other virulence factors that promote growth in a wound. The location of the wound, dead space, compromised circulation, necrotic tissue, patient comorbidities, and the host’s immune system also affect the type and number of bacteria, thus potentially altering the needed volume of lavage to reduce the bacterial burden below 105 CFUs/g of tissue.16 These factors may alter the threshold level of bacteria that would cause infection.

There are several limitations to this study. As discussed, the use of a model to predict outcomes in live tissues is not ideal. However, given the practical and financial challenges associated with trying to perform such a study in clinical patients, the authors feel that this model is useful in providing a baseline recommendation for wound lavage volumes. Due to supply availability within our laboratory, PBS and DPBS are used interchangeably for bacterial suspension. While both solutions are isotonic and not expected to impact the results of this study, this has not been specifically evaluated and thus must be considered a limitation.

Clinical patients compared to the silicone skin model are much more complex. Nevertheless, we believe the trend in reduction of bacterial isolates with lavage volumes used in this model can guide decisions in clinical patients to minimize the bacterial burden. Veterinarians should consider applying at least 2.5 mL/cm of lavage when aiming to decontaminate SC tissues in linear surgical wounds; however, further work in clinical cases is required to evaluate differences in live tissue compared to this model.

Acknowledgments

The authors want to thank the Texas A&M Clinical Microbiology laboratory for the use of their BSL-2 hood and equipment for this project. The authors additionally wanted to thank the Texas A&M Small Animal Operating Room team for ensuring models were plasma sterilized in a timely manner.

Disclosures

The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.

Funding

A Boehringer Ingelheim VSP grant under the 2023 Veterinary Medical Summer Research Training program at Texas A&M University School of Veterinary Medicine and Biomedical Sciences was used to support the first author.

ORCID

Vanna M. Dickerson https://orcid.org/0009-0008-0096-9942

References

  • 1.↑

    Nicholson M, Beal M, Shofer F, Brown DC. Epidemiologic evaluation of postoperative wound infection in clean-contaminated wounds: a retrospective study of 239 dogs and cats. Vet Surg. 2002;31(6):577 581. doi:10.1053/jvet.2002.34661

    • Search Google Scholar
    • Export Citation
  • 2.↑

    Williams RW, Cole S, Holt DE. Microorganisms associated with incisional infections after gastrointestinal surgery in dogs and cats. Vet Surg. 2020;49(7):1301–1306. doi:10.1111/vsu.13495

    • Search Google Scholar
    • Export Citation
  • 3.↑

    Andrews C, Williams R, Burneko M. Use of liposomal bupivacaine in dogs and cats undergoing gastrointestinal surgery is not associated with a higher rate of surgical site infections or multidrug-resistant infections. J Am Vet Med Assoc. 2023;262(1):1–6. doi:10.2460/javma.23.08.0463

    • Search Google Scholar
    • Export Citation
  • 4.↑

    Turk R, Singh A, Weese JS. Prospective surgical site infection surveillance in dogs. Vet Surg. 2015;44(1):2–8. doi:10.1111/j.1532-950X.2014.12267.x

    • Search Google Scholar
    • Export Citation
  • 5.↑

    Nelson LL. Surgical site infections in small animal surgery. Vet Clin North Am Small Anim Pract. 2011;41(5):1041–1056. doi:10.1016/j.cvsm.2011.05.010

    • Search Google Scholar
    • Export Citation
  • 6.↑

    Marshall H, Sinnott-Stutzman V, Ewing P, Bracker K, Kalis R, Khorzad R. Effect of peritoneal lavage on bacterial isolates in 40 dogs with confirmed septic peritonitis. J Vet Emerg Crit Car. 2019;29(6):635–642. doi:10.1111/vec.12893

    • Search Google Scholar
    • Export Citation
  • 7.↑

    Groenen H, Bontekoning N, Jalalzadeh H, et al. Incisional wound irrigation for the prevention of surgical site infection. JAMA Surg. 2024;159(7):792–800. doi:10.1001/jamasurg.2024.0775

    • Search Google Scholar
    • Export Citation
  • 8.↑

    Tobin GR. Closure of contaminated wounds: biologic and technical considerations. Surg Clin North Am. 1984;64(6):639–652. doi:10.1016/S0039-6109(16)43380-3

    • Search Google Scholar
    • Export Citation
  • 9.↑

    Lee H, Lee Y, Park JY, Han JW. Analysis of flow rate and pressure in syringe-based wound irrigation using Bernoulli’s equation. Sci Rep. 2022;12(1):14957. doi:10.1038/s41598-022-19402-2

    • Search Google Scholar
    • Export Citation
  • 10.↑

    Devriendt N, de Rooster H. Initial management of traumatic wounds. Vet Clin North Am Small Animal Pract. 2017;47(6):1123–1134. doi:10.1016/j.cvsm.2017.06.001

    • Search Google Scholar
    • Export Citation
  • 11.↑

    Bowler PG, Duerden BI, Armstrong DG. Wound microbiology and associated approaches to wound management. Clin Microbiol Rev. 2001;14(2):244–269. doi:10.1128/CMR.14.2.244-269.2001

    • Search Google Scholar
    • Export Citation
  • 12.↑

    Saeg F, Schoenbrunner AR, Janis JE. Evidence-based wound irrigation: separating fact from fiction. Plast Reconstr Surg. 2021;148(4):601e–614e. doi:10.1097/PRS.0000000000008331

    • Search Google Scholar
    • Export Citation
  • 13.↑

    Daeschlein G. Antimicrobial and antiseptic strategies in wound management. Int Wound J. 2013;10(suppl 1):9–14. doi:10.1111/iwj.12175

  • 14.↑

    Chisholm CD, Cordell WH, Rogers K, Woods JR. Comparison of a new pressurized saline canister versus syringe irrigation for laceration cleansing in the emergency department. Ann Emerg Med. 1992;21(11):1364–1367. doi:10.1016/S0196-0644(05)81903-1

    • Search Google Scholar
    • Export Citation
  • 15.↑

    Lam M, Migonney V, Falentin-Daudre C. Review of silicone surface modification techniques and coatings for antibacterial/antimicrobial applications to improve breast implant surfaces. Acta Biomater. 2021;121:68–88. doi:10.1016/j.actbio.2020.11.020

    • Search Google Scholar
    • Export Citation
  • 16.↑

    Teot L, Ohura N. Challenges and management in wound care. Plast Reconstr Surg. 2020;147(suppl 1):9S–15S. doi:10.1097/PRS.0000000000007628

    • Search Google Scholar
    • Export Citation
All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 204 204 73
PDF Downloads 153 153 19
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
Issue:
20 May 2025
Advertisement