Effect of Negative Pressure Wound Therapy With Instillation on Bioburden in Chronically Infected Wounds

Introduction. Standard negative pressure wound therapy (NPWT) has been shown to help close wounds despite increasing planktonic bioburden. Both planktonic and biofilm critical colonization are associated with delayed wound healing; therefore, reducing microbial colonization is thought to aid wound healing. The use of NPWT with topical antimicrobial irrigation solution has previously shown reduction in quantitative planktonic bioburden when combined with sharp debridement in chronic wounds. Objective. The goal of this study was to evaluate the effectiveness of NPWT with instillation (NPWTi) on biofilm of chronic wounds. Materials and Methods. A prospective, randomized trial was conducted. Following sharp debridement, 20 patients with chronic wounds were randomized to 1 week of either NPWTi with 0.125% sodium hypochlorite solution (n = 10) or NPWT without instillation (n = 10). Serial wound biopsy was performed predebridement, postdebridement, and after 1 week of study therapy to test for quantitative nonplanktonic or biofilm-protected bacteria. Results. As expected, there was no difference in change in wound size between the 2 groups at 1 week. The NPWTi group had a mean reduction in quantitative biofilm-protected bacteria of 48%, while the NPWT without instillation group had a mean increase of 14% (P < .05). Discussion. Consistent with previous studies, this trial demonstrates that NPWTi with dilute sodium hypochlorite solution is effective at reducing nonplanktonic bioburden of chronically, critically colonized wounds. Conclusion. Therefore, based on this and previously published work, this therapy provides both planktonic and nonplanktonic bioburden reduction as well as NPWT benefits and may be a tool for the preparation of infected wound beds prior to definitive closure.

Chronic lower extremity ulcers are subject to critical colonization by multiple species of bacteria.¹ In addition to motile planktonic bacteria, the presence of biofilm-protected bacteria, a conglomeration of cell-to-cell aggregated bacteria embedded in glycoproteins and debris, has been increasingly recognized as a major role player in colonization.^2,3 Biofilm provides bacteria with an environment resistant to pH, osmolarity, nutrient limitation, debridement, and antibiotic exposure.^4,5 Together, planktonic and biofilm-protected bacteria create a state of chronic inflammation in the local wound environment that impedes tissue healing through an increase in metabolic requirements, reduction in perfusion, persistent neutrophil recruitment and cytokine release, and disruption of existing extracellular matrix.³

Reduction of such bioburden is critical for healing chronic wounds. Aggressive wound debridement alone has been the standard for reducing planktonic bacteria, effectively managing devitalized tissue, and restoring the wound back to a normal healing cycle.⁶ However, such an approach has been shown to be suboptimal for removing biofilm-protected bacteria.⁷ The need for multiple concurrent modalities is increasingly recognized in the management of biofilm-protected bacteria.³ 

One such strategy employed with early success to reduce bioburden is the use of sharp debridement followed by negative pressure wound therapy (NPWT) with instillation (NPWTi).⁸ While NPWT alone effectively increases local wound perfusion and granulation tissue formation, it has been shown to actually increase bioburden in chronic wounds.⁹ However, the new generation of NPWT devices have the capability to irrigate wounds with various solutions between intervals of negative pressure. Early results of NPWTi on chronic wounds have been promising, demonstrating decreases in bioburden.^10,11 However, there is a dearth of clinical studies on the effects of NPWTi on chronic wounds, especially analysis of its impact on biofilm-protected bacteria. 

The aim of this study was to directly examine the effect of NPWTi on bioburden in chronic wounds and compare the change in both planktonic and biofilm-protected bacteria after a standardized treatment protocol of debridement followed by either NPWT or NPWTi.

Study design and participants
An Institutional Review Board-approved, single-center, randomized controlled trial was conducted between January 2014 and November 2014. All patients were recruited from the outpatient vascular and wound care clinic at Mt Sinai St Luke’s – Roosevelt Hospitals in New York City. Inclusion criteria consisted of a patient with a leg or foot ulcer > 40 cm² that would usually be treated with NPWT and the patient would be hospitalized. Patients were sequentially enrolled into either the NPWT group or the NPWTi group in an unblinded fashion. There was no difference in patient characteristics between groups (Table). Systemic antibiotic therapy was not used. 

The investigational NPWT and NPWTi used is V.A.C. Ulta (KCI, an Acelity company, San Antonio, TX). The instillation agent used was Dakin’s Solution, quarter strength (Century Pharmaceuticals, Indianapolis, IN), sodium hypochlorite 0.125%.

Treatment
Both groups underwent sharp surgical debridement and irrigation in the operating room under regional or general anesthesia on day 0. Wound biopsies were taken before and after debridement. Immediately following debridement, NPWT or NPWTi was initiated for a 7-day period. For the NPWTi group, sodium hypochlorite 0.125% was instilled at a volume of 0.2 mL per cm² wound area, allowed to dwell in the wound for 10 minutes, and followed by 60 minutes of negative pressure therapy. A negative pressure of -125 mm Hg was used for both NPWT and NPWTi devices; in the case of the NPWT, the suction was continuous.

On day 7, patients were brought back to the operating room where sharp debridement and wound irrigation were performed again. Wound biopsies were taken before and after debridement. Wounds were then treated with either a split-thickness skin graft or skin substitute followed by a NPWT bolster. Patients remained in the hospital for an additional 4 days before discharge with weekly outpatient follow-up. 

Analysis of bioburden
Tissue samples for planktonic bacteria were serially diluted, plated on tryptic soy agar media, and allowed to incubate (proprietary information to the University of Florida, Gainesville, FL). Number of colonies recovered from each sample was converted to standard colony forming units (CFU) per gram of tissue. Specific bacteriology of organisms was subsequently obtained.

A second tissue sample for biofilm analysis was received in a sterile 15-mL tube containing phosphate-buffered saline (PBS) and Tween-80 (Sigma-Aldrich, St Louis, MO). Samples were then vortexed for 30 seconds, sonicated for 5 cycles of 90 seconds, and vortexed again for 30 seconds. This suspension was then serially diluted with PBS, plated in triplicate on tryptic soy agar, and incubated at 37°C for 24 to 48 hours. The CFUs were then counted, and the biofilm viable cell density was calculated based on the dilution plated and the surface area of the specimen.

Statistical analysis
Data were analyzed using SPSS Version 12 software (SPSS, Chicago, IL). Continuous variables were defined by mean ± standard deviation. Student t test and chi-square test were used where appropriate. Significance level was determined as P < .05.

A total of 19 patients with 20 chronic leg ulcers were included in this study. Patient demographics and wound characteristics are listed in the Table. No statistical differences were found between the NPWT and NPWTi groups.

There was no statistical difference in the initial planktonic bacteria concentration between the 2 groups (P = .86), which was 12.3 x 10⁵ CFU/g ± 28.6 x 10⁵ CFU/g and 10.5 x 10⁵ CFU/g ± 15.1 x 10⁵ CFU/g for the NPWT and NPWTi groups, respectively. Following initial debridement, there was no significant decrease in planktonic bacteria concentration for the NPWT (83.4%; P = .16) and NPWTi (72.5%; P = .32) groups, respectively. For planktonic bacteria no statistical difference between the 2 groups was seen at any time in the study (Figure 1). 

Initial biofilm-protected bacteria concentrations did not differ (P = .48) between the NPWT and NPWTi groups, 8.6 x 10³ CFU/g ± 8.8 x 10³ CFU/g and 12.9 x 10³ CFU/g ± 12.5 x 10³ CFU/g, respectively. Sharp debridement did not produce a significant change in either of the groups. Analyzing the change in biofilm-protected bacteria concentration following 7 days of NPWT or NPWTi shows a significant reduction (43%; P < .05) in the NPWTi group and a nonsignificant increase (14%; P = .46) in the NPWT group (Figures 2, 3). However, between-group analysis did not find a significant difference (P = .11) in biofilm-protected bacteria concentration (Figure 3). Interestingly, pseudomonal biofilms were eradicated easily in both groups, as were methicillin-resistant Staphylococcus aureus (MRSA) biofilms. However, streptococcal and fastidious organisms showed the most resilience independent of the therapeutic group. There were no observable differences between the 2 therapies that were specific to bacterial species (Figure 4).

The presence of bioburden in chronic wounds is a major obstacle to healing. While reduction of planktonic bacteria is achievable through various readily available methods, such as sharp debridement and antimicrobial dressings, control of biofilm-protected bacteria remains a challenge for several reasons. First, detection of biofilm-protected bacteria is not a well-established process and cannot be extrapolated to all settings, and the current general ability to identify the presence of biofilm is limited.^12,13 Results of culture methods have not correlated well with direct detection methods such as peptide nucleic acid-based fluorescence in situ hybridization.¹³ This may be the result of the heterogeneous distribution and slow growth of biofilm-protected bacteria.⁷ It has been suggested that complete surface swabs and multiple biopsies be analyzed by both culture and direct molecular detection methods.¹⁴ However, such a rigorous analysis would be prohibitively expensive as well as not clinically feasible due to pain and time considerations. Furthermore, direct molecular detection methods are only able to provide a qualitative result. Clinically relevant quantities of bacteria would remain unknown, which is another challenge; the mere presence of biofilm does not solely indicate a negative influence on that wound. Whereas 10⁵ CFU/g is the commonly accepted quantity of planktonic bacteria defining clinical infection, such a threshold has yet to be established for biofilm-protected bacteria.¹⁵ As such, it also remains unclear as to what degree of biofilm-protected bacteria reduction must be achieved to elicit a clinical result. 

Beyond detection, effective therapeutic means to reduce biofilm-protected bacteria are not yet available. Antibiotic resistance traits in biofilm-protected bacteria are well documented.^16-18 Not only do bacteria-produced glycoproteins and polysaccharide matrices limit antibiotic penetration, they also change the pharmacokinetics of antibiotic-degrading enzymes such as beta-lactamase.¹⁶ Further, the slow growth rates of biofilm-protected bacteria make them inherently resistant to antimicrobials.¹⁹ 

Therefore, to meet these challenges, novel strategies for biofilm-protected bacteria control are being explored. The general direction of these strategies is towards disrupting biofilm either by biochemical means (ie, lactoferrin, honey, and enzyme cocktails) or novel mechanical means (ie, ultrasound).^7,8 The treatment in the present study is of the latter category. 

Theoretically, NPWT can provide repeated mechanical disruption of biofilm attachments in chronic wounds while continuously removing bacteria-produced exudative precursors to biofilm.^20,21 There is a mechanical component of biofilm disruption that occurs with the 3-times-per-week mechanical disruption of removing the sponge from the wound bed. Although Morykwas et al²² had initial success in decreasing wound bioburden with NPWT in a porcine model of chronically infected wounds, those results have not been replicated on human wounds. In fact, there have been reports of wound bioburden increasing with NPWT compared with standard dressings.⁹ Perhaps, as reported by Rupp et al,²³ the viscoelastic property of biofilms actually increases in areas of high shear stress and thereby resists NPWT disruption.¹⁷ 

The addition of a second modality, fluid instillation and irrigation, may further disrupt biofilm. The sodium hypochlorite 0.125% used in this study has been shown to be efficacious as a bactericidal agent while being nontoxic to native tissue.²⁴ Numerous other wound irrigation solutions are also commercially available, including solutions based on betadine, silver, surfactants, and antibiotics, though direct comparison of their efficacy is not known. This research group has had initial success with NPWTi on planktonic bacteria using sodium hypochlorite 0.125% as the instillation agent and aimed to determine whether that was applicable to biofilm-protected bacteria.²⁵ Of note, by way of discussion, the investigators do not believe this to be the correct agent for all biofilms, as this paper outlines the reduction in pseudomonal and MRSA biofilms with sodium hypochlorite 0.125%. However, there appears to be little effect on streptococcal and fastidious biofilms, indicating a possible need for a different agent if these are identified species.

Sharp debridement has long been the gold standard for reduction of bioburden.²⁶ However, it appears this may not be enough to minimize bacterial burden in the wound bed; pictorially, the wound bed preparation seen in Figure 5 — with the visual difference in the quality of the wound bed accompanied by the data around the reduction in bioburden at the end of a week of therapy — could support the idea that NPWTi is one of the most effective tools to help prepare a wound for closure to date. In the present study, as expected, planktonic bacteria concentration markedly decreased following the initial operative debridement. However, due mostly to small sample size and large variance in data, this did not prove to be statistically significant. Further, planktonic bacteria concentration remained below 10⁵ CFU/g throughout the 7 days of the study in both the NPWT and NPWTi groups with no between-group differences seen. It could be extrapolated that both negative pressure modalities might significantly increase planktonic bacteria following debridement, though without a control group this assertion cannot be made. 

Conversely, sharp debridement did not decrease biofilm-protected bacteria concentration; in fact, a nonsignificant increase was seen. As discussed above, the viscoelastic property of biofilm resists shear stress and, not surprisingly, prevents bacteria removal with debridement alone. Following 7 days of treatment, a statistically significant reduction in biofilm-protected bacteria was seen in the NPWTi group but not in the NPWT group. However, there was no between-group difference seen. These findings are in line with the small number of clinical studies available on NPWTi and chronic wounds. One study that compared NPWT to NPWTi with saline in chronic leg ulcers found that instillation correlated with earlier granulation tissue formation.²⁷ Gabriel et al²⁸ conducted a prospective study on 15 wounds using NPWTi with silver nitrate and compared them with a retrospective control group treated with wet-to-moist wound care, finding the NPWTi group required fewer days of treatment, hospitalization, infection, and wound closure.

The investigators are aware that biofilm often live greater than 5 mm below the wound surface, often using dermal appendages as “safe harbors.” To this end, the study’s debridements, even in the operating room, do not extend below a few millimeters. It is possible that deeper debridements may have a more significant effect on biofilms. The investigators’ current strategy, at least in part, focuses on not allowing the resurgence of bacteria, both planktonic and biofilm-protected, after surgical debridement. Unfortunately, there is too little data concerning the actual effect of such a strategy and too much speculation.

The limitations of this study, largely alluded to above, include small sample size and associated large data variance. Further, the absence of a standardized biopsy schema, given the heterogeneous distribution of biofilm-protected bacteria within wounds, may not properly capture the effects of the treatment modalities. 

The results of this study are line with the growing consensus that a multimodality approach is necessary to treat wounds with biofilm-protected bacteria. Early data suggests NPWTi to be an effective therapy for reducing overall bioburden in chronic wounds. A larger prospective study, possibly including different instillation agents based upon bacterial species, would further this interesting field.

Affiliations: Mt Sinai St Luke’s – West Hospitals, New York, NY; and University of Florida, Gainesville, FL

Correspondence:
John C. Lantis II, MD
1090 Amsterdam Ave, Suite 12
New York, NY 10025
JLantis@chpnet.org

Disclosure: Greg Schultz, PhD, is a paid consultant for Acelity and Smith & Nephew. Dr. Lantis is a paid consultant for Acelity, Smith & Nephew, Kerecis, and Intregra.