Surgical Humidification (HumiGard^TM) Reduces Bacterial Load in an Open Spine Animal Infection Model

Abstract

Study Design

Randomised controlled study.

Objectives

Evaporative cooling of surgical wounds during open surgery was overlooked as a modifiable factor in reducing surgical site infections (SSIs). HumiGard^TM is a system which delivers warm and humidified air to the wound bed intraoperatively. This study investigated the potential for HumiGard^TM to reduce bacterial load in a porcine model of spine surgery.

Methods

Open spine surgery was simulated in a porcine model. The wound surface was inoculated with ∼10⁶ CFU/mL of Staphylococcus aureus and remained exposed for 180 minutes to the ambient theatre conditions (control) or warm humidified air (HumiGard) (n=6 per group). Wounds were closed and re-opened three days post-surgery to collect muscle biopsies for bacterial enumeration and histological analyses.

Results

The growth of Staphylococcus aureus was 84% less in the HumiGard muscles than control at Day-3 post-surgery (p=0.041). Large bacterial colonies infiltrated the deeper muscle tissues in control, while the HumiGard muscles had smaller bacterial colonies confined to the superficial zone. Neutrophil proportion in the HumiGard group (34% ± 5.1%) was lower than control (65% ± 6.1%) (p=0.005). In contrast, HumiGard tissues had a higher proportion of mononuclear cells (66% ± 5.1%) than control (35% ± 6.1%) (p=0.005), implying a transition from the pro-inflammatory to the reparative stage of wound healing.

Conclusions

In this animal study, maintaining physiological temperature and humidity while the wounds were open reduced bacterial growth and advanced the wound healing process. HumiGard may offer a protective benefit by reducing the risk of SSIs following open spine surgery.

Introduction

Surgical site infection (SSI) is a leading cause of revision operations, influenced by factors such as surgical complexity, patient health status, comorbidities, and antibiotic resistance. ¹ Treating SSIs is costly, often prolongs hospital stays, and significantly diminishes patients’ quality of life, underscoring the need for strategies to reduce SSI rates. ² Advancement in surgical techniques and infection control practices, including improved operating room ventilation, sterilisation methods, and the use of antimicrobial prophylaxis, have significantly reduced SSIs over the years. ³ However, the recently reported infection rates for spinal surgery range from 0.7% to 16%, indicating that there remains considerable scope for further refinement to the infection-prevention strategies.^4,5

Open surgical wounds are exposed to contamination from two main sources: exogenous contaminants (such as airborne particles) and endogenous flora (originating from the patient’s body). Airborne particles are capable of carrying pathogenic microorganisms, including viable bacterial cells and spores, fungal spores, and viruses. ⁶ Endogenous flora reside on superficial skin and upper airways and become opportunistic under ideal conditions. ⁷ In orthopaedic SSIs, Staphylococcus aureus (S. aureus) is the most commonly isolated pathogen, accounting for approximately 48% of culture-positive SSIs. ⁸ Any of these sources have the potential to progress to severe infections if they are introduced to the exposed internal tissue during surgery.

The risk of developing an SSI is dependent on the interaction between contaminant dose, pathogen virulence, and host resistance. ⁹ Host’s resistance to bacterial growth is influenced by the temperature of the patient. Temperatures lower than the physiological norm adversely affect tissue perfusion, oxygenation, and the immune response, thereby compromising the body’s ability to defend against infection. ¹⁰ Current SSI prevention guidelines from the National Institute for Health Care Excellence (NICE), Centers for Disease Control and Prevention (CDC), and Association of periOperative Registered Nurses (AORN) emphasise the importance of maintaining intraoperative normothermia as studies have reported that the incidence of SSI rises when the intraoperative core temperature of patients falls below 35.5°C. ¹¹ While surgical staff take measures to uphold intraoperative core temperature, the effects of cooling and drying of the local surgical wound during surgery is generally neglected. Studies of exposed spinal and open abdominal surgical wounds have recorded temperatures below 30°C, even though core temperature remains within the normothermic range.^12,13

Theatre air is cold (approximately 20°C) and dry (around 50% relative humidity), contrasting with the body’s internal physiological conditions of about 37°C and 100% relative humidity. This cold, dry external environment draws heat and moisture from an open wound through evaporative cooling. Active room air exchange and laminar airflow directly above the patient bed exacerbate this condition, leading to the cooling and drying of the exposed tissue. Evaporative cooling begins as soon as the wound is open and the effect of this intensifies with the duration of surgery, which could compromise tissue health and potentially increases the risk of wound infection. ¹⁴

Surgical humidification is the intraoperative delivery of warmed, humidified air to an open wound during surgery to alleviate the adverse effects of exposure to a cold, dry surgical environment. This pilot study investigated the growth of bacteria in the wounded tissue when the surgical site is exposed to the cold, dry theatre air or warm, humidified air using HumiGard during surgery.

Methods

Intervention Device

The HumiGard™ Surgical Humidification system (Fisher & Paykel Healthcare Ltd, Auckland, New Zealand) is a device which delivers warm (37°C) and humidified medical-grade air over an open surgical wound during surgery.

Animals and Husbandry

The study was approved by the University of Auckland (UoA) Animal Ethics Committee and performed in accordance with the UoA Code of Ethical Conduct for Animal Research. Fourteen 3-month-old female Landrace and Duroc crossbred pigs, each weighing approximately 30 kg, were included. The animals were acclimatised and housed under a 12/12 light/dark cycle at a temperature range of 19-22°C for the duration of the study with ad libitum access to food and water. Animals were anesthetised and ventilated before the surgical procedure.

Surgical Procedure and Bacterial Inoculation

Wound infection studies conducted in porcine models typically inoculate bacteria at a dose of 10⁶ – 10⁹ CFU.^15,16 A pilot study was conducted using a streptomycin-resistant strain of S. aureus ¹⁷ to determine an appropriate inoculum for the porcine wound infection model. An inoculum of 10⁶ CFU was found to produce a measurable and clinically tolerable infection (n=2; data not shown), as evidenced by a detectable S. aureus burden following enumeration of wound biopsies collected three days after inoculation, in the absence of severe wound deterioration requiring intervention, systemic illness, or mortality.

Pigs were randomised to the control (without HumiGard) and intervention (with HumiGard) groups (n=6 per group). The sample size was determined using the ‘resource equation’ method. ¹⁸ Given the exploratory nature of this pilot study, the sample size was selected to provide preliminary data on the effect of tissue exposure conditions on bacterial growth. In the control group, a single 160 mm incision was made along the midline lumbar spine with a scalpel and resected to the depth of the lumbar lamina using monopolar diathermy and the wound was exposed to standard operating theatre environment (∼20°C, ∼50% relative humidity with laminar downflow) during the experiment (Figure 1A). In the intervention group, HumiGard was turned on prior to making the incision and warm humidified air was delivered to the wounds for the duration of the procedure (Figure 1B). After the wound was retracted, a baseline muscle biopsy was taken, followed by bacterial inoculation on the wound surface (∼10⁶ CFU S. aureus in 1 mL phosphate buffered saline (PBS)). After 45 minutes, excess solution was removed using a sterile gauze. The wounds were exposed for a further 180 min to the ambient theatre conditions (control) or to warm and humidified air using HumiGard (intervention), as it is not uncommon for spine surgeries to take up to 3 hours (some procedures can be longer). Wound temperature was monitored at 15-min intervals using a FLIR E8xt thermal camera. Five muscle biopsies were obtained from the wound before closure. The wounds were closed using Fibrewire for the fascial layer, Vicryl for the subcutaneous layer, and staples for the skin. HumiGard was turned off once the wounds were closed. Primapore dressing was applied to cover the surgical site.

Figure 1.

Digital and thermal photographs of the experimental setup. (A) Wounds in the control group were exposed to the cold dry theatre environment. The local tissue temperature was depicted by a thermal camera. (B) Wounds in the Intervention group received warm humidified air from HumiGard. The average tissue temperature in the point of focus was at 37.3^oC

Animals were recovered and received appropriate care and pain relief. Three days post-surgery, the wounds were reopened under anaesthesia. Five muscle biopsies were obtained for enumeration. Each muscle biopsy was transferred to a pre-weighed tube containing sterile PBS and 2 mm ceramic beads and was homogenised using a Bead Ruptor 24. The homogenised samples were plated onto agar plates and CFU per gram of tissue was determined. Muscle biopsies were also obtained for histological assessment using Hematoxylin and Eosin (H&E) and Gram staining. The experimental workflow is outlined in Figure 2.

Figure 2.

Overall experimental workflow. B: baseline (time of wound retraction), T0: time of excess bacterial solution removal from wound site, T180: time of wound closure, D3: three days post-surgery

Statistical Analysis

Data were analysed in GraphPad Prism 10. Bacterial load results are presented as median values (IQR: 25^th-75^th percentile) and analysed using a Mann-Whitney U test. Neutrophil and mononuclear cell proportions are presented as mean ± SEM and was compared using an unpaired t-test. Statistical significance was defined at p<0.05.

Results

Bacterial Enumeration

To determine whether surgical humidification has an impact on bacterial growth within the wound post-surgery, S. aureus load in muscle biopsies were quantified at pre-inoculation (baseline), immediately before wound closure (T180), and at Day 3 post-surgery (D3). Baseline tissues contained no measurable S. aureus levels by enumeration for both the Control and HumiGard groups. Three days after surgery, S. aureus load in wounds exposed to cold dry theatre environment (control) was increased by 100-fold (31-185) from T180, while S. aureus load in wounds that received warm humidified air (HumiGard) was increased by 16-fold (6-58) (Figure 3, Supplementary Material 1). The growth of S. aureus was 84% less in the HumiGard group than the control (p=0.041).

Figure 3.

Fold change in S. aureus load in wound tissue on day 3 post-surgery compared to wound at closure on surgery day (T180). S. aureus load was increased by 100-fold (31-185) in the control group and by 16-fold (6-58) in the HumiGard group. Bars are represented as median and inter-quartile range. *p<0.05, Mann-Whitney U test

Tissue Histological Analysis

Histology analysis was performed using H&E and Gram staining to assess the impact of surgical humidification on tissue integrity. On Day 3 post-surgery, wound tissues from the control group had multiple large bacterial aggregates which infiltrated the deeper tissue layers. These regions were accompanied by dense and diffuse inflammatory cell infiltrates (Figure 4A–B). In contrast, tissues from the HumiGard group had small bacterial aggregates which were confined to the superficial layers. Inflammatory cell infiltrates were predominantly superficial, indicating a milder and more localised inflammatory response (Figure 4C–D).

Figure 4.

Representative Hematoxylin and Eosin (H&E) staining and gram staining of wound tissue from control and HumiGard groups. (A)–(B) Control wound tissues had numerous large colonies of Gram-positive bacteria which extended into deeper muscle layers. (C)–(D) HumiGard wounds had smaller bacterial colonies limited to the superficial layer. Red arrows indicate colonies of coccoid bacteria. Red ‘S’ indicates surface of wound tissue. Black asterisks indicate inflammatory cell infiltrates. (A) and (C) Scale bar: 50 µm, (B) and (D) Scale bar: 100 µm

To assess the effect of surgical humidification on infection severity and inflammatory response, the proportion of white blood cell subtypes in wound tissues were assessed at Day3 post-surgery. The proportion of neutrophils in the HumiGard group (34% ± 5.1%) was significantly lower than the Control group (65% ± 6.1%) (p=0.005, Figure 5A). In contrast, the proportion of mononuclear cells (which include macrophages, plasma cells, and lymphocytes) was significantly higher in the HumiGard group (66% ± 5.1%) than the Control group (35% ± 6.1%) (p=0.005, Figure 5B).

Figure 5.

Immune cell distribution in wound tissues collected at Day 3 post-surgery. (A) Proportion of neutrophils was 65% ± 6.1% in control and 34% ± 5.1% in HumiGard tissue. (B) Proportion of mononuclear cells was 35% ± 6.1% in control and 66% ± 5.1% in HumiGard group tissues (66% ± 5.1%). Values are represented as mean ± SEM. **p<0.01, unpaired t-test

Discussion

SSIs remain a persistent challenge in open spine surgery despite recent advancements in infection prevention protocols. ³ Evidence from open colorectal surgery patients indicates that surgical humidification is associated with reduced incidence of SSIs. ¹⁹ A mechanism contributing to this benefit is the deflection of exogenous particles away from the surgical site. ²⁰ However, viable contaminants may still enter the surgical wound, which then the host’s physiological response is essential for suppressing bacterial growth to prevent infections. Host response can be less efficient when internal physiology is disrupted by evaporative cooling during open surgery. ¹⁰ Surgical humidification can also mitigate evaporative cooling of tissues, but whether this has the potential to improve the body’s ability to combat bacteria has not been explored. This proof of concept study investigated whether maintaining physiological conditions in wounds during surgery using HumiGard can improve patients’ resistance against infections.

Surgical humidification reduced bacterial growth in wound tissues, which was potentially attributable to decreased tissue desiccation. The heat and moisture loss from internal tissues during surgery can lead to desiccation or necrosis. Dead tissue may be a source of nutrients to bacteria and create an ideal setting for bacterial growth. ²¹ In this study, wounds which were exposed to cold dry air showed a 100-fold increase in bacterial load on day 3 post-surgery. In contrast, when HumiGard was used intra-operatively, the growth of bacteria was 84% less than the control (Figure 3). Muscle tissues from the HumiGard wound typically had smaller-sized bacterial colonies that were confined to the superficial layer (Figure 4C–D), while the control group tissues had larger bacterial colonies which infiltrated the deeper tissue layers (Figure 4A–B). Surgical humidification may have reduced intraoperative tissue desiccation and limited nutrient availability for bacteria, creating a less favourable environment for growth in the wound. ²²

The composition of immune cells in wound tissues on Day 3 post-surgery differed noticeably between the HumiGard and control groups, a response that may be attributable to temperature-related effects. Temperatures below the physiological norm induce vasoconstriction, which reduces tissue perfusion and restrict immune cell arrival to wound site. ¹⁰ In addition, temperature affects the offloading of oxygen from haemoglobin. ²³ At temperatures ranging from 37°C to 42°C, haemoglobin exhibits a lower affinity for oxygen, allowing it to offload readily into the surrounding tissue. ²³ Sufficient tissue oxygenation is crucial for accelerating the wound healing process as it recruits neutrophils to the wound site and activates mononuclear cells. ²⁴ Neutrophils are the first responders during the initial inflammatory phase which clear pathogens, while mononuclear cells (including macrophages and lymphocytes) are involved in later-stage immunity and wound healing. ²⁵ In particular, macrophages play a key role in the healing process by producing growth factors which promote cell proliferation and angiogenesis. ²⁶ Therefore, the stage of inflammation and the healing process can be characterised by the relative abundance of different white blood cell populations. Wounds which were exposed to cold, dry air had a significantly higher proportion of neutrophils than the wounds which were exposed to warm humidified air (Figure 5A). A persistently elevated neutrophil count on the third day post-surgery may indicate delayed and/or ongoing immune activity and the need for further clearance of bacteria. This is supported by numerous large colonies of bacteria observed in the deeper layers of the wound tissues which were exposed to cold dry air (Figure 4A–B). In contrast, tissues from the HumiGard group only had small bacterial colonies which were confined to the superficial layer of wound tissues (Figure 4C–D). This may be attributable to the lower proportion of neutrophils and a higher proportion of mononuclear cells in this group (Figure 5B). Delivery of warmth to the surgical site may have therefore improved tissue perfusion, oxygenation, and therefore enhanced immune function against bacteria growth.

While this study established proof of concept and generated promising preliminary data, several limitations should be considered when interpreting the findings. Firstly, the restriction on collecting biopsies at multiple post-operative timepoints narrowed the assessment of bacterial load effects on wound healing over time. It is well established that the inflammatory response peaks around 3 days post-injury. Therefore, the day 3 biopsies in this study demonstrated a shift in neutrophil to macrophage ratio with surgical humidification, indicating a faster progression through the inflammatory phase. Other key processes involved in tissue repair, such as granulation tissue formation, fibrosis, and elastin deposition, typically begin at around day 7 and may continue for up to several weeks. ²⁷ Collecting biopsies at this later stage would have provided valuable insights into the reparative phase of wound healing. However, repeated opening and tissue collection from the same wound site wound introduce additional trauma and disrupt the healing process. While this study provides important data on the early inflammatory response, future investigations could aim to evaluate the effects of surgical humidification on wound repair and tissue remodelling when bacterial growth is minimised. An additional limitation is that this study exclusively tested S. aureus, one of the most common causative pathogens of SSI. Consequently, the findings may not be generalisable to infections caused by other SSI-relevant pathogens, such as Gram-negative pathogens, which may differ in their virulence, host interactions, and response to treatment.

The findings from this study offer valuable insight into how surgical humidification can further improve SSI prevention. HumiGard intends to provide benefits that are not offered by existing protocols by reducing the risk of viable contaminants developing into an infection. This effect may be mediated through improved tissue perfusion and enhanced immune response, thereby increasing patients’ resistance to infections.

Conclusions

In this animal infection model, the use of HumiGard during open surgery was associated with reduced S. aureus growth and enhanced early wound healing. These findings support a potential role for maintaining physiological wound temperature and humidity within the surgical wound in reducing the bacterial burden. Future studies should evaluate the impact of these findings on longer-term clinical outcomes and their potential to reduce the risk of SSI following open spine surgery.

ORCID iDs

John Cunningham https://orcid.org/0000-0002-0018-9769

Minah Kim https://orcid.org/0000-0002-2556-7092

Joseph F. Baker https://orcid.org/0000-0002-8518-8780

Ethical Considerations

This study was approved by the University of Auckland (UoA) Animal Ethics Committee (approval no. AEC25228) and performed in accordance with the UoA Code of Ethics Conduct for animal research.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.*