Table of Contents
DFUs and postoperative superficial and deep wounds can cause adverse outcomes for patients leading to hospital admissions, delayed healing, poor outcomes, pain, amputations, and death [1, 2]. One in four patients with diabetes will develop a DFU in their lifetime with 50% of those with a DFU developing an infection [3]. A DFU has a major impact on physical functioning, morbidity and exact a high human and financial cost, with cost exceeding that of lung, prostate and breast cancer collectively [4].
Similarly SSIs are among the most common and most expensive health care–associated infections and result in a significant psychosocial and financial burden for both patients and the healthcare system [5]. They have been reported to represent 31% of all hospital acquired illness and are the most common nosocomial infection [6]. Foot and ankle surgery (FAS) has been reported to have higher than average post-surgical infections when compared to other orthopaedic subspecialties [7, 8], with SSI being one of the most troublesome complications after FAS [9]. Post FAS SSI can lead to serious consequences including bone union related issues and joint dysfunction [10]. Additionally, the presence of diabetes complications (defined as the presence of PVD and/or neuropathy) and the presence of neuropathy in non-diabetes patients has been shown to further increase the risk of SSIs following FAS compared with the risks for patients with or without diabetes and/or neuropathy [10]. Wukich and colleagues [10] reported a 7.25-fold increased risk of SSI in patients with complicated diabetes compared with patients without diabetes. Pin site infections are also a well-documented complication of external fixation for correction of Charcot deformity in-patient with diabetes, with infection rates of up to 40% [11].
Staphylococcus aureus (S. aureus), Gram-positive cocci, is a major human pathogen and a predominant cause of SSIs [12, 13]. In a relatively recent research study on the bacteriological profile of SSIs, S. aureus had a prevalence of over 50% and Gram-negative isolates comprised 49.6% of all aerobic bacterial isolates, with Escherichia coli (E. coli) being the most common Gram-negative bacteria, followed by Pseudomonas aeruginosa (P. aeruginosa). Similar bacteriological profiles have been identified for chronic wounds including DFUs with S. aureus being the predominant bacterial species and the most frequently identified pathogen being P. aeruginosa [14]. Co-infection with P. aeruginosa and S. aureus is believed to express virulence factors and surface proteins affecting wound healing [15].
Infectious diseases globally are still the second biggest cause of morbidity and mortality due in part to the increase in drug resistance among large numbers of common infecting organisms [16]. For all antibiotic classes, including the major last resort drugs, resistance is increasing worldwide, which poses a serious threat to public health [17]. Increasingly more alarming is the fact that few new antibiotics have been developed in recent decades [18]. Controversy exists around the efficacy of routine use of perioperative antibiotics to prevent infection with evidence indicating that this does not affect wound complications or infection rate [1, 19]. There is therefore a need to investigate additional alternative strategies for wound management to improve patient outcomes through the prevention of infection, decreasing the need for antibiotic therapy [20].
Investigating various aspects of the wound environment and the alterations that occur during the various stages of the healing process may be the way forward for detecting new strategies to reduce the risk of infection. Wound pH has a significant role to play in both directly and indirectly affecting the cellular processes in the wound and has been shown to be one of the critical factors involved in the wound healing process of both chronic and acute wounds [21]. Wound pH is believed to affect matrix metalloproteinase activity, keratinocyte proliferation, fibroblast activity, microbial proliferation, biofilm formation and immunological responses [18]. Strohal and colleagues in their pilot study on 30 wounds identified that wounds, which presented with a highly alkaline pH (9) at the start of the study progressed to the mean pH decreasing significantly over time with the ulcers having an almost neutral pH as the wound progressed towards healing [22]. Researchers reported that decreased wound size correlated significantly with a reduction in the pH of the wound [22]. In the same study [22] successful control of infection and a reduction in bioburden correlated with a statistical signification pH change from an alkaline towards an acidic wound environment. Agrawal and colleagues in their study on 100 infected wounds similarly noted the role of pH in wound healing with an acidic pH inhibiting the growth of bacteria [23]. Shukla and colleagues [24] in a much earlier study concurred with the aforementioned. They observed that improvements in wound status were associated with reductions in wound pH with decreases during the study period being associated with wounds progressing from ‘unhealthy’ towards a ‘granulating’ or ‘healthy’ status.
Research to date could thus indicate that lowering wound pH may provide an opportunity to create an environment that allows the wound to progress towards healing. Medical grade honey (MGH) known to have a pH of four may potentially halt the growth of most common bacteria [22, 23, 25]. Honey is believed to present with high levels of antimicrobial compounds including methylglyoxal and bee defensin-1, and glycoside derivatives, all known to effectively inhibit viable bacteria of resistance strains [16, 25, 26]. Due to the antimicrobial effects of honeys from the simultaneous action of pH coupled with the many active compounds present, bacteria are deemed unlikely to develop resistance to this substance [27]. Additionally, its ability to accelerate wound healing and its low-cost production make it an attractive option in wound care [28].
Topical agents and wound dressings form an important part of all wound management plans regardless of wound etiology, and their therapeutic availability has increased tenfold in the last decade [29]. While the aforementioned research indicates that the wound pH changes during the healing process and during times of infection, it does however remain unclear if wound pH affects the efficacy of such topical agents like MGH (Activon).
Aim
The aim of this research is to investigate the antibacterial effects of MGH when altered to pH environments known to exist in wounds prevalent to the foot and ankle, including DFUs and post-surgical wounds, and exposed to typical bacteria commonly found in said wounds.
Methods
The in vitro research used a broth culture assay of Tryptone Soya Broth (TSB, Oxoid, Basingstoke, UK), a well-characterized and standardized medium known to support the growth of the below organisms) and honey (sterile medical grade Manuka honey) (Advancis Medical, UK) adjusted to pH values known to exist in wounds (pH 6, pH 7, and pH 8). This broth culture was used to investigate the effects of pH changes and honey on the growth of Gram-negative and Gram-positive bacterial species/strains isolated from post-surgical wounds.
Microorganisms used for the research
Common foot and ankle wound pathogens were sourced from the National Collection of Type Cultures (NCTC) and isolated from wounds. These included; P. aeruginosa (NCTC10782), E. coli, (NCTC10418), S. aureus (NCTC10655) and Staphylococcus epidermidis (S. epidermidis) (NCTC5955). Stock cultures were created by inoculating 10 ml of Tryptone Soya Broth (TSB) (product code: CM0129) with 100 µL of said bacterial isolates which were incubated overnight.
Dispensing bacteria into each solution
Experimental inoculums were obtained by transferring 100 µL of approx. 1012 CFUs/ml of P. aeruginosa, E. coli, S. aureus, and approx. 109 CFUs/ml of S. epidermidis individually into each pH adjusted honey and TSB solution and the positive and negative controls. Single-celled communities of bacteria were investigated to explore if the presence of said organisms had an impact on the antibacterial properties of honey.
Liquid and solid media preparation
TSB and Tryptone Soya Agar (TSA) (product code: CM0003), and Phosphate Buffered Saline (PBS) solution (product code: BR0052) (Oxoid, Basingstoke, UK) was prepared by suspending the dry ingredients in double-distilled water. Following this, the broths and agars were brought to boiling point and dispensed into tubes or bottles, then heated at 121 °C for 15 min in an autoclave to ensure sterilization. TSA is prepared by a similar method, but aseptically dispensed into sterile disposable plastic Petri dishes while still molten and allowed to set overnight to form sterile agar plates.
pH adjusted TSB and Medical Grade Honey (MGH)
TSB was prepared to a clinically relevant range of final pH values of pH 6, pH 7, and pH 8 using hydrochloric acid and sodium hydroxide (Fisher, UK). Activon has been chosen for this study as it contains 100% sterile MGH and is currently used topically on wounds within the National Health Service (NHS) to aid in the healing process. A method previously described by Schneider et al. [30] with final honey concentrations of 75% (w/v) honey was used for this research. Briefly, 7.5 g of MGH was added to TSB to a final solution of 10 ml. Due to honey having an acidic pH (4), the pH of the TSB and honey solution was measured with an electronic pH meter (Fisher Scientific Accumet AE150), and either hydrochloric acid or sodium hydroxide was added accordingly to gain the optimum pH values (pH 6, 7, and 8). Following this, 100 µL of the experimental inoculums, as explained above, of each single bacterium was aseptically dispensed into the TSB and honey solutions and incubated in an orbital incubator at 100 RPM for 24 h at 37 °C.
Controls used in the experiment
Controlled experiments ran concurrently with the above pH adjusted TSB and honey solutions. The controls included pH-altered TSB (with no honey) with final pH values of pH 6, pH 7, and pH 8, as well as the natural occurring pH value of honey (pH 4). As stated above, the pH was altered using hydrochloric acid and sodium hydroxide (Fisher, UK). A further positive control using honey without pH adjustment and a negative control of TSB with no bacteria or honey was also used. One hundred microliters of 108 CFUs/ml of each organism were aseptically dispensed into all controls and incubated for 24 h at 37 °C.
Serial dilutions and plate counts
Serial dilutions and plate counts are the most frequently used method for estimation of bacterial numbers [31]. In this procedure, bacterial cultures or suspensions are serially diluted in 9 ml isotonic diluent. Triplicate samples were used from each member of the dilution series (containing single cells), spreading cells over the surface of all agar plates (e.g. Tryptone Soya Agar, TSA, Oxoid). The plates were then incubated for 24 h, and individual cells or clumps (defined as colony forming units, CFUs) multiplied to form optically visible and countable colonies on the agar surface. Surviving viable cell numbers were estimated by counting agar plates with CFUs between 30 and 300. The reason for being, if there are too few colonies (< 30), the count may not be accurate and too many colonies (> 300) it is difficult as well as time-consuming to distinguish the individual colonies on a plate [32]. Multiplication of the number of colonies by the dilution factor provides an estimate of the bacterial number in the original culture/suspension, usually reported as log 10 CFU/ml [31].
Data analysis
All tests were carried out in triplicate, with the negative control and altered honey and pH experiments being repeated on three separate occasions. The data was recorded as mean ± standard error of the mean and was analyzed in Microsoft Excel 2016. The data was compared to the relevant controls using a two-tailed independent student t-test as used in previous research [25]. A p-value of ≤ 0.05 was considered statistically significant.
