Prevalence of Biofilm in Chronic Wounds: Systematic Review With Meta-Analysis

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Chronic wounds are lesions that do not follow the natural healing process, remaining open and stagnant at a certain stage of the healing process for a prolonged period, usually longer than 3 months. These wounds are often associated with underlying conditions such as diabetes, vascular disease, and prolonged difficulty mobilizing, which compromise the body’s ability to heal tissue effectively. Chronic wounds include pressure injuries, venous ulcers, and diabetes mellitus-related ulcers, each with specific characteristics related to their etiology.¹ 

Inadequate and incomplete healing of these wounds can lead to serious complications such as infections and amputations. In addition, these complications require prolonged and complex care and compromise patients’ ability to perform daily activities, significantly decreasing their quality of life.² 

Treating these lesions requires significant health care resources, involving specialized care, long periods of treatment, and recurrent hospitalizations.³ The high prevalence and challenges associated with treating these wounds highlight the need for effective prevention strategies and therapies worldwide.⁴ 

There are several intervening factors that delay healing; of these, the presence of biofilm stands out. Biofilms play an important role in wound chronicity, significantly hindering the healing process. The presence of biofilms in chronic wounds requires specific and more intensified therapeutic approaches because conventional treatment strategies, such as the administration of antibiotics, are often ineffective because of the resistance conferred by the protective matrix of the biofilm.⁵ 

Biofilms are structured communities of microorganisms that adhere to living or inanimate surfaces and are encased in a matrix of extracellular polymeric substances.⁶ This matrix—composed primarily of polysaccharides, proteins, and nucleic acids—protects the microorganisms from the harsh conditions of the external environment, such as the action of antimicrobials and the host’s immune system response.⁷ 

Techniques such as debridement, the application of topical antimicrobials, and the use of technologies that promote biofilm disruption—such as laser therapy and ultrasound—are being explored to combat these resistant microorganisms.⁸ The effective management of biofilms in chronic wounds is critical to improving clinical outcomes, reducing the burden of infection, and accelerating the healing process.

Thus, it is important to evaluate the prevalence of biofilm in chronic wounds in order to reveal its effect on lesions and communicate to health professionals, especially stomal therapy nurses, the need for complex care to combat it. 

To this end, the objective of this systematic review with meta-analysis was to estimate the prevalence of biofilms in chronic wounds.

Of note, a review on this topic was published in 2017.⁹ The current study is an update that includes international research until September 2024.

This systematic review of prevalence studies was registered in the International Prospective Register of Systematic Reviews (PROSPERO) and carried out on March 30, 2024 (registered in PROSPERO (CRD420244526479). The review and meta-analysis were structured according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). 

The methodological stages of the Joanna Briggs Institute were followed for systematic prevalence reviews; this included the definition of the protocol title and review; definition of the review question; and definition of inclusion criteria and search strategy screening and selection of the study, evaluation and methodological quality, and extraction and synthesis of data.¹⁰

The research question that directed the study was constructed using the PCC mnemonic: Problem (biofilm), Concept (prevalence), Context (complex wounds). Studies that presented the prevalence of biofilm in humans, in any time frame without limitation of language and place of research, were included. Thus, no delimiter filters were used in any of the databases. Review studies, case studies, abstracts, dissertations, and theses were excluded.

Scopus (Elsevier), Web of Science (Clarivate), Embase (Elsevier), and MEDLINE/PubMed (National Institutes of Health) databases were searched on April 11, 2024, by 2 independent researchers in order to answer the following research question: What is the prevalence of biofilm in chronic wounds? For Scopus, Web of Science, and MEDLINE/PubMed, the Medical Subject Headings (MeSH) descriptors were used; Emtree descriptors were searched in Embase. A manual search of the list of references of the studies and gray literature was also conducted. Only articles from the databases were included because no studies were identified through the other searches.

All articles selected at that time were imported into Rayyan (Rayyan Systems, Inc.) for screening by the evaluators. To create the data strategy, the ECCCU search equation¹¹ was used: extraction, conversion, combination, construction, and use. Due to the particularities of access to each database and in order to cover the largest number of studies, it was necessary to adjust the search strategy in certain research sources. The Box describes the strategy adopted in each of the databases. 

After the initial articles were selected, the evaluators read the titles and abstracts to identify studies that were beyond the scope of the research. Studies that did not present primary data on the prevalence of biofilm in chronic wounds were excluded. This decision aimed to ensure that only research with specific quantitative information on the occurrence of biofilm was included, allowing for a more accurate and consistent analysis in the meta-analysis. Additionally, literature reviews and case studies were excluded.

 Two independent evaluators participated in the selection of the studies. Any conflicts were later resolved by the 2 evaluators so that there was no need for a third evaluator. 

A data collection instrument was used to capture the following information from each study: author and year of the research, total number of lesions evaluated, number of lesions with biofilm (prevalence), which bacteria was detected, level of biofilm, country where the research was developed, and methodological quality. The articles were read in full, and the data were collected and organized in an Excel spreadsheet (Microsoft). 

The Newcastle-Ottawa Scale was adapted to evaluate the cross-sectional studies and consider their methodological quality. This instrument considers the following items for evaluation: sample representativeness, sample size, non-responder rate, how the outcome was evaluated, and statistical analyses performed. A score is given for each evaluated item, where the item that evaluates the outcome can reach up to 2 points, totaling from 0 to 6 points (very good studies: 6 points; good studies: 4-5 points; satisfactory studies: 3 points; unsatisfactory studies: < 2 points). To compare the groups in relation to methodological quality in the meta-analysis, up to 3 points were categorized as satisfactory quality and less than 3 as unsatisfactory.¹²

A meta-analysis was performed to analyze the prevalence of biofilm (primary outcome) using the CI and percentage rate. The Cochran’s Q test and I² statistics were used to assess heterogeneity. When evaluating the homogeneity of the studies, high heterogeneity was considered when I² was greater than 50 and Cochran’s Q P value was less than .10, thus applying the random effects model. Metaregression was performed to identify the cause of the heterogeneity of the studies.

Publication bias was assessed through visual inspection of the funnel plot as well as calculation of the Egger’s test, where the result to determine the absence of bias was indicated by a P value of greater than .05.¹³ For variables that could not be determined by the meta-analysis, qualitative synthesis was performed. 

Subgroup analyses were performed for bacteria detected in the biofilm and for the continents where the research was carried out. The prevalence between moderate and high levels of biofilm in the lesions was also compared. Statistical analysis was performed using the R 2024.4 software (The R Foundation for Statistical Computing).

A total of 281 articles were identified by manual search in the databases. Duplicates (n = 83) were removed, resulting in 198 studies for evaluation. After analysis of the titles and abstracts, 148 additional studies were excluded for not meeting the objective of the research. The remaining 50 studies underwent full-text evaluation. Twenty-four studies from 12 countries were included in the meta-analysis (Figure 1).

The number of wound samples analyzed totaled 2666, ranging from 20 to 355 samples evaluated in each study, and the biofilm was isolated between 5 and 277 times in the lesions. Eleven studies also classified their samples as having biofilm-forming capacity; these were classified as strong, moderate, or weak biofilm producers.

In the biofilm-level analysis performed in 11 studies, a higher chance of high biofilm levels (odds ratio [OR]: 0.70; 95% CI, 0.34-1.43) was recorded (Figure 2).

Of the studies that were included in the review, 2 presented good methodological quality,^14,15 17 were classified as satisfactory,^16-32 and 5 were classified as unsatisfactory.^33-38 The synopses of the studies are shown in the Table.

The country with the most research on the prevalence of biofilm in wounds was Iran with 9 studies,^{20,21,24,26,29,31,32,35,36} followed by India^27,28,33 and Nigeria^16,17,37 with 3 studies each, and Iraq with 2.^19,30 All other represented countries (the United States,²⁵ Saudi Arabia,¹⁸ Poland,³⁴ Sri Lanka,²² Pakistan,¹⁵ and Cameroon¹⁶) had 1 study each. A study that investigated the prevalence of biofilm in wounds in France and Benin was also included.²³ 

The overall biofilm prevalence was 68% (95% CI, 58%-79%), ranging from 23% to 100% (Figure 3).

Of the 24 articles, 9 identified the presence of biofilm formed by Pseudomonas aeruginosa (P aeruginosa)^{14,15,17,20,19,24,31,32,37} and 6 indicated Staphylococcus aureus (S aureus).^{16,18,26,30,36,21} Two studies investigated the presence of biofilm formed by Acinetobacter baumannii (A baumannii).^29,35 Biofilms formed by Enterococcus spp³⁴ and Candida spp²⁷ were each searched in 1 study (Figure 4). The remaining studies did not look for biofilms formed by specific microorganisms.

In a study carried out in Iran with strains of S aureus, 99 of 105 samples had biofilm formation, 25 of which were classified as strong biofilm-forming, 59 as moderate biofilm-forming, and 15 as weak biofilm-forming.²⁶ In another study from Iran, this time with strains of A baumannii, 76 of the 89 isolates had biofilm, 39 of which were classified as strong, 27 as moderate, and 10 as weak biofilm-forming—these strains were multi-drug-resistant to antimicrobials.²⁹ Another Iranian study examining the prevalence of biofilms formed by A baumannii found 95 positive samples out of 117.³⁵

In a study in India, 159 out of 355 samples were positive for the presence of biofilm. The search was not performed on samples of a given microorganism, and the strains were multi-drug-resistant to antimicrobials. Of these strains, 94 were found to be strong biofilm-forming and 65 were found to be moderate biofilm-forming.²⁸ In another study in India, 131 out of 190 samples showed the presence of the biofilm, with 45 being strong and 86 being moderate biofilm-forming.³³

In a study that examined Candida spp samples in isolates collected from the feet of people with diabetes mellitus in India, all 75 samples were positive for biofilm; 54 were classified as strong to moderate biofilm-forming.²⁷ A Polish study developed 100 samples of Enterococcus spp and found 71 to be biofilm-positive: 27 strong biofilm-forming, 11 moderate biofilm-forming, and 33 weak biofilm-forming.³⁴

In the only study conducted with patients in more than 1 country, France and Benin, the mean age was 64 years and 26 of 111 samples were positive for biofilm.²³ In the study from the United States, 30 of the 50 samples of chronic wounds showed the presence of biofilm.²⁵ Furthermore, in the Sri Lankan study, 20 out of 20 samples were positive for the presence of biofilm.²²

In Nigeria, studies looking for biofilms formed by P aeruginosa¹⁷ and S aureus¹⁶ showed the presence of biofilm in 5 of 28 and 26 of 55 samples, respectively. In another Nigerian study, out of 303 samples of P aeruginosa, 277 were positive for the presence of biofilm.³⁷ A study from Pakistan showed the presence of P aeruginosa in 35 out of 44 samples.¹⁵ Of 65 samples with P aeruginosa collected from wounds in Iraq, 25 had biofilm.¹⁹ Another Iraqi study, this time concerning S aureus, found 123 out of 280 samples to have biofilm.³⁰

In a study that collected P aeruginosa samples in Cameroon, 38 of 38 samples revealed the presence of biofilm. Of these, 19 were classified as strong biofilm-forming, 14 as moderate biofilm-producing, and 5 as weak biofilm-producing.¹⁴ 

The Saudi Arabian study included 55 samples of S aureus, 31 of which presented biofilm;¹⁸ of these, 18 were strong biofilm-forming, 7 were moderate biofilm-forming, and 6 were weak biofilm-forming.¹⁸ 

Several studies from Iran evaluated burn patients specifically. Dad et al showed that 68 out of 70 samples of S aureus had biofilm.²¹ Moghadam et al found 54 out of 65 isolated samples of S aureus to be positive for biofilm.³⁶ Jabalameli et al showed the presence of biofilm in 93 of 96 samples of P aeruginosa; 46 were strong biofilm-formers, 25 moderate, and 22 weak.²⁴ Asadpour found 61 of 90 samples of P aeruginosa to have biofilm.²⁰ Radhi et al³⁰ and Tahmasebi et al³² both examined P aeruginosa and found biofilm in 107 of 176 and 26 of 73 samples, respectively.

Subgroup analysis was also performed in relation to the continent where the studies were carried out (Figure 5). The meta-analysis showed significant differences in prevalence between the continents where the studies were published (P = .02), and the Asian continent showed the highest prevalence (73%; 95% CI, 62%-84%).

Figure 6 evaluates the risk of biases in the studies. The funnel plot shows asymmetry between the surveys confirmed by the Egger’s test (P = .000). Thus, the main meta-analysis showed a prevalence of 68%, but after the publication bias was verified, the model was adjusted by the trim-and-fill method, revealing that the actual prevalence reaches up to 98%, suggesting that the result of our main analysis is under publication bias. The meta-regression could not explain the causes of heterogeneity of the studies (P > .05).

In this systematic review, the results indicate an overall prevalence of 68% of biofilm in chronic wounds. Thus, an intensive therapeutic approach is needed to prevent and treat this condition, as the presence of biofilm in wounds prolongs the healing time of chronic wounds, increasing health expenses and decreasing the patients’ quality of life.⁶

It should be noted that the time for biofilm development in the wound bed varies because of the different structures of each bacterium, so the stomal therapist must be aware of the clinical signs of the presence of biofilm in order to institute adequate therapy. Products based on Ag/Fe₃O₄ nanocomposites, propolys, tryptophan and enzymes, silver coatings, negative pressure therapy with silver, or polyhexamethylene instillation can reduce or remove biofilm.³⁹ 

There was no significant difference between the types of bacteria isolated in the lesions, although S aureus had a higher prevalence (71%), corroborating a study that also shows a higher prevalence of S aureus than that of P aeruginosa.⁴⁰

S aureus presents a significant challenge because of its large capacity for virulence-factor production and frequent resistance to available antibiotics.⁴¹ Nevertheless, P aeruginosa, another microorganism frequently isolated in the articles of this study, is also known for its intrinsic resistance to existing antimicrobials and for producing virulence factors.⁴²

It is noteworthy that the studies in the review presented analyses of monospecies in the lesions, and that an in vitro study⁴³ demonstrated the possibility of double bacterial installation, revealing an increase in the maturation of double biofilms, a 50% increase in tolerance to antibiotics, and a polymer matrix more resistant to drug penetration. This demonstrates that in-depth understanding of bacterial communities, as well as investment efforts in effective prevention and treatments, are increasingly emerging.⁴³

Nevertheless, the potential of biofilm to negatively impact public health, regardless of the microorganism responsible for its formation, is remarkably relevant. This context is further intensified when considering the high levels of biofilm, which result in increased antimicrobial resistance, greater chronicity of wounds and infections, and increased virulence factors.^12,44 

The criteria used to determine what is considered to be a high level of biofilm may vary according to the context and method used for measurement. Microtiter plate assays can measure biofilm mass, and this was one of the main forms of measurement observed in the studies of the present research. By this method, levels of biofilm are considered to be high when there is a dense and extensive biofilm coverage on the tested surfaces.¹² Clinically, high levels of biofilm are identified when infections are difficult to treat with standard antibiotic regimens, requiring higher doses or alternative therapies.⁴⁵

Notably, there was a significant difference in the prevalence of biofilm between continents, with a remarkable 73% prevalence in chronic wounds on the Asian continent. The authors of the current study were not able to find any studies to justify such a finding; however, it is possible that the other continents are lacking comparable studies on biofilm prevalence. In addition, the difference may be the result of conditions such as socioeconomic status, demographics, race, and organization of health services, among others, that were not accounted for in this study and that directly impact the development and control of lesion infections. Lack of access to health services and individual perception can contribute to a delay in seeking care, facilitating complications in people with lesions.⁴⁶

The methodological quality of the studies should also be considered. There was a small number of studies with very good or good methodological quality; however, the vast majority of studies were classified as satisfactory, followed by some as unsatisfactory. Additionally, one must consider the differences found between them. 

This review confirms the high prevalence of biofilm in chronic wounds and presents a global overview of the research on this topic, thus stimulating the development of more research to understand and update the mechanisms of biofilm development for the purpose of producing effective therapies, as well as encouraging the stomal therapist to spare no effort in the correct treatment of chronic lesions. The inclusion of methodologically unsatisfactory studies has been mentioned as a limitation of this research.

Thus, more research—with satisfactory methodological quality—is needed to accurately define the prevalence of biofilm in wounds and guide the development of strategies aimed at its control and prevention. Traditional culture techniques may present a significant bias as a diagnostic tool, favoring the detection of microorganisms that grow easily, such as S aureus, while hindering the identification of hard-to-culture bacteria, such as anaerobes. Therefore, it is necessary to expand research to compare the results obtained with conventional cultures with those provided by high-resolution molecular techniques. 

To care for wounds with biofilm, stomal therapist nurses must understand the nuances and action of the biofilm, including preventive management as well as antimicrobial and antibiofilm agents. Care for chronic wounds is a component of stomal therapy, a specialty exclusive to nurses who work in the care of people with wounds, incontinence, and ostomies.

This study has limitations that should be considered. The geographic concentration of studies, particularly in Asia and the Middle East, limits the generalization of the results to other regions. Moreover, the absence of longitudinal studies prevents the assessment of causality between the presence of biofilm and chronic wounds. Publication bias, evidenced by the asymmetry of the funnel plot and Egger’s test, suggests that the actual prevalence of biofilm may be higher than reported.

Other limitations include the quality of the methods used, the prevalence found, and the results obtained from each study, which highlight the importance of investigating the methodological quality in review studies. When considering the methodological quality of the studies, this systematic review with meta-analysis is justified to make visible the heterogeneity of the results found.

This systematic review analyzed 24 articles on biofilm in chronic wounds, showing an overall prevalence of 68%. A considerable majority of the studies came from the Asian continent, and from Iran in particular. There was a higher prevalence of S aureus. It is important to note that the studies showed publication biases, so it is possible that the overall prevalence of biofilm may reach up to 98%.