The Pathogen Isolates in Chronic Wound Infections in Poland

Regardless of the constant development of techniques for chronic wound treatment and the availability of more and more modern dressings and antibacterial products, wound care is a medical, organizational, and economic challenge (EWMA 2008). In Germany, in 2012, 1.04% of insured patients were diagnosed with a wound, of which the vast majority were leg ulcers (0.7%) (Heyer et al. 2016). In turn, 739,000 patients were found to have leg ulcers in the UK in 2019 (Graves et al. 2022). There are no precise data on the number of patients with chronic wounds in Poland. Some authors say it may amount to 500,000 (Sopata et al. 2020).

Most frequently observed chronic wounds include diabetic foot syndrome, venous leg ulcers, wounds during critical limb ischemia, pressure ulcers, neoplastic wounds, but also postoperative wounds. Infection is one factor that hinders or prevents wound healing and deteriorates the patient’s prognosis. It is estimated that clinically significant infection in the course of diabetic foot syndrome occurs in up to 30% of patients (Mrozikiewicz-Rakowska et al. 2021).

Accurate diagnosis of infection features and proper microbiological tests are crucial for introducing appropriate local and often systemic treatment. Antibacterial management and appropriate causal treatment lead to infection control and initiation of the healing process. Proper debridement before diagnostic material sampling and the recommended best form of culture by deep-tissue biopsy is rarely performed, mainly in specialist centers that treat chronic wounds (Tedeschi et al. 2017; García et al. 2020).

The selection of antibiotics in the empirical therapy of chronic wounds is based on standards developed on data collected abroad. Knowledge of the epidemiology of chronic wounds in Polish patients is necessary to create national standards of empirical therapy that should be applied before the results of bacterial cultures are obtained. Improving the quality of initial treatment in this area can significantly affect the infection control rate and the time when regeneration occurs in damaged tissues and reduce the risk of severe complications, including sepsis or limb amputation.

The aim of the study was a descriptive analysis of the microorganisms found in infected chronic wounds in patients from Poland, consulted on an outpatient basis at a wound care center from February 26, 2013, to June 29, 2021.

In the analyzed population of patients with infected chronic wounds, the largest group (n=446; 36.6%) were patients with all types of DFS and patients with VLU (n = 225; 18.4%). The quantitative distribution of the material used for microbiological examination depending on the type of wound is presented in Table II.

Soft tissues were used in 85.4% (n=3,345) of cultures. Bone scrapings accounted for 14.3% (n = 562) of cultures. This material was collected when bones were visualized at the bed of an infected wound, most often in the case of ischemic DFS (26.7%) and the group of wounds defined as foot deformity with neuropathy (22.1%).

Table I also includes microbiological tests for which the material is not specified. They accounted for 0.3% (n = 10) of all tests and concerned cultures for which the type of material was not specified in the available documentation or the material was not collected by deep-tissue biopsy due to the small amount of available material or anatomical conditions.

Overall, 2,837 aerobic (72.4%), 1,062 anaerobic (27.1%), and 18 mycological (0.5%) cultures were performed.

Among all performed tests, every third culture (n = 1,235; 31.6%) was negative, i.e., one in which no microorganism was grown. A quantitative summary of negative cultures depending on the type of wound is presented in Fig. 1.

Fig. 1.

Noteworthy is a relatively large number of negative cultures obtained from chronic post-traumatic wounds (42.5%), other infections of soft tissues (40.2%), and post-operative wounds (38.2%). Among the aerobic cultures, only 12.7% of the results were negative, and in the group of anaerobic tests, the lack of bacterial growth was reported in as many as 81.2% of the tests. In the remaining tests, from 1 to 7 pathogens were isolated. The quantitative distribution of tests depending on the number of isolated microorganisms in relation to the type of culture (aerobic, anaerobic, or mycological) is presented in Table III. On average, 1.2 pathogens were grown from one culture. After the exclusion of negative cultures from the analysis, an average of 1.75 pathogens was obtained.

Following the standards in force in the microbiological laboratory in which all the analyzed tests were performed, samples from which four or more isolates were cultured were marked as suspected of contamination. In the analyzed material, 4 and more isolates were cultured in 153 samples (all in aerobic tests) which constituted 5.4% of the entire group of aerobic cultures. It was confirmed that all standards of wound debridement for sampling and the sampling itself were met. After consultation with the microbiology laboratory, it was concluded that the cultured bacteria could be considered an actual component of the infected wound microbiome. There was no suspicion of contamination in the description of the anaerobic culture results. Most often, four or more organisms were cultured from wound material in VLU (7.6% of all studies in this group), critical ischemia of the lower extremities (5.5% of all studies in this group) and neuropathic DFS (2.9% of all studies within this group).

The distribution of the number of cultured microorganisms in one culture depending on the type of wound is presented in Fig. 2.

Fig. 2.

A total of 120 different species of microorganisms were identified in the materials tested. The 15 most frequently isolated microorganisms from the analyzed material are presented in Fig. 3. Staphylococcus aureus was found in 31.3% of aerobic cultures, Enterococcus faecalis in 26.3%, Escherichia coli in 13.3%, and Pseudomonas aeruginosa in 12.5%.

Fig. 3.

The most frequently isolated organism was S. aureus. Eighty-six percent of these isolates were sensitive to methicillin (MSSA – methicillin-sensitive Staphylococcus aureus). In the remaining 14% of cases, S. aureus resistant to methicillin (MRSA – methicillin-resistant Staphylococcus aureus) were isolated. S. aureus was most common as an etiological agent of chronic wound infection in nail fold wounds (71.4%), mixed leg ulcers (35.6%), lymphatic leg and/or foot ulcers (35.5%), and VLU (33.9%). Further in the frequency of occurrence, according to the type of wound, this microorganism was a component of the microbiome in infections 34.9% of wounds during critical ischemia of the lower limbs, 34.4% of post-traumatic wounds, and 32.9% of wounds in neuropathic DFS.

In addition to S. aureus, coagulase-negative Staphylococci (CoNS) were observed in the materials studied. The presence of Staphylococcus epidermidis (7.3%), Staphylococcus simulans (1.9%), Staphylococcus lugdunensis (1.6%), and Staphylococcus hemolyticus (0.7%) was most frequently found. Considering the whole group of coagulase-negative staphylococci, we isolated them from 13.7% of aerobic cultures.

The second most common microorganism was E. faecalis. Vancomycin-resistant Enterococcus (VRE) was found in 2.4% of these isolates. E. faecalis was the most common component of the wound microbiome in ischemic DFS (39.1%), neoplastic wounds (37.9%), pressure ulcers (34.8%), leg and/or foot lymph ulcers (29.0%), and VLU (28.0%).

It is worth noting that another microorganism frequently found in infected pressure ulcers (33.9%), as well as in infected neoplastic wounds (27.6%) was E. coli.

In turn, P. aeruginosa was most often isolated from the material obtained from infected leg ulcers of mixed (42.5%) or venous (24.8%) etiology.

The analysis presented above indicated that the most common etiological factor of infections in the course of neuropathic DFS was one of the four following microorganisms S. aureus (32.85%), E. faecalis (26.96%), E. coli (9,75%), Streptococcus agalactiae (8.66%). In the mixed DFS, the microorganisms most often cultured were: E. faecalis (28.00%), S. aureus (22.86%), E. coli (15.14%), S. epidermidis (11.43%). The most common cultures in the wounds in the course of critical ischemia of the lower limbs were S. aureus (34.92%), E. faecalis (25.62%), P. aeruginosa (13.38%), E. coli (12.24%), and E. cloacae (6.6%). The most common cultures in the wounds in patients with venous leg ulcers were S. aureus (33.64%), E. faecalis (28.04%), P. aeruginosa (24.77%), and E. coli (15.89%).

The data presented above concerned the cumulative results of microbiological tests carried out in outpatients from 2013 to 2021. However, it was observed that in the following years, the microbiological profile of infections and the percentage share of individual pathogens underwent visible changes. Fig. 4 presents the dynamics of these changes concerning eight microorganisms most frequently found in the results of microbiological tests in the analyzed period. Throughout the period covered by the analysis, the most frequently detected microorganisms were S. aureus (31.3%) and E. faecalis (26.3%).

Fig. 4.

Treatment of chronic wounds requires knowledge of their microbiome to implement appropriate antibacterial treatment. Recommendations for empirical antibiotic therapy are often based on data from centres located in remote parts of the world with different climates and concern other types of microorganisms detected in locally infected wounds (Abdu et al. 2019; Schaumburg et al. 2022).

In addition to the significantly smaller number of patient groups analyzed in such studies, collecting material for microbiological tests often needs to meet the current standards. To the best of our knowledge, the material presented in this study is the largest database of cultures taken by deep-tissue biopsy from a single center in Poland. Special care was given to the material quality sent to the microbiology laboratory analyzed in this study. Standard technique used to collect the culture in patients with chronic wounds is deep-tissue biopsy (Hryniewicz et al. 2012; Tedeschi et al. 2017; García et al. 2020). It is worth emphasizing that the material for microbiological tests was collected only from wounds with clinical signs of infection. Colonized wounds were excluded from the evaluation.

In the analyzed material, a relatively large number of negative cultures was observed, obtained from chronic post-traumatic wounds (42.5%) and postoperative wounds (38.2%). Gitajn et al. (2016) described 9% of negative cultures among patients with infected postoperative wounds after bone fracture stabilization procedures. Rondas et al. (2015) found 18.9% of negative cultures taken from chronic wounds with clinical signs of infection.

In the presented material, only 12.7% of the tests were negative among aerobic cultures. In contrast, in the group of cultures performed for anaerobic bacteria, no growth was found in as many as 81.6% of the tests. It may result from a low prevalence of anaerobic microbiota in the etiology of diagnosed infections. However, the impact of using non-optimal transport media in the microbiology laboratory serving the center cannot be ruled out. Routine use of specialized tubes (e.g., Venturi Transystem^®) or liquid transport media (e.g., eSwab^®) may increase the detection of anaerobic microorganisms responsible for infections of chronic wounds (Hudspeth et al. 1997; van Horn et al. 2008; Tyrrell et al. 2016; Demuyser et al. 2018).

In the analyzed material, four or more types of microorganisms (maximum seven) were isolated in 153 aerobic culture samples (5.4% of the tested sample). Such a small percentage may indicate a significantly good debridement of wounds for culture collection and a sufficiently high data reliability. On average, 1.2 pathogens were isolated from one culture. In studies by Han et al. (2011), standard bacteriological cultures from chronic wounds showed the presence of an average of three pathogens. Interestingly, high-throughput next-generation sequencing of the same material detected an average of 17 microbes in each wound. Although the cited work was based on analyzing samples from only 15 patients, it made us realize how complex and diverse the wound microbiome can be, potentially responsible for its infection (Han et al. 2011).

A total of 120 different microorganisms were identified in the analyzed material. S. aureus (31.3%) had the largest share of infections of chronic wounds. This observation is consistent with the data presented in the literature. In the meta-analysis by Howell-Jones et al. (2005) S. aureus and coagulase-negative staphylococci were the dominant pathogens isolated from infected wounds.

S. aureus was isolated from infected leg ulcers with an incidence of 43%, while S. epidermidis was detected in 14% of VLUs and 20.6% of DFS cultures (Howell-Jones et al. 2005). Similarly, coagulase-negative Staphylococci (CoNS) were relatively common in our analysis. The entire CoNS group accounted for 13.7% of all isolated microorganisms in our material. The most common CoNS were: S. epidermidis, S. simulans, S. lugdunensis, and S. hemolyticus.

In the study by Puca et al. (2021), S. aureus was present in 38.5% of samples collected using the “Z” technique, obtained from wounds in outpatients. Also, in our material, S. aureus was the most common microorganism, and its incidence in the examined cultures was similar (31.3%).

In the already cited study, Rondas et al. (2015) isolated most frequently: S. aureus (51.1%), S. agalactiae (12.2%), P. aeruginosa (8.9%), E. coli (7.8%), and P. mirabilis (7.8%) regardless of the type of wound. S. aureus, E. coli, and P. aeruginosa were also dominant in our material. Significant discrepancies concerned S. agalactiae. This microorganism was detected most often among all streptococcal in our material but significantly less often than in the cited publication (5.7% vs. 12.2%). E. faecalis was the second pathogen after S. aureus and was present in 26.2% of the tested samples from our center, compared to 6.7% of pathogens isolated from infected chronic wounds in the study by Rondas et al. (2015). Also, MacDonald et al. (2002) observed a higher incidence of S. aureus (42%) and E. faecalis (29%) in infected wounds during DFS.

In our material, methicillin-resistant S. aureus (MRSA) accounted for 14.0% of all S. aureus isolates. Data from the literature indicate a much higher percentage of these isolates in infections of chronic wounds. In the study by Yates et al. (2009), MRSA accounted for as much as 39.6% of all isolated Staphylococci aureus, and in Kasithevar et al. (2017) this percentage was 48.1%.

Anaerobic organisms were also observed among the microorganisms detected in infected chronic wounds. Positive cultures for anaerobes were obtained in 18.8% of cultures. These affected 13.2% of VLU samples and 18.9% of DFS wound samples (all types combined), respectively. Trengove et al. (1996) detected anaerobes in a quarter of samples from infected and uninfected VLU. In the observations of MacDonald et al. (2002), they accounted for only 6% of isolates from infected wounds in the course of DFS, while in the work of Rondas et al. (2015), anaerobic bacteria were isolated from only 2% of chronic wounds. In this context, an interesting observation was made by Bowler and Davies (1999), who found anaerobes in as many as 82% of infected leg ulcers. Noteworthy, this study dates back to 1999, when different standards of wound debridement for material collection and microbiological examination were in force. The wounds included in this study were only cleansed with saline before the culture was taken. The cultures were collected using techniques similar to Levin’s, including necrotic tissue. Since the results of the cited study differ significantly from those presented by other authors and us, it should be assumed that they reported wound-colonizing microorganisms and not those responsible for the infection.

Although proper surgical debridement of the wound is of crucial importance for treating chronic wounds, in some clinical cases, it is also necessary to include antimicrobial treatment. The idea is then to use targeted therapy. However, this is possible only after obtaining the microbiological test result. In many cases, the rate of infection-induced inflammation causes rapid tissue loss. Hence the need for immediate antibacterial treatment. In selecting antibiotics in the empirical therapy of chronic wounds, we rely on standards developed based on data from outside Poland. The constantly growing population of patients with infected wounds, the growing resistance of microorganisms, and the shortage of optimal systemic solutions require several actions to organize the management of this group of patients (WHO 2019; World Union of Wound Healing Societies 2020).

Further analysis of the extensive database of reliable cultures obtained by deep-tissue biopsy is crucial to create new recommendations for empirical antibacterial treatment of infected chronic wounds, especially in terms of drug susceptibility of isolated microorganisms.

CoNS

DFS

IDSA

MRSA

MSSA

VLU

VRE