Volume 26 Number 3

Key message

• The paper evaluates the microbiological patterns associated with diabetic foot infections in hospitalised patients and their relationships with clinical outcomes, particularly focusing on amputation rates. The goal of the manuscript is to identify the prevalent bacterial profiles in DFI and to analyse their correlation with infection severity and amputation outcome in patients with diabetes. The study found that MSSA was the most commonly isolated bacterium, with 37.8% of cases exhibiting polymicrobial infections. While the overall amputation rate was significant, clinical severity, as indicated by elevated WBC counts, was the primary factor influencing outcomes, rather than the specific types of bacteria.

Introduction

Diabetic foot disease (DFD) stands as a silent killer among diabetic complications, significantly contributing to amputations worldwide. Its impact extends beyond amputations, as DFD leads to increased hospitalisations, reduced quality of life (QoL), and greater disability burdens. Alarmingly, DFD accounts for 2% of the global disease burden, ranking as the 13th most impactful health issue among over 350 leading conditions.¹

Patients with diabetes can develop a diabetic foot ulceration (DFU) in 19–34% of cases during their life, and 60% of DFUs, can develop a concomitant infection (DFI).^2,3 Many patients with DFI often require hospitalisation to manage their infective framework,⁴ thus risking minor or major amputation of the affected limb in a high percentage of cases.^3-7 DFI significantly increases health care costs when compared to uninfected DFU,⁶ and DFI is a well-known independent risk factor for mortality and amputation in diabetic patients. In the case of late referral or management, DFI may degenerate into more severe conditions, such as wet gangrene, necrotising fasciitis, necrosis of the skin’s soft tissue or osteomyelitis.⁷ The above-mentioned conditions require antibiotic therapy in combination, or not, with surgical treatment to control the infected site.⁷ Antibiotic therapy should initially be prescribed according to the clinical framework, and adapted to respond to the majority of the bacteria, including gram-positive bacteria, such as staphylococci and streptococci, which are often present in less severe infections.⁸ In the case of moderate or severe infection, or if patients have recently received antibiotic therapy, broad-spectrum therapy should be initiated, acting on both gram-negatives and anaerobes.⁸ Performing microbiological sampling of infected tissues is recommended to identify the pathogens responsible for the infection and then to continue targeted antibiotic therapy according to the isolate.⁸ To study and know the microbial agents involved in DFI is also helpful to know the microbiological pattern, the potential presence of polymicrobial DFI. To prescribe the best antibiotic therapy, knowing the potential microbiological pattern could be useful since the achievement of microbiological results can sometimes take several days.

This concept is important for patients requiring hospital admission due to moderate or severe infections, and where adequate empirical therapy is mandatory to reduce the evolution of infectious processes.

For the reasons mentioned above, the authors considered that it may be useful to know the local DFI epidemiology of hospitalised patients and the association between microbiological features and specific clinical patterns. Therefore, the aim of this study was to evaluate the epidemiology and characteristics, both clinical and microbiological, of patients admitted for a DFI.

Methods

Patient selection

This study is a retrospective observational investigation conducted at a single center. We included consecutive diabetic patients hospitalised for diabetic foot infection (DFI) in the Endocrinology and Diabetes Unit of University Hospital Fondazione Policlinico Tor Vergata, Rome, Italy, from January to September 2023. Only patients with confirmed DFI were included, while those without clinical signs of foot infection were excluded.

For all patients presenting with clinical signs of DFI, a sample of soft tissue or bone was collected for microbiological analysis. If the initial sample did not yield bacterial growth, a second sample was taken. Patients were excluded from the study if two consecutive samples tested negative for bacterial isolation. Additionally, patients were excluded if they had received antibiotics within the two weeks prior to consulting our Diabetic Foot Service (DFS), had an indication for primary major amputation upon admission, were undergoing immunosuppressive therapy, had neoplastic disease, or had a reduced life expectancy. (Table 1).

 

Table 1. Inclusion and exclusion criteria at the assessment

 

All patients were managed by a multidisciplinary diabetic foot team through a pre-set limb salvage protocol following the International Working Group on Diabetic Foot (IWGDF) guidelines.⁹ Treatment also included lower revascularisation in case of ischaemic/neuro-ischaemic DFU and, if required for specific cases, surgery, including necrosectomy, minor amputation and bone removal, along with negative pressure therapy and/or dermal substitute application.

Additionally, metabolic dysfunction, diabetic complications, and associated comorbidities were promptly addressed, and key principles of patient education were shared with patients, their relatives and caregivers. Demographic and clinical data, along with the characteristics of each DFU, were thoroughly documented during the initial assessment.

Clinical assessment

Type of diabetes, duration and metabolic control were noted. Hypertension was considered if the patient was currently undergoing antihypertensive therapy; hypercholesterolemia was considered if the patient was on statin therapy or if there was evidence of elevated low-density lipoproteins (LDL) (>55 mg/dL) at the assessment, thus necessitating statin therapy.¹²

The presence of ischaemic heart disease (IHD) was defined by a history of previous acute coronary syndrome or coronary revascularisation, the presence of angina, and significant electrocardiographic changes (such as ST elevation or depression, q waves, T wave inversion, or new left bundle branch block).

Patients were classified as smokers only if they were actively smoking at the time of assessment. The prevalence of patients with end-stage renal disease (ESRD) who required dialysis was also reported.

DFU assessment

Ulcer characteristics were documented during the presentation and the initial assessment at the DFS based on IWGDF definitions.⁹ Included DFUs were categorised as ischaemic infected wounds or neuropathic infected wounds, based on the presence or absence of peripheral arterial disease (PAD).

Assessment of peripheral neuropathy was based on the loss of peripheral sensitivity detected through vibration perception (128 Hz tuning fork) and/or Semmes‐Weinstein 10-g monofilament⁹.

Vascular assessment included the classification of neuro-ischaemic/ischaemic DFUs based on criteria, such as the absence of palpable distal pedal pulses, the ankle-brachial index (ABI)<0.5, and/or transcutaneous oxygen pressure (TcPO2)<30mmHg, requiring lower limb revascularisation. Bedside tests for vascular assessment were performed exclusively by expert professionals (interventional radiologists/vascular surgeons) to minimise inter-observer variability. Morphological evaluation of the vascular tree was conducted in all patients using ultrasound duplex. Computed tomography or MRI was employed only when uncertainty regarding vascular plaques and the type of vascular intervention arose.⁹

The diagnosis of infection adhered to the IWGDF guidelines, requiring the presence of at least two of the following criteria: local swelling, induration, erythema >0.5 cm around the wound, local tenderness, pain, increased warmth and purulent discharge.⁹

DFUs classified as infected for this study were considered as severe infections, defined by the number of WBC >12000x10³µL.⁸

Osteomyelitis was diagnosed in cases in which a deep ulcer involved the bone (exposed or positive probe-to-bone procedure), confirmed by X-ray or Magnetic Resonance Imaging, and positive microbiological analysis of a bone biopsy.⁸

The clinical framework was also reported: soft tissue infection was considered in the case of wet gangrene, necrotising fasciitis, and phlegmon/abscess/cellulitis, while osteomyelitis was considered in the case of bone infector as described above.

For all included patients, a sample for microbiological analysis was collected, involving a punch biopsy of deep tissues for soft tissue infection and a bone sample (percutaneous or surgical approach) for suspected or confirmed osteomyelitis. Microbiological analysis of the samples, performed in the hospital microbiology lab followed EUCAST criteria, and recorded the type of bacteria, presence of single or polymicrobial infection.¹⁰ Polymicrobial infection was considered by the isolation of at the least two bacteria in the microbiological analysis.

The rate of in-hospital amputation (minor and major) was  reported. In addition, the characteristics of amputees and non- amputees were also analysed and compared, evaluating the microbiological features of both groups. Minor amputation was defined as any amputation below-the-ankle, and major amputation was recorded for any amputation above-the-ankle.

The association between clinical patterns of DFUs (moderate or severe infection, according to IDSA classification,⁸ and type of isolate (monomicrobial or polymicrobial) was reported.  Furthermore, the association between neuropathic and ischaemic DFUs with monomicrobial or polymicrobial isolates, and IDSA severity was examined.

Statistical analysis

Statistical analysis was performed by SAS (JMP12; SAS Institute, Cary, NC, USA). Continuous variables were expressed as the mean±SEM. Comparisons between group characteristics were made with an X2 test (frequency data) or analysis of variance (continuous data). The P value <0.05 was considered statistically significant.

Results

A total of 123 patients were included in the study. The average age was 67.8±10.6 years, with 74.5% being male and 97.5% diagnosed with type 2 diabetes. The mean HbA1c value was 66±21 mmol/mol, and 98.4% of the patients had dyslipidemia (Table 2). Among these patients, 22% had osteomyelitis, while 78% had soft tissue infections. The most frequently isolated bacteria were Methicillin-susceptible Staphylococcus aureus (MSSA) (26%), Enterococcus faecalis (12.2%), Pseudomonas aeruginosa (10.6%), and Methicillin-resistant Staphylococcus aureus (MRSA) (7.3%). Polymicrobial infections were present in 37.8% of cases.

 

Table 2. Clinical and demographic characteristics at the assessment.

 

Table 3. Isolate strains among infected ulcers.

 

The in-hospital minor amputation rate was 56.3% (69 patients), while the major amputation rate was 4.2% (5 patients). A comparison between patients who underwent major and minor amputations during hospitalisation revealed that those who underwent any amputation were older (70.1±8.9 vs 60.9±12.1 years, p=0.006), had a higher prevalence of ischemic diabetic foot ulcers (91.7% vs 70.6%, p=0.002), and exhibited elevated WBC counts (10.9±6.5 vs 8.3±3.4, p=0.01) compared to non-amputees (Table 4).

 

Table 4. Comparative analysis between clinical features and amputation.

 

According to a multivariate analysis no correlation was found between any in-hospital amputations (minor and major) and the type of bacteria, the presence of polymicrobial infection, and the type of infection (bone or soft tissue). Only severe infection (vs moderate infection), defined by presence of WBC>12000 x10³µL at the time of admission, resulted as an independent predictor of amputation.

No relationship was found between the risk of any kind of amputations and microbiological pattern.

Discussion

The present study, including a cohort of patients hospitalised for DFI, showed that the majority of admitted patients were male with type 2 diabetes. A significant number of patients also showed the presence of end-stage renal disease and ischaemic heart disease.

Most DFIs were related to soft tissue infections (78%), while 22% of cases presented as osteomyelitis.

DFI remains a pressing clinical concern, elevating the risk of amputations and morbidity among patients with DFUs. It is widely recognised that osteomyelitis and gangrene independently increase the risk of non-healing, minor amputations, major amputations and hospitalisation.^11,12 The risk of non-healing and amputation is more closely related to the presence of a concomitant peripheral arterial disease.¹² In our population we found that more than 80% of patients were affected by peripheral arterial disease, defining the population as very hard to be managed.

Regarding the microbial patterns, the most isolated strains in the current study were MSSA (26%), Enterococcus faecalis (12.2%) and Pseudomonas aeruginosa (10.6%) respectively.

These data are similar to results of an epidemiological analysis performed in the same local area by Cipriano et al, which found the most isolated strain was Staphylococcus aureus, followed by Pseudomonas aeruginosa and Enterococcus faecalis.¹³ The results are similar although the abovementioned study was carried out only in outpatients.

Another study by Alvaro Alfonso et al showed a clear prevalence of Staphylococcus aureus in DFI.¹⁴ In that study, results were similar to those found in our study, although they recruited only patients affected by osteomyelitis and few patients with ischaemic ulcers.

A recent review on DFIs confirmed that Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa are generally the most prevalent strains.¹⁵ This data is relevant from an epidemiological point of view since it was an observational analysis on 765 cases of DFU, even though only outpatients were considered in this research.

The same study found that in mild superficial infection, aerobic Gram-positive cocci were also the predominant organisms, and Staphylococcus aureus and Streptococci were the most frequent isolates.¹⁵ Staphylococcus aureus being the predominant strain in mild and moderate-severe infections (such those included in our study) may suggest that the high prevalence rate of this bacterium is, on the one hand, not closely related to the severity of DFI and, on the other hand, that the same bacterium could contribute to different frameworks of infection severity in relation to the clinical condition in which it develops.

In the current study, 37.8% of polymicrobial infection and 62.2% of monomicrobial infections were found. The polymicrobial infection seems to be lower than that encountered in a similar paper of Al Bewan et al on DFI in a cohort of hospital subjects, which were found to present in 75% of cases a polymicrobial flora.¹⁶

Gadepalli et al found 82.5% of polymicrobial episodes in 80 patients with DFU and severe infections.¹⁷ In another study Zubair et al described 65% cases of polymicrobial etiology in patients treated for DFI.¹⁸ The different data in comparison to those we found in our current study could be explained by several factors, but the main reason may be the different sampling methodology (biopsy vs sample swab), which could result in the identification of a different flora⁸. The microbiogical analysis of sample culture in patients DFI could be impaired by the potential contamination of commensal bacteria.¹⁹ Accordingly, in the current study, as in clinical practice, authors performed culture biopsy of soft tissue or bone based on the characteristics of the DFI.

In a similar study on DFI where the biopsy was used as the sample methodology, the rate of polymicrobial infections was comparable to those found in our population.¹⁴

Referring to the in-hospital outcomes, the rate of minor amputations was 56.3%, whereas the rate of major amputations was 4.2%. Such positive results, namely the low rate of major amputations in such complex patients with DFI and often ischaemia, could be partly related to a multidisciplinary approach including early lower limb revascularisation of ischaemic subjects and aggressive management of DFI according to a preset local limb salvage protocol adopted in the respect of guidelines.²⁰

Comparing patients who had amputations (both major and minor) to those who reported foot and limb salvage, the group of amputees showed a higher rate of peripheral ischemia when compared to non-amputees (91.7% vs 70.6%), a higher rate of severe infections and more cases of ESRD. Unsurprisingly, these data confirmed that peripheral ischemia, severe infections and ESRD are related to higher risk of amputations.^{12,21,22,23,24}

Nonetheless, there are no significant differences in terms of microbiological pattern between amputees and non-amputees, considering both the types of isolated bacteria and the presence of polymicrobial and monomicrobial flora.

The multivariate analysis on the outcome of interest (amputation) showed that the only factor which might predict in-hospital amputation was the presence of severe infection (based on IDSA classification and definition).

This data seems to confirm a close association between severe infection and hospital outcomes (amputation and mortality) in patients hospitalised for ischaemic ulcer infection, as reported by a recent study.²³ The same study highlighted a significantly  higher value of procalcitonin in patients with worse outcomes, a lab value usually higher in severe infection than moderate/mild infections.²³

It appears that microbiological characteristics, such as the type of bacteria, the presence of polymicrobial flora, or the location of infection, soft tissue or bone, did not significantly impact on the prognosis of admitted patients, including the possibility of digit or limb salvage. The severity of the clinical pattern (severe vs moderate) seems to have had the greatest influence in patients with DFI. The close correlation between severe infection and amputation may be bilateral, on the one side a septic status can reduce a clinical response to the treatment, as well the reduction of distal blood perfusion which is necessary to promote wound healing; on the other side, when a severe infection is present, the local framework is already advanced and a large part of tissue could be necrotic or not salvageable from a surgical point of view. These findings urge us to enhance the clinical approach to these patients. The risk of severe infection necessitates the optimisation of clinical management, which includes avoiding late referral, as late referral is a well identified risk factor for negative outcomes. Late referral is key factor in the risk of advancement of non-infected or mildly infected wounds to a more severe clinical framework, such as severe infection and sepsis^25; while early referral allows immediate surgical management and aggressive antibiotic approaches which are mandatory to limit the evolution of local infections.²⁵

From this perspective, the knowledge of our local microbial flora identified in hospitalised patients gives us foresight in patient management in daily clinical practice while awaiting culture results. This data suggests the prevalence of different kinds of bacteria and how antibiotics should be prescribed before obtaining the microbiological data.

Based on this data, antibiotic therapy should be primarily targeted to the most prevalent bacteria including Staphylococcus aureus (both MSSA and MRSA), Enterococcus Faecalis and Pseudomonas Aeruginosa.

To the best of our knowledge, the current study is among a few to specifically assess the microbiological pattern of hospitalised patients and evaluate the correlation between microbiology and outcomes. The data shows that the severity of DFI is not related only the specific bacteria, but moreover to the field (the wound bed) where they grow. It also confirmed that vascular status and comorbidities influence the chances of limb salvage in patients with DFI. Therefore, it becomes mandatory to manage the vascular aspect, as well as general health status.

Limitations

The study has some limitations. It was a monocentric study and retrospective observational study, which inherently could limit the establishment of causal relationships. Additionally, the sample was not so large, although it remains acceptable compared to similar studies on this topic. The study did not assess the presence of antibiotic resistance, despite previous research having documented that antibiotic resistance does not significantly affect clinical outcomes. Lastly, associations between isolates, clinical presentation, and other factors, such as soft tissue involvement, gangrene, or necrotising fasciitis, were not characterised.

Conclusion

In the context of our study, Staphylococcus aureus, both in the methicillin-sensitive and methicillin-resistant types, emerged as the most frequently isolated bacteria, exerting a predominant influence within the clinical environment. In addition, in most cases the flora was monomicrobial instead of polymicrobial. However, what emerges as a significant reflection is that microbiological characteristics alone did not prove to be independent predictors for in-hospital amputation, underscoring the complexity of infectious disease mechanisms. Rather, it was the clinical severity of the infection that emerged as the determining factor in the decision-making process regarding amputation, thus highlighting the crucial role of thorough clinical evaluation and comprehensive management in complex medical situations such as this one.

Author contributions

Meloni, Mazzeo, Bellizzi, made a substantial contribution to the design of the work; Meloni, Bellizzi, Mazzeo and Cipriano  made a substantial contribution to the acquisition of data; Meloni, Mazzeo and Bellizzi made a substantial contribution to the analysis and interpretation of data; Mazzeo and Bellizzi  wrote the manuscript; Ruotolo, Andreadi, Romano, Giurato, Bellia, Uccioli and Lauro revised it for intellectual content.

Conflict of interest

The authors declare no conflicts of interest.

Funding

The authors received no funding for this study.

Author(s)

Luca Mazzeo¹, Aikaterini Andreadi¹, Ermanno Bellizzi¹, Valeria Ruotolo¹, Maria Romano¹, Rossana Cipriano², Laura Giurato³,
Alfonso Bellia⁴, Luigi Uccioli³, Marco Meloni¹*, Davide Lauro^1
1Department of Systems Medicine, University of Rome Tor Vergata, Italy; University Hospital Fondazione Policlinico Tor Vergata, Rome, Italy
²Figebo Centro Polispecialistico, Cassino, Frosinone, Italy
³CTO Andrea Alesini Hospital, Division of Endocrinology and Diabetes, Department of Systems Medicine, University of Rome Tor Vergata, Italy
⁴Department of Systems Medicine, University of Rome Tor Vergata, Italy; University Hospital Fondazione Policlinico Tor Vergata, Rome, Italy

*Corresponding author email meloni.marco@libero.it

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