Prevalence of Microorganisms and Antibiotic Sensitivity Among Patients with Diabetic Foot Ulcer in Sulaimani City, Iraq

Copyright © 2020 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Background As a serious complication associated with diabetes, diabetic foot can seriously affect the quality of life in patients.1 According to a report of the World Health Organization (WHO), the incidence of diabetes mellitus (DM) is on a steady rise, particularly in developing countries. Thus, determining what microorganisms are involved in such infections is essential to determining an appropriate antimicrobial therapy.2 The burden of diabetic foot disease is expected to rise given the increasing global prevalence of type 2 diabetes mellitus (T2DM).3 Diabetic foot ulcer (DFU) causes significant morbidity and mortality in those patients who develop this complication.4 About 382 million people worldwide suffer from diabetes, and it has been estimated that this number will increase to 592 million by the year 2035.4 It has also been stated that 15% of patients with DM will develop DFU at some point in their lives.3,5 Clearly, patients with uncontrolled diabetes are at high risk for developing complications like pedal ulcer, diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy. Moreover, if DFUs are not treated promptly, they may frequently lead to amputations.3,6 DFUs are responsible for more days of costly hospitalization than any other diabetes-related complication and are the major cause of medical lower limb amputations.7 DFUs are commonly classified according to Wagner’s classification system, which assesses ulcer depth and the presence of osteomyelitis or gangrene (Table 1). Depth of the ulcer is the most important risk predictor Abstract

of amputation and is usually caused by neuropathy, peripheral arterial disease, decreased immunity, and pressure overload. 5,9 Other factors associated with amputation include age, male gender, and long duration of diabetes. 10,11 The wound surface can be colonized by a wide range of microorganisms, and it may be polymicrobial. 3 In DFUs, correct antibiotic usage based on cultures and sensitivity is needed for proper management. 5 Good management of the infection can reduce the incidence of infection-related morbidities, the need for and duration of hospitalization, and the incidence of major limb amputation. Early identification of the lesions, prompt initiation of appropriate antibiotic therapy, aggressive surgical debridement of necrotic soft tissue and bone, and modification of host factors are all equally important for a successful clinical outcome. [12][13][14] Patient knowledge and practice of foot care are crucial to preventing foot ulcer formation. 15,16 However, several studies found that most patients do not receive adequate information regarding foot care practices. 4 Today, there are specialized diabetic foot units where proper management of DFU (like empiric antibiotic therapy) is carried out after antibiotic sensitivity testing and while the results of culture and sensitivity testing is pending. 15 The most common isolates from DFUs are gram-positive cocci, especially Staphylococcus aureus. The advised empirical treatment is a narrow spectrum for low-grade ulcers and a broader spectrum for higher grades. 16

Objectives
The present study aimed to examine the prevalence of microorganisms in patients with DFUs and evaluate their drug sensitivity patterns.

Patient Selection; Inclusion and Exclusion Criteria
This cross-sectional study was carried out on 92 cases with DFUs admitted to medical and orthopedic wards of Shar Teaching Hospital and Shahid Hemin Hospital in Sulaimani, the Kurdistan region of Iraq, from September 2018 to April 2019. Patients who were diagnosed with type 2 or type 1 DM (HbA1c more than 6.5% based on American Diabetic Association criteria, 17 treated with oral anti-diabetic medication or insulin, and had DFUs were included in the study. Cases who were treated with antibiotics before hospital admission, those who had and tissue sampling for gram stain, fungal, and cultural sensitivity. Sterile cotton swabs, syringes, and the sterile containers were used to take samples following disinfecting (for grade 1) of the skin surrounding the ulcer. Pus samples were taken from the deepest parts of the base of the ulcer by rubbing it with a sterile swab in a circular motion. All procedures were performed aseptically. Sampling was not done during the holidays.

Transportation
Samples were immediately transported to the microbiology laboratory for processing without storage.

Processing
Before smear staining, the samples were cultured to avoid contamination culture. This procedure included: 1. Aerobic culture at 37℃ on blood and McConkey agar, CO2 enhanced culture on chocolate agar for fastidious bacteria and Sabouraud Dextrose agar as special fungal isolation media (at both 37℃ and 25℃); 2. Anaerobic enhanced culture in thioglycolate-enriched broth (supportive for anaerobic and microaerophilic agents); 3. Part of the swab, pus, or tissue sample was put inside a container with thioglycolate to support anaerobic growth. All samples were incubated for 48 hours on Petri dishes in each atmosphere. Thioglycolate broth was subcultured after 48 hours of incubation for another 48 hours' duration on similar agars.
Each positive sample was processed based on the following logic: 1. Gram stain on a colony to determine whether it is gram-positive or gram-negative. 2. Using VITEK II identification and antibiotic sensitivity testing (AST). 3. AST panel items were used based on the availability of (automated VITEK II AST card) with different codes that may include different items that are not uniform.

Degree of Adherence to Medications
The degree of adherence to medications was determined according to the Morisky Green Levine Medication Adherence Scale (Table 2). 18 Scores gained from the MGLS range from 0-4, and each of the four items is in a (yes/no) format. One point is scored for each positive response; 1 point is given for a "yes" answer, and 0 point for a "no" answer. A score of 0 indicates high adherence; a score of 1 or 2 illustrates intermediate adherence, and a score of 3 or 4 indicates low adherence.

Patients' Knowledge about Foot Care and Hygiene Practices
This study employed a diabetic foot care questionnaire designed by Hasnain and Sheikh (2009) which comprised a set of 15 yes-or-no questions about knowledge and practices (Table 3). Each 'yes' answer carried one (1) point and each 'no' answer carried zero points. The level of knowledge and practice, whether good or poor, was determined based on the median score of each category. Scores higher than the median were considered good, and scores lower than the median were considered bad.

Laboratory Investigations
Laboratory investigations were performed during admission after blood samples were taken aseptically from upper limb peripheral veins and included whole blood count, HbA1c, and lipid profile (triglycerides, cholesterol, low-density lipoprotein) after 12 hours of fasting.
Based on the criteria of the American Diabetes Association, 17 an HbA1c value less than 7% was regarded as controlled and more than 7% as uncontrolled. According to the WHO, 20 hemoglobin levels of less than 12.9 g/ dL for males and 11.9 g/dL for females were regarded as anemia. Based on criteria from the American College of Endocrinology, 21 triglycerides of less than 150 mg/dL, total cholesterol of less than 200 mg/dL, and low-density lipoprotein (LDL) of less than 130 mg/dL were considered normal.

Results
This study included 92 diabetic foot patients with a mean age of 59.5 ± 12.1 years. There were more male patients than females (56.5% vs. 43.5%). The majority of the patients (95.6%) had T2DM. The mean DM duration was 14.6±7.9 years. The mean body mass index of diabetic foot patients was 25.6 ± 4.1 kg/m 2 . The main treatment was drugs (76.08%), followed by insulin (17.3%) and combined drugs and insulin (6.5%). Patient adherence to treatment was commonly low (64.1%), and smoking history was positive in 26.08% of cases. Past medical history was positive in 61.9% of the patients, and history of an ulcer was positive in 52.2% of them (Table 4). Most DFUs were in grades 2 (39.1%) and 3 (28.2%), respectively. Ulcer site in most cases (73.9%) was the right foot. The knowledge of foot care was bad in 63.04% of diabetic foot patients (Table 5).
A total of 100 positive microbial agents were isolated from swabs taken from the diabetic foot patients. Two diabetic foot patients had no growth, while 10 had polymicrobial infections. The gram-positive microorganisms represented 53% of the microorganisms isolated, with the first three most frequent of them being S. aureus (54.7%), Staphylococcus epidermidis (22.6%), and Enterococcus faecalis (15.1%). Gram-negative microorganisms represented 47% of the total isolated microorganisms, with the first three most frequent of them being E. coli (32%), Klebsiella (17%), and Proteus mirabilis (17%) ( Table 7).
Regarding the severity of the diabetic foot lesions, there was a highly significant association between the isolated gram-negative microorganisms and higher grades of DFUs (P < 0.001). Regarding the site of foot ulcers, no significant differences were observed between diabetic foot patients with gram-positive microorganisms and diabetic foot patients with gram-negative microorganisms (P = 0.2). A highly significant association was observed between patients' bad knowledge and hygiene practices and gramnegative microorganisms (P < 0.001) ( Table 9).

Discussion
As the most severe complication of diabetes, foot ulceration affects diabetic patients who are not restricted to certain superficial underlying subcutaneous tissue. If diabetes is not controlled or if there is poor health self-care, diabetic foot ulceration may result. 22,23 It is essential to identify the microorganisms responsible for DFU in order to determine the treatment for DFUs so as to prevent amputation. 24 In the present study, more than half of the patients were men, and slightly over 6% of them were younger than 40 years of age. The fact that over 93% of patients were older than 40 years may be due to the fact that today, various factors, such as professional activities and lifestyle, that would cause the feet to tolerate more pressure have changed, and consequences are more commonly seen in middle-aged and older people. 3 The present study showed that most of the patients were of an elderly age and the male gender. Consistently, Sari et al documented that being elderly and being male were associated with DFUs. 25 The mean duration of diabetes in the present study was 14.6 years, which is close to the mean duration of diabetes of 13.5 years reported in Iranian diabetic patients by Farshad et al. 26 Mohammed et al stated that older age, long diabetes duration, multiple anti-diabetics, hard physical activity, and bad foot care are the common risk factors for the development of DFUs in Iraq. 27 In the present study, DFU cultures revealed that 53% and 47% of the microorganisms were gram-positive and gramnegative, respectively. This finding is inconsistent with the      28 However, the findings of the present study are similar to the results of Al-Hemedawi and colleagues' study in Iraq which found that gram-positive bacteria, particularly S. Aureus, were most common. 29 Differences in microorganism cultures between different studies might be due to the variance in risk factors in developing foot ulcers among diabetic patients. The current study showed that S. aureus was the most prevalent gram-positive bacteria and E. coli was the prevalent gram-negative bacteria responsible for DFUs. These findings are consistent with the results of studies carried out by Al-Hemedawi in Iraq and Sekhar et al in India. 29,30 In the current study, microorganisms showed higher sensitivity to imipenem, moxifloxacin, teicoplanin, daptomycin, vancomycin, and meropenem, while resistance was high for erythromycin, tetracycline, ampicillin, and ceftriaxone. These findings are similar to the results of a recent Iraqi study which found that microorganisms isolated from DFUs were highly sensitive to imipenem and vancomycin and resistant to doxycycline and ceftriaxone. 31 The current findings are also similar to the results of Perim et al, whose study in Brazil revealed that imipenem was an effective antibiotic against grampositive and gram-negative bacteria of DFUs. 32 Previous use of broad-spectrum antibiotics is reported as a common risk factor for the development of highly resistant bacteria. 33 The higher rate of multidrugresistant bacteria present in the DFUs can be explained by patients experiencing antibiotic therapy prior to surgical debridement. 34 Antibiotic resistance can also be explained by exposure to multiple courses of antibiotics, which is common among patients with DFUs, because they are usually hospitalized multiple times. 35 Therefore, the potential presence of such resistant strains puts emphasis on the significance of obtaining optimal specimens from DFUs for culture and sensitivity testing as well as the need to avoid excessive antibiotic therapy which promotes this resistance. 36 Differences in the results reported by many studies indicate that the patterns of microbial infection are not consistent in patients with diabetic foot infections; therefore, repeated evaluation of microbial characteristics and antibiotic sensitivity is necessary for the selection of appropriate antibiotics. 37 In the present study, antibiotics such as imipenem, moxifloxacin, teicoplanin, daptomycin, vancomycin, and meropenem caused the highest rate of sensitivity. In terms of the application of imipenem and vancomycin, the current results were in line with those of the study carried out by Perim et al who reported that the prepared cultures were much more sensitive to these antibodies. 32 Moreover, erythromycin, tetracycline, ampicillin, and ceftriaxone caused the highest rate of resistance to the prepared culture. This finding is in agreement with those reported by Perim et al, who mentioned that erythromycin, tetracycline, and ampicillin antibiotics cause the highest rate of resistance. 32 In addition, in a similar study, Sivanmaliappan and Sevanan reported that 100% of Pseudomonas aeruginosa isolates were resistant to ampicillin and norfloxacin, 83.3% to tetracycline, 66.6% to gentamicin and imipenem, and 16.6% to cefotaxime. 34 In the current study, a highly significant association was observed between the isolated gram-negative microorganisms and higher grades of DFUs (P < 0.001). This finding is in agreement with the results of a study by Otta et al in India. 38 Additionally, the DFU patient's bad knowledge and hygiene practices were significantly associated with gram-negative microorganisms (P < 0.001). This finding is in agreement with reports from Kwon and Armstrong's study in South Korea, which documented that bad knowledge and practices of diabetic patients are risk factors for developing foot ulcers with poor prognoses especially if infected with gram-negative bacteria. 39 In the present study, the mean Hb of diabetic foot patients was 11 g/dL, and 48.9% of diabetic foot patients had anemia. The results of a study by Olgun et al in Turkey revealed that 85.3% of diabetic foot patients had anemia. 40 Anemia is common among patients with DFUs and is associated with advanced morbidity of diabetic foot, regardless of its severity. 41 As seen in HbA1c values, 95.7% of diabetic foot patients in the present study had poor glycemic control. This finding is similar to the results reported by Chuan et al. 42 The present study had some limitations. The most remarkable one is related to its small sample size; therefore, generalizing the findings should be done with much caution. The second limitation is related to the fact that cultures for anaerobic bacteria could not be performed. The third limitation is that in studies with a cross-sectional design, it is impossible to infer causality or untangle bidirectional relationships. Future studies are recommended to resolve these limitations.

Conclusion
DFUs are predominantly caused by gram-positive bacteria, such as S. aureus. Moreover, gram-positive bacteria like S. aureus were more prevalently isolated from DFUs than gram-negative bacteria like E. coli. In addition, imipenem was an effective antibiotic against microorganisms causing DFUs. Various studies on the bacteriology of DFUs have reported results that vary and are often contradictory; therefore, the application of molecular techniques may lead to more accurate microbial characterizations and targeted antibiotic therapy. Thus, it is necessary to evaluate different microorganisms causing DFUs on a routine basis and to know the antibiotic susceptibility patterns of isolates from infected wounds in patients with such lesions. This knowledge is crucial for planning the treatment of these patients with the appropriate antibiotics, reducing resistance patterns, and minimizing healthcare costs.