Analysis of diabetic foot ulcers in a tertiary ca

　　　　 作者：Shazi Shakil， Shaista Alvi， Mujahid Beg， Asad U. Khan

【摘要】 　　Objective:To assess potential risk factors for diabetic foot ulcers infected with multidrugresistantorganisms (MDROs) and to investigate antibiotic susceptibility patterns and extendedspectrumβlactamase (ESBL)production in bacteria isolated from these ulcers. Methods: Seventyseven diabetic foot ulcerpatients were studied in a consecutive series. Fortyfour study factors were recorded for each patient. Specimens were obtained by scraping the ulcer base or the deep portion of the wound edge with a sterile curette. The soft tissue specimens were quickly sent to the laboratory and processed for microbial pathogens. Gramnegative bacterial isolates were tested for ESBL production by doublediscdiffusion method. The association of study variables with MDRO and nonMDRO infection status of ulcers was tested by Student's t test or Fisher's exact test using SPSS (version 11.5， Chicago).Results: An alarming number of patients were MDROpositive (72.7%). ESBLpositive strains constituted 19.8% of total isolates. Staphylococcal isolates identified as methicillinresistant Staphylococcus aureus (MRSA) and vancomycinresistant Staphylococcus aureus (VRSA)， were 3.7% (n=8) and 0.5% (n=1) respectively. Twentytwo study factors were found to be significantly associated with MDRO infection status of ulcers in the univariate analysis. Multiple logistic regressions proved that MDRO status was the only significant， independent predictor of glycemic control (Odds ratio = 4.22， P&<0.01).Conclusion:It is concluded that MDRO infected patients have poor glycemic control. Imipenem and linezolid can be suggested as the drugs of choice in the described setting.

【关键词】 Diabetic foot ulcers; Glycemic control; Multidrug resistant organisms

　　INTRODUCTION
　　
　　Foot ulcers are a very common complication of type 1 and type 2 diabetes［1］. In developing countries like India， the matter is worse because of poor sanitation that creates an infectionprone environment［2］. The prevalence of diabetes in Indian population is increasing［3］. Infections with multidrug resistant strains of bacteria are becoming a major problem in the whole world［4，5］ and inpiduals with diabetes are known to be at a greater risk of foot infections［6］ than the inpiduals without diabetes. Over 20%40% of diabetic wounds have been found to be methicillinresistant Staphylococcus aureus (MRSA) infected by many studies［7，8］.
　　
　　Studies related to multidrugresistantorganisms (MDROs) infections in diabetic foot ulcers may be helpful as they provide basis for empirical antimicrobial therapy. There was an urgent need for a study that could deal with the association of different factors with MDRO and nonMDRO infection status of ulcers in due detail， from this region. The main aim of this study was to assess potential risk factors for diabetic foot ulcers infected with MDROs. The association of study variables with MDRO and nonMDRO infection status of ulcers has been analyzed. Both univariate and multivariate analyses have been performed.

　　MATERIALS AND METHODS

　　Clinical setting and data collection

　　Seventyseven diabetic patients having clinically infected foot ulcers admitted to the endocrinology ward at the JN Medical University， Aligarh， over a period of 2 years were studied in a consecutive series. Wagner classification was employed to grade the ulcers［9］.

　　Fortyfour study factors were recorded for each patient. BMI i.e. Body Mass Index (&<18.5 kg/m2=underweight; 18.522.9 kg/m2=normal weight; 23.024.9 kg/m2=overweight)， presence of nephropathy (creatinine ≥150 μmol/L or presence of micro or macroalbuminuria)， neuropathy (absence of perception of the SemmesWeinstein monofilament at 2 of 10 standardized plantar sites on either foot) and peripheral vascular disease (ischemic symptoms and intermittent claudication or rest pain， with or without absence of pedal pulses)， were some of them. Osteomyelitis was diagnosed on suggestive changes in the radiographs and bone scans. If a patient had multiple ulcers， specimens from all the ulcers were taken; and the patient was grouped in the MDRO infected category if any of the ulcers was found to be MDRO positive. Fasting high density lippprotein (HDL) and very low density lipoprotein (VLDL) were measured and low desnsity lipoprotein (LDL) was calculated by subtracting their sum from total cholesterol.
　　
　　Clinical assessment for signs of infection (swelling， exudate， surrounding cellulitis， odor， tissue necrosis， crepitation， and pyrexia) was made. Ulcer size was determined by multiplying the longest and widest diameters and expressed in centimeters squared. Each patient was minutely tracked until discharge or death during the stay. Informed consent was obtained from all subjects， and clearance was obtained from the institute's ethics committee.

　　Shazi Shakil et al. Analysis of diabetic foot ulcers in a tertiary care hospitala clinicomicrobiological perspectiveMicrobiological procedures
　　
　　Culture specimens were obtained at the time of admission， after the surface of the wound had been washed vigorously by saline， and followed by debridement of superficial exudates. Specimens were obtained by scraping the ulcer base or the deep portion of the wound edge with a sterile curette. The soft tissue specimens were quickly sent to the laboratory and processed for microbial pathogens. Standard methods for isolation and identification of aerobic and anaerobic bacteria were used［10，11］.

　　Antimicrobial susceptibility testing

　　Antimicrobial susceptibility testing of aerobic isolates was performed by the standard disc diffusion method as recommended by the Clinical and Laboratory Standards Institute［12］. All anaerobic isolates were tested for susceptibility to metronidazole and amoxyclav by microbroth dilution test［13］. Gramnegative bacilli were tested for extendedspectrumβlactamase (ESBL) production by the double disc diffusion method and Staphylococcus species were tested for methicillin resistance by using oxacillin 1μg disc， oxacillin HiComb minimum inhibitory concentration (MIC) test as per manufacturer's instructions (Himedia， Mumbai， India)， oxacillin screen agar (6 μg/mL) and cefoxitin 30μg disc［12］. Only the isolates， which were simultaneously positive for all these tests， were designated as methicillinresistant. Staphylococci were also tested for vancomycin resistance by HiComb MIC test as per manufacturer's instructions (Himedia， Mumbai， India) and confirmed by vancomycin screen agar (6μg/mL). MRSA， vancomycinresistant Staphylococcus ayreus (VRSA)， ESBLpositive Gramnegative bacteria， and all the Gramnegative bacteria that were resistant to at least four of the test antibiotics were considered as MDROs in our study.

　　Antibiotic treatment of patients

　　As an empiric treatment at admission， amoxyclav (1.2 g i.v. every 8 h) was given to all patients， intravenously which was switched to oral administration (625 mg p.o. every 8 h). If cellulitis or gangrene was also present， metronidazole (500 mg i.v. every 8 h) was added to the regimen. To target specific pathogens， drug regimen had to be modified in accordance with the culture susceptibility reports， as appropriate.

　　Statistical analysis

　　Qualitative variables were expressed as percentages and quantitative variables were expressed as means±SD (Standard Deviation). The association of study variables with MDRO and nonMDRO infection status of ulcers was tested by using Student's t test or Fisher's exact test as appropriate. The odds ratios (ORs) with 95% CIs (Confidence Intervals) for having MDROassociated ulcers were calculated. Multiple logistic regressions were employed to identify independent predictors of MDRO infections and predictors of glycemic control. A twotailed P value of&<0.05 was taken as significant. Statistical analysis was performed using SPSS (version 11.5， Chicago).

　　RESULTS

　　General results

　　Patients had ulcers with Wagner's grade 25. Fortynine (63.6%) patients had grade2 ulcer. Grade5 was found only in 1 patient belonging to the MDRO group. Males were predominant (80.5%) in the study. Fiftysix (72.7%) subjects， out of 77 had MDRO infected ulcers. Nine (60%) out of 15 females had MDRO infected ulcers. All the MDRO infected patients were 5059 years old， had diabetes from 1019 years with ulcer duration&>3 months. It might be merely a coincidence. The majority of subjects (90.9%) had type 2 diabetes. Ulcer was necrotic in 16 (20.8%) subjects. Fiftynine (76.6%) patients had diabetes for&>10 years. Sixtysix (85.7%) were hypertensive， 25 (32.5%) had retinopathy， 47 (61%) had nephropathy， 42 (54.5%) had neuropathy and 44 (57.1%) had peripheral vascular disease. Twelve (15.6%) patients had osteomyelitis. Fiftyfive patients (71.4%) had undergone surgical treatment for foot. Only 7 (9.1%) subjects were taking diabetic diet. Three patients died during the hospital stay.

　　Microbiological results

　　A total of 217 isolates were detected from the 95 samples obtained from 77 patients. Percent occurrence of different bacterial isolates is detailed in Table 1. ESBLpositive strains constituted 19.8% of total isolates. The highest ESBL production was noted in P. aeruginosa (32.8%)， followed by E. coli (27.5%) and K. pneumoniae (23.7%). Staphylococcal isolates identified as MRSA and VRSA， were 3.7% (n=8) and 0.5% (n=1)， respectively. The numbers of anaerobic isolates obtained was only 13(6%). The anaerobes belonged to Veillonella species， Bacteroides species， Peptostreptococcus species and Clostridium species.
Table 1 Percent occurrence of bacterial strains isolated from infected foot ulcers in diabetic patients' specimens (略).

　　Results of susceptibility testing

　　Antimicrobial susceptibility patterns of the isolates are shown in Table 2. Linezolid was found to be the most potent drug against S. aureus isolates. Staphylococci exhibited a high level of resistance against erythromycin (88.9%); tetracycline， ciprofloxacin， levofloxacin and cotrimoxazole (81.5% each). All the Gramnegative isolates showed 100% susceptibility to imipenem. Quinolones were effective against Proteus species (80% susceptibility to ciprofloxacin). Amikacin was the most effective aminoglycoside against the Gramnegative organisms. Tetracycline and piperacillin were uniformly met with a high level of resistance by these isolates. After imipenem， piperacillin/tazobactam was the most potent option against Escherichea coli and Klebsiella pneumoniae strains. Pseudomonas aeruginosa exhibited a high level of resistance (72.1%) against gentamicin. Cefepime and quinolones were equally potent against Acinetobacter species (80% susceptibility to each). Imipenem and linezolid were the most potent antimicrobials in our study. All the anaerobes were susceptible to metronidazole and amoxyclav.

　　Results of statistical analysis (univariate)

　　Study factors such as duration of ulcer， ulcer size， fasting and postprandial plasma glucose， triglycerides， LDL， VLDL， phospholipids， hypertension， surgical treatment history for foot， smoking and glycemic control at discharge were found to be very closely associated with MDRO and nonMDRO infection status of ulcers (all P&<0.01; for phospholipids P&<0.05). A moderate association was observed for factors such as mean age， Wagner grade and fever (all P&<0.05); duration of diabetes， systolic blood pressure， HDL， neuropathy， diet (all P&<0.05); and lymphocyte count (P&<0.05). Table 2 Antimicrobial susceptibility/resistance pattern of bacterial strains isolated from infected foot ulcers in diabetic patients（略）

　　Results of statistical analysis (multivariate)

　　As in the univariate analysis many study factors were found to be significantly associated with MDRO infection status of ulcers， we went for multivariate analysis. Multiple logistic regressions showed a high degree of interaction between presence of neuropathy and ulcer size. Taking the absence of neuropathy as a baseline (OR =1.00)， presence of neuropathy with an ulcer size &<4 cm2 showed an OR of 0.45 (95% Wald CI 0.082.41) while the same with an ulcer size &>4 cm2 showed an OR of 17.4 (95% Wald CI =2.55118.88). Thus， an ulcer size of &> 4 cm2 with the presence of neuropathy could be a strong indicator of MDRO infections. We found that in the multivariate analysis， the presence of neuropathy and the ulcer size &>4 cm2 were still significantly associated with MDRO infections. Taking the achievement of glycemic control at discharge (yes/no) as the outcome， the association of various study variables with this outcome was examined by multiple logistic regression. We found that MDRO status was the only significant， independent predictor of glycemic control (OR =4.22， P&<0.01). Hence our main finding is that， patients with MDROinfectedulcers have poor glycemic control during the hospital stay.

　　Comparison of "glycemic control" in the two groups of diabetic patients having infected foot ulcers
　　
　　Patients with MDROinfected ulcers had significantly higher blood glucose levels， both fasting and postprandial， on the day of admission. Regarding a fasting blood glucose level of&<110 mg/dL and/or postprandial level of&<160 mg/dL as glycemic control， the number of patients achieving glycemic control over the hospital stay was compared between those with and without MDRO infections. The trend in the average blood glucose levels (both fasting and postprandial) for the two groups during the hospital stay is shown in Figure 1　Open trianglesMDRO 2h; filled squaresMDRO fasting; open cirlcles-non MDRO 2h， filled triangles-non MDRO fasting.

　　DISCUSSION

　　This study presents a clinicomicrobiological analysis of infected diabetic foot ulcers in hospitalized patients in due detail. We regret that we could not use bone biopsy sample in case of osteomyelitis. Bone biopsy may be traumatic to patient and is not routinely performed in our hospital. Moreover， Pellizzer et al［14］. have reported that samples taken from the base of the wound after debridgement are adequate to identify the infecting organism. Gramnegative bacteria were dominant in diabetic foot ulcers in our study contrary to the previous studies［15，16］. These variations might be due to the differences in the study setting， agesex composition， ulcer grades etc. between our study subjects and those of previous studies. Nonetheless， our results from the northern region of the country are in harmony with many akin studies from the southern parts of India［7］， which also concluded gramnegative bacteria to be the dominant pathogens in diabetic foot ulcers. A high prevalence of multidrug resistant P. aeruginosa reported from south India， is in accord with our findings. P. aeruginosa was the most frequently isolated pathogen in our study (28.1%). This raises a serious concern， as P. aeruginosa may sometimes be quite an aggressive pathogen［17］.
　　
　　MDRO infection in hospitalized patients with diabetic foot ulcers has become remarkably common. This finding presented by our study is in agreement with the report of Heurtier et al［18］. The incidence of both MRSA isolates and ESBLproducing Gramnegative bacteria in diabetic foot ulcers has been reported by many previous studies［18］. Ours is a tertiary care hospital with widespread usage of broadspectrum antibiotics. High selection pressure might be the reason for high rates of antibiotic resistance observed in the present study. Compared with earlier reports， we recovered fewer anaerobic species［19］. Our patients did not have chronic draining wounds， and very few had gangrene associated with their infections. This may be an indication of fewer anaerobic species among nonthreatening lowerextremity infections， which has been reported earlier［20］. We could not obtain previous hospitalization records (including antibiotic usage) for the same wound in our patients. This information could have helped in explaining the reasons for the high prevalence of MDROs in our patients.
　　
　　The escalating occurrence of MDROs is alarming as infection with these bacteria restricts the options of antibiotic treatment and multidrug resistant bacteria are increasingly difficult to treat［21］. We found similar duration of hospital stay in both MDROs and nonMDROs. This is contrary to reports that state that MDRO infections might increase hospital stay and cost［22］. It is well established until now， that those patients with diabetic foot ulcers whose blood glucose levels are poorly controlled exhibit higher mortality rates［23］. Our results indicate that patients with MDRO–infected ulcers have poor glycemic control. Hence， MDROs might lead to higher mortality among diabetic foot infections. This is the first study to report on MDRO infections in diabetic foot ulcers from this region of India (Uttar Pradesh). We do not claim that these 77 patients represent the entire region. Hence， similar studies from this region and across the globe， should be encouraged.
　　
　　In conclusion， this study reveals that there is a high frequency of occurrence of MDRO infections in diabetic foot ulcers. MDRO infected patients have poor glycemic control. Antibiotic treatment policies can be updated with the information obtained from such studies ensuring a better patient management. Imipenem and linezolid can be suggested as the drugs of choice in the described setting.

　　ACKNOWLEDGEMENTS

　　This study was supported by the central facilities and internal funds of Interdisciplinary Biotechnology Unit， AMU， India which are highly acknowledged. Shazi Shakil thanks the Department of Biotechnology (DBT)， Government of India for his research fellowship. The authors declare that they have no conflicts of interest.

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