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Clinico Microbiological Spectrum of Diabetic Foot Ulcer with Multidrug Resistant Organisms at Tertiary Care Hospital, Jaipur

1,2,3,5 Department of Microbiology, SMS Medical College, Jaipur, Rajasthan 4 Department of Surgery, SMS Medical College, Jaipur, Rajasthan *Corresponding Author Rajni Sharma SMS Medical College, Jaipur, Rajasthan, India Abstract Background: Diabetes is one of the major health problems that have become a big burden in all over the world. Amongst the diabetic population, about 8– 20% experience foot ulcer in life time. DFIs are predominantly polymicrobial and multidrug-resistant (MDR) with the ability to form biofilm, which is an important virulence factor and results in treatment failure .


Introduction
Diabetes is one of the major health problems that have become a big burden in all over the world.
Diabetes mellitus (DM) is a serious and complex illness that affects almost every vital organ in the body. There are approximately 415 million people http://jmscr.igmpublication.org/home/ ISSN (e)-2347-176x ISSN (p) 2455-0450 DOI: https://dx.doi.org/10.18535/jmscr/v8i7.18 worldwide suffering from diabetes. It has been estimated that this number will increase to 642 million by 2040 1 . Amongst the diabetic population, about 8-20% experience foot ulcer in life time. It is one of the most common causes of hospital admissions in diabetics. Diabetic foot ulcers have 15 -45% higher risk of amputation of limb as compared to foot ulcers secondary to other aetiologies 2 . Diabetic foot involvement including infections and foot ulcers are one of the frequently seen and disabling complications of diabetes leading to significant morbidity and mortality [2][3][4] . Diabetic neuropathy and micro or macro ischemia are the two main risk factors that cause diabetic foot ulcer (DFU).
Impaired microvascular circulation limits the access of phagocytic cells to infected area, and this result in poor concentration of antibiotics in infected tissue 5 . DFIs are predominantly polymicrobial and multidrug-resistant (MDR) with the ability to form biofilm, which is an important virulence factor and results in treatment failure 6 . Diabetic foot infections are often polymicrobial. Escherichia coli, Proteus spp., Pseudomonas spp., Staphylococcus aureus and Enterococcus spp are the most frequent pathogens contributing to progressive and widespread tissue destruction.

Methicillin-resistant
Staphylococcus aureus (MRSA) has been commonly isolated from 10-40% of the diabetic wounds 3,4 . Infection with MDR pathogens is also responsible for the increased duration of hospitalization, cost of management, morbidity and mortality of the diabetic patients 5, 7,8 . Therefore this study has been performed to determine the common etiological agents of diabetic foot infections and in vitro susceptibility to routinely used antibiotics and to assess the magnitude of infection by Multidrug resistant organisms (MDRO'S) like Extended spectrum beta lactamase (ESBL) and Methicillinresistant Staphylococcus aureus and their correlation with type of ulcer and severity of diabetes.

Materials and Methods
Study Population: A total of 73 pus and exudates sample were collected from diabetic foot patients attending hospital both OPD and IPD were included in the study. Specimens were collected from under the margin of the ulcer and base of the ulcer with a sterile swab stick from each patient in a sterile container under aseptic conditions by standard protocols. The study has been carried out at department of Microbiology, S.M.S Medical College Jaipur. Inclusion criteria: i) Diabetic patients complicated by diabetic foot ulcer ii) Patient who gives a valid consent. Exclusion criteria: i) Diabetic patients without complicated by diabetic foot ulcer Sample Processing Samples were taken using two swab sticks, first swab stick subjected to gram staining to observe for the presence of pus cells and organisms. From second swab stick, sample was inoculated on Nutrient agar, Blood agar, Mac-Conkey agar and Thioglycollate broth. The inoculated media were incubated aerobically at 37⁰ C for 18-24 hours and were observed for growth.

Significant
isolates were identified by conventional methods according to the standard laboratory protocol, including colony morphology, gram staining and biochemical reactions. All gram negative bacilli were identified to species level by their characteristic appearances on the media, Gram's stain, Oxidase test, Motility and biochemical reactions as per standard laboratory protocol. All gram positive organisms were identified to species level by their characteristic appearances on the media, Gram's stain, and Catalase test followed by Coagulase test. Enterococcus was identified by Bile Esculin disc test and it was also confirmed by Salt tolerance test (6.5% NaCl.)

Antimicrobial Susceptibility Testing
The antibiotic sensitivity test was performed by modified Kirby Bauer disc diffusion technique 9 with commercially available Hi-Media antibiotic discs according to Central Laboratory Standard Institute (CLSI) guidelines on Mueller Hinton agar plates. The antibiotics which were used in our study were based on the standard protocol of the hospital and departmental policies as per CLSI guideline 10 . Detection of extended spectrum betalactamases 11 Detection of extended spectrum beta-lactamases was done according to CLSI guidelines by two methods Phenotypic Confirmatory Disc Diffusion Test (PCDDT) and Double Disc Synergy Test (DDST) Phenotypic confirmatory disc diffusion test (PCDDT) Lawn culture of isolate was made on Muller Hinton Agar (MHA) plate; discs of ceftazidime and ceftazidime + Clavulanic acid were placed on the surface of MHA and incubate overnight at 37˚C. An increase of ≥5mm in zone diameter of ceftazidime + Clavulanic acid in comparison to the zone diameter of ceftazidime alone confirmed the organisms to be ESBL producers ESBL detection by Double Disc Synergy Test (DDST)-Lawn culture of isolate was made on Muller Hinton Agar (MHA) plate. Disc of ceftazidime (30µg), cefotaxime (30µg) and co-amoxyclav (20µg Amoxycillin and 10 µg Clavulanic Acid) were placed at a distance of 20mm from centre to centre in a straight line, with co-amoxyclav disc in the middle. The plate was incubated aerobically overnight and the results were read on the following day. Isolates which shows an enlargement of the zone of inhibition greater than 5mm on the co-amoxyclav side of the disc compared to the result seen on the side without co-amoxyclav was confirmed as ESBL producers. ESBL production was interpreted if the inhibition zone around the test antibiotic disc increased towards the co-amoxyclav disc.
Screening of MRSA was done by cefoxitin disc diffusion test: All strains were tested with 30 μg cefoxitin discs (Hi-Media) on Mueller-Hinton agar plates 12 . The bacterial suspension for each strain was adjusted to 0.5 McFarland. The zone of inhibition was calculated after 16-18 hr incubation at 35 °C. Zone size were defined according to CLSI criteria (23)(24): susceptible, ≥22 mm; resistant, ≤21 mm. ATCC 25923 MSSA was used as negative control. Statistical Analysis: The qualitative data were expressed in proportion and percentages and the quantitative data expressed as mean and standard deviations. The difference in proportion was analyzed by using chi square test. Significance levels for tests were determined as 95% (P< 0.05)

Results
A total of 73 clinically suspected cases of diabetic foot ulcer were enrolled in our study among that 65 (89.04%) were significant growth and 03 (4.1%) were sterile while contaminants grown in 05 (6.8%) samples. Out of 65 significant growth 42 (64.61%) were male while 23 (35.38%) were female. The maximum number of diabetic foot cases 30(46.15%) were seen between age of 51-60 years of age followed by elderly people ≥ 60 years of age (32.30%) and more number of cases seen in patients with duration of 4-8 weeks of foot ulcer. According to Wagener's classification the maximum number of cases were observed in Grade 2 of diabetic foot ulcer 24(36.9%) followed by 16       The monomicrobial nature of infection is associated with the duration of the ulcer and antimicrobial treatment. Earlier in the infection, the monomicrobial state prevails and as the infection progresses with time, a polymicrobial state arises. In this study we found that the age group most commonly affected by the diabetic foot ulcer was 51 -60 years (46.15%), followed by 61 or more years (32.30%) which is consistent with the study conducted by Jitendranath et al 3 , P. Ramakant et al 13 , Khare at al 16 and Patil .S et al 17 who also reported the same findings. The number affected increases in elderly age group and less common in younger age group. In this study most cases of diabetic foot ulcer were of 4-8 weeks duration i.e 30 cases (46.15%) followed by 2-3 weeks in 21 cases(32.30%). Most commonly right foot was involved. Our results are concordance with Khare J et al 16 and Katherivel M et al 18 who also reported most cases of diabetic foot ulcer were of 4-8 weeks duration. In this study maximum no. of cases were observed in Grade 2 of diabetic foot ulcer(36.9%) followed by Grade 3 (24.6%) which is similar with the study conducted by Jitendranath A et al 3  We also observed that maximum monomicrobial growth was isolated from grade 2 followed by grade 3 as the grade progresses polymicrobial wound infection was found to be more common. It can be interpreted that to start with, there is a poor response to injury in a patient with diabetes. This may be because of several factors like vascular insufficiency, neuropathy and poor diabetic control. The poor defence leads to a rapid increase in the number of microbes, with progressive deterioration of diabetic foot wound. In this study Pseudomonas species(25.65%) was predominant among gram negative bacilli and Staphylococcus aureus (16.84%) was the predominant among gram positive cocci .Our results are consistent with study conducted by Ramakant et al 13  We also found 24.6% of ESBL producers in our study which is similar with the study conducted by Shanmugam et al 22 21 where MRSA were reported 41%, 40%, 44.2% and 40% respectively. The MRSA displayed high level of resistance to Erythromycin (88%), Penicillin (75%) and Quinolones (88%). This study presents a comprehensive clinical and microbiological profile of infected diabetic foot ulcers, especially by multidrug-resistant organisms. Appropriate selection of antibiotics based on the anti-biograms of the isolates from the lesions is most critical for the proper management of these infections.

Conclusion
Diabetic foot ulcers are one of the most common and dreaded complications of diabetes. Thus it is utmost important to screen all elderly patients for diabetes and educate them about foot care. Early identification of the risk factors and timely institution of appropriate treatment indispensable to avoid amputations. Clinicians should switch to culture report-based use of narrower spectrum therapy. This would improve the overall antibiotic efficacy and reduce the emergence of multidrug resistant organisms in diabetes.