Identi(cid:2510)ication of bio(cid:2510)ilm-producing microorganism’s and drug susceptibility pattern from diabetic foot ulcer patients at Puducherry

Diabetes mellitus is a signi(cid:977)icant health problem worldwide that affects approximately 171 million people; severe complications lead to the develop-ment of diabetic foot ulcers. Diabetic ulcer infections are mainly polymicrobial in nature and multidrug-resistant (MDR), which is capable of forming a bio(cid:977)ilm, which is the important virulence factor results in treatment failure. The main objectives of this study to investigate the etiologic agents of diabetic foot infections, their antimicrobial resistance and bio(cid:977)ilm formation. A total of 200 patient samples were taken from diabetic foot ulcer patients between September 2015 and February 2016. Isolation and identi(cid:977)ication of microorganism were made according to standard microbiological procedures. Antibiotic Susceptibility testing performed by Kirby Bauer disc diffusion method and the bio(cid:977)ilm production was performed by the tube method and Congo Red Method. Out of 200 samples processed, 110 (55%) were polymicrobial, 50 (25%) monomicrobial and 40(20 %)culture Sterile. The most common organism isolated were 82(39%) Pseudomonas aeruginosa,45(21%) Staphylococcus aureus, 48(23%) Candida sp followed by others. Bio(cid:977)ilm production was seen in 112 (53%) of the isolates . Antimicrobial drug resistance was higher among 92(82%) bio(cid:977)ilm producers than non-bio(cid:977)ilm 20(18%) producing microorganisms. Organisms isolated from chronic diabetic foot ulcers cases were multidrug-resistant and bio(cid:977)ilm producers. Our study shows the importance of bio(cid:977)ilm screening with the usual antibiogram, as a routine technique in diabetic foot ulcers patients for effective treatment.


INTRODUCTION
Chronic wounds in diabetic foot ulcer affect 1 -2% of the population, persistent morbidity with delay in the wound healing process (Gjødsbøl et al., 2006;Davis and Brown, 2016).
Bio ilm is a structured consortium of bacteria and extracellular matrix, which is essential for interconnecting the bacteria and can be composed of polysaccharides, proteins, and extracellular DNA (Whitchurch, 2002).
Almost 50-60% of chronic wound specimens were characterised as bio ilm containing, whereas only 6%-10% of acute wounds, bio ilms were more prevalent in the chronic wound than acute wounds (Malone and Swanson, 2017;James et al., 2008).
Bio ilm producing organisms exchange the genetic material which altered phenotype and delays the wound healing process (Murali et al., 2014). Bio ilm is a crucial virulence factor as it creates a suitable environment for them to survive and evade antibiotics.
Pseudomonas aeruginosa, Staphylococcus aureus and Candida sp are the most causative microorganism in chronic wounds, as a bio ilm producer, that delaying the wound healing process and responsible for resistance to antimicrobial therapy (Davis and Brown, 2016;Mantero et al., 2017) They are dif icult to eradicate using antibiotics, identi ication of bio ilm-producing organism among clinical isolates lead to better management of Diabetic Foot ulcer instead of antibiotic treatment failure (Schierle et al., 2009) The aim of our present study to identify the microorganisms from diabetic foot ulcer patients and their antimicrobial susceptibility pattern and bio ilm formation.

MATERIALS AND METHODS
This prospective study was conducted at the Department of Microbiology, in a tertiary care hospital, Puducherry during six months from September 2015 to February 2016. A total of 200 samples from Diabetic Foot ulcer patients attending the surgery outpatient department were included in this study. Institutional ethical clearance was taken, and informed consent was obtained.
Samples were collected using a sterile cotton swab from the deeper parts of the wound and sent to the laboratory. Samples were processed according to standard microbiological procedures. Antimicrobial susceptibility testing was performed by disc diffusion method according to the Clinical Laboratory and Standards Institute Guidelines.

Bio ilm formation by tube method
A loopful of pure culture on Tryptone soya agar was inoculated into 2ml of Tryptone soy broth in Falcon tubes. The tubes were incubated for 24 hours at Phosphate buffered saline and left to dry. 2 ml crystal violet (1%) were added for 15minutes to stain the Cells. Tubes were washed with water to remove the unbound dye for drying place the tubes in Inverted Position. Presence of a stained ilm on the sides of the tube was positive of bio ilm formation. Bio ilm formation was reported as strong, moderate or weak.

Congo Red method
Congo Red Plates were inoculated and incubated aerobically for 24-48 hours at 37 • C. Bio ilm forming bacteria shows.

Positive -Black colonies with a dry crystalline morphology
Negative -Pink, dark at the centre of the colony

Bio ilm Production of Candida
Isolated organisms inoculated into a tube containing 10ml Sabouraud's liquid medium strains with glucose. The tubes were incubated at 37 • C after 24 hours broth was discarded and tubes walls were stained with safranin. Bio ilm formation was scored as negative (0), weakly positive (1+), moderately positive (2+) and strongly positive (3+).

RESULTS
Total of 200 samples was collected from patients with diabetic foot ulcers, 124(62%) were male, and 76(38%) were females, age group ranges from 45-85 years.
In Table 5 out of 210 isolates, the positive result was noted in 116(55%) isolates formed black colonies with a dry crystalline morphology, whereas 94 (45%) shows negative result. P. aeruginosa were the predominant bio ilm producer followed by Staphylococcus aureus.

DISCUSSION
Foot ulcer infection is the major public health problem in diabetes; if left untreated, it results in limb amputation (Singh et al., 2005). The main objective of the present study, to identify the microorganisms and their antimicrobial susceptibility pro ile and bio ilm formation in diabetic foot ulcer patients.
A total of 200 patient samples with diabetic foot infections of both sexes, age between 35-80 years were examined through a period of six months. Among them, 36 (72%) were male, and 14 (28%) were females. Males were more infected in diabetic foot ulcer than females due to increased outdoor work, a similar study conducted by (Yerat and Rangasamy, 2015).
Based on the microbial growth, most organisms isolated were Polymicrobial in our study correlates with (Manisha et al., 2012) study.
Bio ilm formation makes microorganisms much more resistant to antimicrobial therapy. It involves penetration of antibiotics through the bio ilm matrix, reduced oxygen and nutrient, decreased growth rates and metabolism (Keren et al., 2004).
Among 210 isolates tested for bio ilm production by tube method.112 (53%)isolates by tube method, and 116(55%) isolates by Congo red method were bio ilm producers, 98(47%) isolates by tube method and 94(45%) isolates by Congo red method isolates were non Bio ilm producers. These results Correlate with (Swarna et al., 2012)study.
Among all isolates Pseudomonas aeruginosa, Staphylococcus aureus, E.coli was the most strong bio ilm producer in our study. Bio ilm formation by P. aeruginosa more readily in the diabetic wound environment report by (Damir, 2011).
The antimicrobial resistance was higher in bio ilmproducing isolates than in non-bio ilm producers. Among 112 bio ilm producers in our study, 92(82%) isolates which were bio ilm producers were drugresistant strains, whereas 20(18%) bio ilm-forming isolates were Sensitive to all drugs.
Bio ilm enhances the emergence of MDR in chronic foot ulcer infections. A drug-resistant pattern in bio ilm formation correlates with a previousstudy (Banu et al., 2015).

CONCLUSIONS
Diabetic foot ulcer infections are problematic issues due to their polymicrobial nature, antimicrobial resistance, and bio ilm formation. Our study suggests a routine screening test for bio ilm production and antibiograms will help surgeons to formulate effective treatment options for these patients.