This prospective study was conducted from February 2015-August 2016 in the Department of Microbiology, Pt. BD Sharma PGIMS, Rohtak, Haryana, India. During the study period, 240 pus samples submitted in the laboratory were processed. The study was approved by the institutional ethical committee.

Skin was cleaned by 2% chlorhexidine and 70% alcohol. Pus was either aspirated in syringe or collected on sterile swab and sent to laboratory.

Non-repetitive pus samples obtained from various types of wounds and ulcers were included in the study. Isolates other than the members of Enterobacteriaceae were excluded from the study. Samples were subjected to microscopy and culture. Cultures were performed on blood agar and MacConkey agar plates. The colonies grown on the blood agar and MacConkey agar plates were processed further for the identification of the organisms by colony morphology, Gram staining and biochemical reactions as per standard microbiological protocol [12-14].

AST was done by Kirby-Bauer disc diffusion method in accordance with CLSI guidelines 2016 [15]. The antimicrobial drugs tested were gentamicin (10μg), amikacin (30μg), amoxicillin/clavulanic acid (20μg/10μg), piperacillin/tazobactam (100μg/10μg), ciprofloxacin (5μg), imipenem (10μg), meropenem (10μg), trimethoprim/sulfamethoxazole (1.25μg/23.75μg), ceftazidime (30μg), doxycycline (30μg). An isolate was considered as Multi-Drug Resistant (MDR) if it was resistant to atleast three classes of antimicrobial agents [16].

Tissue Culture Plate (TCP) method: This method was described by Christensen GD et al., [11].

Test organism was inoculated in Brain Heart Infusion (BHI) broth (Hi media laboratories, Mumbai) supplemented with 2% sucrose dispensed in test tubes and incubated overnight at 37°C. This broth was diluted in the ratio of 1:100 with fresh broth. A 200μL of this diluted culture broth was added to 96 well- flat bottom, non-adherent, non-treated polystyrene tissue culture plates (Hi media laboratories, Mumbai). These tissue culture plates were further incubated for 24 hours at 37°C. After incubation, the contents of the wells were removed and wells were washed four times with 0.2 mL of phosphate buffered saline. Adhered biofilms were fixed with 2% sodium acetate for 30 minutes and stained with crystal violet (0.1% w/v) for 30 minutes. Excess stain was rinsed off with distilled water. After drying, Optical Densities (OD) was determined by an automated micro ELISA reader at wavelength of 570nm.

Modified Tissue Culture Plate (MTCP) Method [17]

The standard TCP method was modified slightly by adding one step in the end. After staining with 0.1% crystal violet and drying, 160μL of 33% glacial acetic acid was added into the microwells. After 15 minutes, OD was taken by an automated micro ELISA reader at wavelength of 570nm. These OD values were considered as an index of bacterial adhesion and biofilm formation. The classification of bacterial adherance by TCP and MTCP method is shown in [Table/Fig-1].

Biofilm production by both the methods was graded as weak/none, moderate and strong. High and moderate biofilm production by each method was considered positive and weak/none biofilm production was considered negative. Based on the literature, TCP method was considered as the gold standard method of biofilm detection and parameters like sensitivity, specificity, negative predictive value, positive predictive value and accuracy of MTCP method were calculated. Association of two or more set of variables was analysed using Chi-square test. A p-value <0.05 was considered as statistically significant. SPSS version 20.0 was used for data analysis.

A total of 160 isolates were obtained out of 240 pus samples. Bacteriological profile includes Klebsiella spp. (n=51), Escherichia coli (n=41), Citrobacter spp. (n=32), Proteus spp. (n=26), Enterobacter spp. (n=10). Out of these, 52.5% isolates showed biofilm production by TCP and 65.6% by MTCP method. The rate of biofilm detection by two methods was found to be statistically significant (p-value =0.004). Quantification of biofilm production has been shown in [Table/Fig-2]. In our study, biofilm production as per standard TCP method was more prevalent in Klebsiella spp. (32, 62.7%) followed by Proteus spp. (15, 57.7%), Enterobacter spp. (5, 50%), E. coli (19, 46.3%) and Citrobacter spp. (13, 40.6%). The magnitude of biofilm production by individual bacterial spp. is depicted in [Table/Fig-3].

Antibiogram of the isolates revealed high resistance to routinely administered antibiotics like ciprofloxacin, co-trimoxazole, gentamicin, ceftazidime and doxycycline while carbepenems were found to be the most effective class of antimicrobials. High resistance by biofilm forming isolates was observed against ceftazidime (73, 87%) followed by doxycycline (70, 83.3%), co-trimoxazole (64, 76.1%), gentamicin (60, 71.5%), amoxyclav (56, 66.7%), ciprofloxacin (54, 64.3%), amikacin (47, 55.9%), piperacillin+tazobactum (36, 42.8%), meropenem (31, 37%) and imipenem (7, 8.3%). The antimicrobial resistance pattern of biofilm producing and non-biofilm producing isolates is shown in [Table/Fig-4]. MDR was seen in 55.6% (n= 89) isolates. Out of which 79.7% (n=67) biofilm producing isolates and 29% (n=22) of non- biofilm producing isolates were MDR [Table/Fig-5]. Sensitivity of MTCP method was found to be 95.45% while specificity was 78.26%. Positive Predictive Value (PPV) was 84.84%, Negative Predictive Value (NPV) was 93.1% and accuracy was found to be 87.89%.

The finding of biofilm in the wounds has great relevance in the wound management. Early identification of biofilm producing strains and appropriate antibiotic selection might help to prevent relapse of such infections. Bacteriological profile in the current study showed Klebsiella spp. to be most common bacterial isolate followed by Escherichia coli. In an Indian study by Subramanian P et al., it was found that the most commonly isolated microorganism included Klebsiella pneumoniae (40.2%) followed by E. coli (30.9%) [18]. This is similar to the present study.

Other studies conducted by Fatima S et al., and Zubair M et al., showed Escherichia coli as the most common bacterial isolate [19,20].

Fatima S et al., found rate of biofilm production to be 33.3% in E. coli followed by 22.9% in Proteus spp., 12.5% in Klebsiella spp., 6.3% in Citrobacter spp. and 2.1% in Enterobacter spp. [19]. Zubair M et al., observed biofilm production in 59.4% isolates. The prevalence of biofilm production was highest in Klebsiella spp. (70%) [20]. Subramanian P et al., found 59.2% bacterial isolates were biofilm producers. The rate of biofilm production was maximum in K. pneumoniae (55.9%) [18]. This was similar to present study.

In the study by Subramanian P et al., resistance pattern of biofilm positive isolates showed 62%, 20%, 74%, 60%, 03%, and 03% resistance to gentamicin, amikacin, ceftriaxone, ciprofloxacin, piperacillin-tazobactum and imipenem respectively as compared to 29%, 11.6%, 37.7%, 24.6%, 1.4%, and 2.9% resistance shown by biofilm non-producers for the same antibiotics [18]. Fatima S et al., also compared rate of biofilm production and drug susceptibility pattern of gram negative isolates. They observed that 69% biofilm producing isolates were MDR while only 41.5% non-biofilm producers were MDR [19]. Similarly, Zubair M et al., also found biofilm producing isolates to show high degree of resistance for routinely administered antibiotics [20].

In the current study, two phenotypic methods for detecting biofilm formation were used and their results were compared to find out most appropriate method for demonstrating biofilm formation. Out of the 160 isolates, the TCP method could detect biofilm in 84 isolates (52.5%). The MTCP method detected biofilm in 105 isolates (65.6%). The present study showed MTCP method to be more sensitive than TCP method (p-value < 0.05). Stepanović et al., evaluated 30 clinical isolates of staphylococci for biofilm formation. In their study, TCP method identified 73.3% isolates as biofilm producer while MTCP method detected 83.3% isolates to be biofilm producer. The authors compared the results of both the methods and found the difference to be statistically significant [21]. Another investigator Babapour E et al., studied 156 clinical isolates of Acinetobacter spp. On comparison, the rate of biofilm formation was 66.7% and 73.7% by TCP and MTCP methods respectively. The authors concluded MTCP method to be more accurate than TCP method in evaluating biofilm formation [22]. The result of present study is in accordance with the above mentioned studies.

This study is based only on phenotypic methods of biofilm detection. Molecular methods identifying gene responsible for biofilm production were not included due to lack of facility.

High rate of biofilm formation shown by the members of Enterobacteriaceae suggests it to be one of the important mechanisms of anti microbial resistance. MTCP method is better than TCP method for biofilm detection and quantification. This is a simple, reliable accurate method and can be utilized for biofilm screening.