Prevalence of biofilm producing aerobic bacterial isolates in burn wound infections at a tertiary care hospital in northern India

Summary

Burn wounds frequently get infected due to a break in skin integrity and prolonged hospitalization. Microbial flora originating from the patient’s own flora colonize and infect the burn wounds. Bacterial biofilms in particular are postulated as the culprit for the development of non-healing burn wounds by inducing chronic inflammation in these patients. In the present study, 190 wound isolates obtained from patients admitted to the burn ward at the Pt. B.D. Sharma PGIMS, Rohtak, were evaluated for biofilm formation along with Antimicrobial Susceptibility Testing (AST). Biofilm detection was done by modified Tissue Culture Plate method and AST was done by Kirby-Bauer disc diffusion method. A total of 190 isolates were studied, which included Staphylococcus aureus, Coagulase negative Staphylococcus, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella spp., Proteus spp., Citrobacter spp., Escherichia coli and Enterobacter spp. Of these, 68.9% isolates showed biofilm formation. Biofilm formation was more common in Pseudomonas aeruginosa followed by Klebsiella spp. and Staphylococcus aureus. Biofilm producing isolates showed greater multidrug resistance than non-biofilm producers. In our study, a high rate of biofilm formation and antimicrobial drug resistance was seen.

Introduction

Burn injury is one of the most common types of trauma that requires urgent medical attention.¹ Normal protective defense mechanisms of the skin are lost after a burn injury, resulting in rapid colonization of the wound surface. Initially, gram-positive organisms derived from skin commensals colonize the wound bed, followed later by gram-negative organisms and yeasts.² Staphylococcus species and Pseudomonas aeruginosa are two of the most frequently isolated microorganisms from burn wounds around the world.³ Wound sepsis is considered the most frequent cause of mortality and morbidity in such patients.⁴

A biofilm is an assemblage of microbial cells that is irreversibly associated with a surface and enclosed in a matrix of primarily polysaccharide material.⁵ Biofilm producing microbial flora manifests an altered growth rate and transcribes genes that provide them with inherent resistance to antimicrobials and the host immune system. This allows them to survive in a relatively harsh environment. Thus, biofilms are reported to be a major factor contributing to many chronic inflammatory diseases.⁶ As burn wounds are chronic, non-healing in nature and difficult to treat, this study was planned to evaluate the role of biofilm producing aerobic bacteria in causing burn wound infection.

Material and methods

A prospective study was conducted from March to August 2015 on 133 pus samples from burn patients received in the Department of Microbiology, Pt. B.D. Sharma PGIMS, Rohtak, Haryana, India. Gram stain was done, followed by inoculation of the sample on MacConkey agar and blood agar plates. The inoculated media were then incubated overnight at 37̊°C aerobically. Identification of the organisms was done by colony morphology, and gram staining and biochemical reactions as per standard microbiological protocol.^7-9 Antimicrobial Susceptibility Testing was done by Kirby-Bauer disc diffusion method in accordance with CLSI guidelines 2015.¹⁰ The antimicrobial drugs tested for gram-positive bacteria included erythromycin (15μg), penicillin (10units), cefoxitin (30μg), cephalexin (30μg), trimethoprim-sulfamethoxazole (1.25/23.75μg), linezolid (30μg), doxycycline (30μg), clindamycin (2μg), vancomycin (30μg) and amoxicillin/clavulanic acid (20μg/10μg). For gram-negative bacteria they were gentamicin (10μg), amikacin (30μg), amoxicillin/clavulanic acid (20μg/10μg), piperacillin/tazobactam (100μg/10μg), cefepime (30μg), ciprofloxacin (5μg), imipenem (10μg), meropenem (10μg), trimethoprim/sulfamethoxazole (1.25μg/23.75μg), aztreonam (30μg), ceftazidime (30μg), netilimicin (30μg) and doxycycline (30μg). An isolate was considered as multi-drug resistant (MDR) if it was resistant to at least three classes of antimicrobial agents.¹¹

Biofilm production was detected by Modified Tissue Culture Plate (MTCP) method.

Modified Tissue Culture Plate (MTCP) method¹²

Isolates from fresh agar plates were inoculated in brain heart infusion (BHI) broth supplemented with 2% sucrose dispensed in 3ml in test tubes and incubated overnight at 37°C aerobically. This cultured broth was diluted in the ratio of 1:100 with fresh medium. 200μl of this diluted cultured broth was then added to 96-well flat bottom, non-adherent, non-treated polystyrene tissue culture plates. These tissue culture plates were further incubated for 24 hours at 37°C. After incubation, the content of the wells was removed by gently tapping the plates and was washed four times with 0.2ml of phosphate buffered saline (PBS). Lastly, the wells 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, the wells were then treated with 160μL of 33% glacial acetic acid for 15 min at room temperature to solubilize the dried crystal violet stain which was adherent to any biofilm. Optical densities (OD) were then determined by an automated micro ELISA reader at a wavelength of 570nm. These OD values were considered as an index of bacterial adhesion and biofilm formation. Biofilm formation was considered as weak/no biofilm formation if OD value was less than 2.63, moderate if OD value was between 2.66-5.32 and strong when OD value was greater than 5.32.

Results

A total of 190 bacterial isolates were obtained from 133 pus samples. Of these, 82 (61.6%) showed monomicrobial infection while multimicrobial infection was seen in only 51 (38.4%) samples. Rate of isolation of gram-negative bacteria was found to be 76% while gram-positive microorganisms were only 24%. P. aeruginsa (32.1%) was the most commonly isolated bacteria followed by Klebsiella spp. (16.3%) and Staphylococcus aureus (14.7%). The bacteriological profile has been depicted in Table I.

Table I. Aerobic bacteriological profile of burn wound isolates.

The gram-positive bacterial isolates showed 93.5% resistance to penicillin, 67.4% to cefoxitin, 60.9% to cephalexin, 56.5% to co-trimoxazole, 47.8% to amoxicillinclavulanate, 45.7% to erythromycin, 43.5% to doxycycline, 32.6% to clindamycin and 10.9% resistance to linezolid. No resistance was observed against vancomycin. This has been depicted in Table II.

Table II. Antibiotic resistance pattern of gram-positive isolates.

P. aeruginosa isolates showed 70.5% resistance to ceftazidime, 59% to gentamicin, 54.1% to netilmicin, 49.2% to ciprofloxacin, 39.4% to cefepime, 37.7% to aztreonam, 36.1% to amikacin, 13.1% to piperacillin-tazobactam, 11.5% to meropenem and 03.3% to imipenem. Antibiogram of Acinetobacter spp. isolates showed 80% resistance to ciprofloxacin and co-trimoxazole, 70% to doxycycline, 60% to ceftazidime and gentamicin, 50% to piperacillin-tazobactam and aztreonam, 40% to meropenem and amikacin, and 20% resistance to imipenem. The antibiogram of non-fermenters has been depicted in Table III.

Table III. Antibiotic resistance pattern of gram-negative isolates (nonfermenters).

Antibiogram of members of the Enterobacteriaceae family showed 79.5% resistance to ceftazidime, 74% resistance to doxycycline, 69.9% to gentamicin, 68.5% to co-trimoxazole, 54.8% to amoxicillin-clavulanate, 46.6% to ciprofloxacin, 37% to amikacin, 32.9% to piperacillin-tazobactam, 27.4% to meropenem and 04.1% to imipenem. This is shown in Table IV. In the current study, 68.9% of isolates showed biofilm production by modified tissue culture plate method. Maximum biofilm production was shown by P. aeruginosa (73.8%) followed by S. aureus (71.4%). Rate of biofilm production by individual bacterial isolates is shown in Table V. Strong biofilm production was seen in 23.7% isolates while 45.3% were moderate biofilm producers. The rest were weak/non biofilm producers. Grading of biofilm formation is shown in Table VI.

Table IV. Antibiotic resistance pattern of gram-negative isolates other than non-fermenters.

Table V. Rate of biofilm formation by MTCP method.

Table VI. Grading of biofilm formation in aerobic bacterial isolates by MTCP method.

Of the 190 isolates, 105 (55.6%) showed multi drug resistance (MDR). The rate of MDR isolates in biofilm positive isolates was 66.4% while only 30.5% non-biofilm producing isolates showed MDR. This difference in resistance pattern was found to be statistically significant (p< 0.05) (Table VII).

Table VII. Comparison of MDR among biofilm forming (BF) and non-biofilm forming (NBF) isolates.

Discussion

The extent of thermal injury along with the types of microorganism colonizing the burn wound influence the future outcome of the wound. In the present study, 61.6% burn wounds had monomicrobial etiology while 38.4% were due to multimicrobial infection. Similar findings were obtained by Al-saad et al., who studied 100 pus swabs obtained from burn patients. They found 76.4% were due to single species infection and 23.6% were due to mixed species infection.¹³ In another study, Alharbi et al. found single species infection in 78.9% of samples while mixed species infection was seen in 21.1% of cases.¹⁴

In the current study, gram-negative bacterial isolates were predominant, accounting for 76% as compared to 24% grampositive bacterial isolates. Similar findings were observed by Alharbi et al., where 65.7% of the isolates were gram-negative and 34.3% were gram-positive cocci.14 Similar results revealing gram-negative predominance have been observed by various researchers.^15-18

In the current study, P.aeruginosa was the most frequently isolated bacteria, followed by Klebsiella spp. and St. aureus. Agnihotri et al. observed that P. aeruginosa was the most common bacterial isolate obtained from burn wounds, followed by St. aureus.¹⁶ Similar findings were also obtained by Alharbi et al.¹³ and Singh et al.¹⁷ However, two separate studies conducted by Mehta et al. and Ramakrishnan et al. showed Acinetobacter spp. to be the second most common bacterial isolate after Pseudomonas species.^15,18 This may be due to the emergence of acinetobacter as a nosocomial pathogen. Since burn patients are admitted to hospital for long periods, they are at increased risk of being infected by this organism.

In our study, biofilm producing bacterial isolates exhibited a high level of resistance to all drugs that are commonly prescribed, like fluoroquinolones, cephalosporins, aminoglyosides and tetracyclins. Vancomycin and carbepenems were found to be the most effective antimicrobial drugs against gram-positive and gram-negative bacterial isolates respectively. Mehta et al. also observed high resistance to aminoglycosides, fluroquinolones and cephalosporins by gram-negative bacterial isolates, and netilmicin and erythromycin by gram-positive bacteria.¹⁵ In the study by Alharbi et al, amoxycillin, ampilcillin and cefaclor showed low activity. The authors attributed this to the extensive use of these drugs in their centre.¹⁴ A similar observation of high antimicrobial resistance by biofilm producing bacterial isolates has been made in other studies.^16-18

In our study, biofilm formation was seen in 68.9% isolates while 31.1% bacterial isolates showed no biofilm production. In the study by Al-saad et al., the rate of biofilm production was 54.2% while the rest were non/weak biofilm producers.¹³ Ramakrishnan et al. observed a slightly low rate of biofilm production, where 43% of isolates were biofilm producers and 57% were weak/non biofilm producers.¹⁸

Conclusion

We observed a high rate of antimicrobial resistance and biofilm production in bacterial isolates obtained from burn patients. It was also observed that the majority of MDR pathogens were also biofilm producers. Thus, early identification of infection caused by biofilm producing strains might help to modify treatment and outcome in burn patients.