Abstract

Introduction: Biofilm is an organized colony of bacterial cells that are adjoined by a self-produced polymeric matrix. These biofilms play a role in the pathophysiology and clinical manifestations of many illnesses, often leading to treatment failures.  Microorganisms that can produce biofilm express high resistance to antimicrobial agents compared to non-biofilm-producing microbes.The study seeks to evaluate the rate of biofilm-producing aerobic bacteria in burn wounds and determine the rate of multi-drug resistant bacteria from burn wound infections.

Materials and Methods: The study was a cross-sectional study conducted at the Department of Microbiology at the Maharishi Markandeshwar Institute of Medical Science and Research (MMIMSR) Mullana, Ambala, Haryana. In this study, a total of 50 burn wound swab samples were collected from patients along with their detailed clinical history and proceeded further for bacterial identification by standard microbiological methods. An antimicrobial susceptibility test was performed for all bacterial isolates using the disc diffusion method of the modified Kirby-Bauer technique using Mueller Hinton agar plates and commercially available antimicrobial discs. Biofilm-forming bacteria were identified using the modified Congo red agar method and the Tube Adherence method.

Results:  56.8% of the isolates obtained were biofilm-forming bacteria. Out of the biofilm-producing bacteria, 48% were multi-drug resistant. Marked resistance was seen for commonly used antibiotics like Quinolones, Cephalosporins, and Cotrimoxazole. Staphylococcus aureus isolates were resistant to Ofloxacin, Penicillin G, and Amikacin; Klebsiella spp. isolates were highly resistant to Ampicillin, Ceftazidime, Trimethoprim-sulfamethoxazole, Tetracycline, and Chloramphenicol; Pseudomonas aeruginosa isolates were highly resistant to Trimethoprim-sulfamethoxazole; Acinetobacter spp isolates were resistant to Cefotaxime, Ceftriaxone, Cefixime and Trimethoprim-sulfamethoxazole. For Gram-positive bacteria, Staphylococcus aureus showed 100% susceptibility to Linezolid, Vancomycin, and Netilmycin while coagulase-negative Staphylococci (CoNS) isolates were sensitive to all antibiotics. For Gram-negative bacteria, carbapenem antibiotics showed the highest sensitivity to Klebsiella, Proteus, Pseudomonas aeruginosa, and Acinetobacter spp.

The study calls attention to the increasing rate of multi-drug resistant bacteria in burn wound infections and the need for effective infection control measures and treatment methods to combat these biofilm-forming multi-drug resistant bacteria.

Introduction

Biofilm is an organized colony of bacterial cells that are adjoining by a self-produced polymeric matrix. Microorganisms that can produce biofilm express high resistance to antimicrobial agents compared to non-biofilm-producing microbes [1]. The National Institutes of Health (NIH) states that microbial films may result in nosocomial infections around 65% and 80% of all microbial infections and chronic illnesses respectively [2]. Biofilms are involved in the development of infections in burn wounds. For example, one of the studies discovered that 90% of burn wound samples had positive bacterial cultures, with 46.6% of the isolates developing biofilms. The most commonly isolated bacterium related to biofilm development were Pseudomonas aeruginosa, Klebsiella species, Proteus species, and Methicillin-resistant Staphylococcus aureus [3], [4], [5]. Biofilms have a crucial role in pathophysiology as well as clinical manifestation of many illnesses, promoting the establishment of multi-drug resistance organisms (MDRO) and treatment failures. These biofilms act as a barrier, preventing antimicrobial agents and host immune system defenses from penetrating [3]. Wounds as a result of burns have been ranked among the most devastating forms of injuries in the last decade. Statistics on burns showed that 11 million people require medical treatment and 300,000 die globally each year [6]. More than 75% of the death cases as a result of sustaining burns are caused by bacteria infection of the wound. Burn patients lose their natural barrier (skin), and have prolonged hospital stays and therapeutic procedures, making them susceptible to a variety of infections [7]. Burn wound infections, especially in low- and middle-income countries, lead to high morbidity and mortality rates and remain a challenge in most hospitals [8]. These infections are caused by biofilm-producing bacteria that have high resistance to antimicrobial treatments due to their biofilm-forming nature [9]. Reports suggest that these biofilms are a major contributor to inflammatory diseases which are long-lasting [10] by increasing the ability of the pathogen to evade both host defenses and antibiotics. Although efforts are being made to manage burn wound infections, the emergence of MDRO strains further complicates treatment options leading to failed therapeutics and adverse clinical outcomes [11]. This study seeks to assess the rate of biofilm-producing aerobic bacteria in burn wounds and determine the rate of multi-drug resistant strains from burn wound infections. The information on the prevalence of MDRO and their association with biofilm production will provide insights into the challenges faced in the clinical management of burn wounds and the importance of infection control measures.

Methods

This was a cross-sectional study conducted at the Department of Microbiology at the Maharishi Markandeshwar Institute of Medical Science and Research (MMIMSR) Mullana, Ambala, Haryana.  The study included 50 swabs collected from burn patients admitted to the hospital alongside detailed clinical histories of the patients.

Sample collection

Burn wound swabs were collected from each patient aseptically and stored in a sterile test tube containing normal saline. Samples were then transported in a sterile container to the laboratory for culturing on 5% Blood agar and MacConkey agar, which were then incubated overnight at 37 °C aerobically for 24 h (Figure 1).

Laboratory analysis of swabs:

As per standard microbiological protocol, the different colonies were first identified through an examination of colony morphology and culture characteristics. Bacteria colonies on the blood and MacConkey agar were gram-stained and tested with biochemical reactions and VITEK® 2 Compact Automated Systems to identify bacteria species present.

Staphylococcus aureus

Staphylococcus aureus was isolated from blood agar after an overnight incubation as a beta-hemolytic microbe with colonies surrounded by zones of clear beta-hemolysis. The identification was confirmed through microscopic examination, which revealed Gram-positive cocci organized in clusters when observed after Gram staining. A catalase test was carried out to distinguish between Staphylococcus, which is catalase positive, and Streptococcus, which is catalase negative. A coagulase test was also carried out to differentiate Staphylococcus aureus (coagulase positive) from other Staphylococcus species (coagulase-negative Staphylococcus).

Pseudomonas aeruginosa

Pseudomonas aeruginosa was isolated from MacConkey agar medium which was incubated overnight. The identification was confirmed by the characteristic appearance as colonies appear round, flat, and colorless, indicating that the organism is a lactose non-fermenter and microscopically as Gram-negative bacilli after being stained by Gram stain. An oxidase test was also performed, in which the bacteria was positive.

Klebsiella spp

Klebsiella spp was isolated from MacConkey agar medium with growth appearing as mucoid and pink in color due to lactose fermentation. Gram staining showed that isolates were Gram-negative, encapsulated, and rod-shaped bacteria. A lactose fermentation test was performed, in which the bacteria was positive.

Acinetobacter spp

Acinetobacter spp was isolated from MacConkey agar medium with colonies appearing small, translucent, and shiny. The colonies were stained using the Gram staining technique and were microscopically identified as Gram-negative bacilli. A lactose fermentation test was carried out, in which the bacteria was negative.

Proteus spp

Proteus spp was isolated from MacConkey agar medium with colonies appearing as smooth and colorless (no swarming growth). After Gram staining, the colonies appeared as Gram-negative rod-shaped. The bacteria was a negative lactose fermenter after the lactose fermentation test.

Antimicrobial susceptibility test: Disc diffusion method of modified Kirby-Bauer technique using Mueller Hinton agar plates and commercially available antimicrobial discs was used to perform antimicrobial susceptibility test on all the bacterial isolates. The procedure was performed based on Performance Standards for Antimicrobial Disk Susceptibility Tests according to the Clinical and Laboratory Standards Institute (CLSI) M02 document [12]. Mueller Hinton agar was prepared by emulsifying the starch in a small amount of cold water and then poured into beef infusion and casein hydrolysate. Agar was then added. The volume was made up to 1 liter with distilled water. The constituents were dissolved by heating gently at 100°C with agitation. The mixture was then filtered and the pH adjusted to 7.4. The mixture was dispensed into screw-capped bottles and sterilized by autoclaving at 121°C for 20 minutes. Mueller Hinton agar plates were labeled according to the different bacteria isolates. A direct broth suspension was made using 3-5 isolated colonies from an 18-24 hour non-selective agar plate with turbidity equivalent to a 0.5 McFarland standard. The inoculum was applied to the agar within 15 minutes by dipping a sterile cotton swab into the suspension and streaking the entire agar surface in three overlapping streaks, rotating the plate 60 degrees each time. Antimicrobial discs were then placed aseptically on the inoculated agar while ensuring even distribution and sufficient spacing to prevent overlapping zones of inhibition. The plates were inverted and incubated at 35°C for 16-18 hours. The zones of inhibition were measured using a ruler above a black and interpreted according to CLSI M100 breakpoint tables [12]. The CLSI M100 document provides breakpoint tables that categorize bacterial isolates as susceptible, intermediate, or resistant based on the diameter of the zones of inhibition. For each antimicrobial agent, the breakpoint tables specify the zone diameter range for each category.

Identification of multi-drug resistant strain: Multi-drug resistant (MDR) strains were identified based on resistance to at least one antibiotic agent in three or more antimicrobial categories. [13]

Detection of biofilm-forming bacteria: The tube adherence method and Congo red agar method [14], [15] were employed in the detection of biofilm. The modified Congo red agar method was performed by inoculating isolates on a specially prepared solid medium, Blood agar base (BAB)-2, supplemented with glucose and Congo red. Congo red was prepared as a concentrated aqueous solution and autoclaved at 121°C for 15 minutes separately from other medium constituents. Agar was then added at 55°C. This medium was incubated aerobically for 24 to 48 hours at 37°C. Isolates were then inoculated onto the Modified Congo red agar.

For the Tube adherence method, isolates were inoculated on Brain heart infusion broth with 2% sucrose in a glass tube and incubated for 24 hours at 37 ° C. After 24 hours, the supernatant was decanted and sediments washed with PBS (pH7.3) and dried. Dried tubes were stained with 0.1% crystal violet. Excess stain was removed and tubes were washed with distilled water three times. Tubes were then dried in an inverted position and were observed for biofilm formation. Biofilm formation was considered positive when visible film climbed the wall and bottom of the tube.

The data were statistically analyzed and presented in the form of tables, graphs, percentages, and tests of significance.

Results

Culture positive: Out of the 50 samples, 82% were culture positive for bacteria while 18% of the sample showed no growth (Figure 2).

Gram-negative and Gram-positive bacteria isolates: For Gram-positive cocci, 11.4% were Staphylococcus aureus and 2.3% were coagulase-negative staphylococci (CoNS). Out of the Gram-negative bacteria, 45.5% were Pseudomonas aeruginosa, 34.1% were Acinetobacter spp., 4.5% were Klebsiella spp. and 2.3% were Proteus spp. (Figures 3, 4)

Antibiotic susceptibility of Gram-positive and Gram-negative bacteria:

S. aureus showed 100% susceptibility to linezolid, vancomycin, and netilmicin while showing high resistance to ofloxacin, penicillin, cefoxitin, ampicillin, and amikacin antibiotics.

CoNS isolates were sensitive to all antibiotics. For Gram-negative bacteria, carbapenem antibiotics showed the highest sensitivity to Klebsiella, proteus, pseudomonas aeruginosa, and Acinetobacter spp. Proteus spp. isolate was sensitive to all the antibiotics. Klebsiella, Pseudomonas aeruginosa, and Acinetobacter isolates were highly resistant to trimethoprim-sulfamethoxazole, ampicillin, and ceftazidime antibiotics.  Klebsiella isolates were 100% resistant to ampicillin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, and ceftazidime. Pseudomonas aeruginosa isolates were highly resistant to cephalosporin antibiotics, ampicillin, amoxicillin-clavulanate, trimethoprim-sulfamethoxazole, chloramphenicol, gentamicin, and netilmicin. Acinetobacter isolates were highly resistant to tetracyclines, sulfonamides, cephalosporins, penicillin, and quinolones classes of antibiotics used (Table 1).

The frequency of Methicillin-resistant Staphylococcus aureus was 60%. In Gram-negative bacteria, 45% of Pseudomonas aeruginosa, 46% of Acinetobacter spp., and 50% of Klebsiella spp. were multi-drug resistant.

Detection of biofilm producers

The biofilm detection method resulted in 25 (56.81%) biofilm-producing bacteria and 19 (43.81%) non-biofilm-producing bacteria (Table 2, Figure 5).

Statistics on multi-drug resistant strains (MDR): For the multi-drug resistance distribution, 3 out of 5 Methicillin-resistant staphylococcus aureus, 9 out of 20 Pseudomonas aeruginosa, 7 out of 15 Acinetobacter spp., and 1 out of 2 Klebsiella spp. were multi-drug resistant (Table 3).

Discussion

In our study, 86.37% of the isolates were gram-negative, while 13.63% were gram-positive. Among the gram-negative isolates, Pseudomonas aeruginosa (45.46%) was the most prevalent, followed by Acinetobacter spp (34.10%), Klebsiella spp. (4.54%) and Proteus spp. (2.27%). Out of the 44 bacterial isolates, the biofilm detection rate was 56.81%. Gram-positive isolates were predominantly Staphylococcus aureus (11.36%). A similar study by Ramakrishna et al. [16] reported that P. aeruginosa (33.3%) was the most common burn wound isolate, followed by species of Acinetobacter (23.3%) and Staphylococcus aureus (16.6%). The frequent occurrence of Pseudomonas aeruginosa in our study can be attributed to its ability to thrive in moist environments [17]. It is also known to grow in common antiseptics due to its inherent resistance to these substances [18]. Acinetobacter has emerged as a significant nosocomial pathogen, largely due to its existence as a normal skin flora, ease of transmission, and ability to survive in hospital environments as a result of its resistance to treatment. Acinetobacter is a water-loving microorganism that specifically inhabits aquatic environments [19].

Staphylococcus aureus was the predominant pathogen with a 100% sensitivity rate to linezolid, vancomycin, and netilmicin and was highly resistant to ofloxacin, penicillin, cefoxitin, ampicillin, and amikacin antibiotics.  Similarly, Chaudhary et al [20] and E Abou Warda et al [21] found that Staphylococcus aureus isolates were susceptible to vancomycin and linezolid. El Hamzaoui et al [22] also found in their study that S. aureus was highly resistant to ampicillin, penicillin, cefoxitin, ofloxacin, ciprofloxacin, and erythromycin. They also observed a high degree of sensitivity to vancomycin, amikacin, gentamycin, and chloramphenicol. Among the gram-negative bacilli, Pseudomonas aeruginosa showed the highest sensitivity to imipenem accounting for 100% susceptibility, followed by meropenem and amikacin antibiotics. A similar finding reported by Sabetha et al [23] showed high susceptibility to imipenem (98% -100%). Also, Abdi et al [24] reported high sensitivity to imipenem (88.9%), meropenem (77.8%), and amikacin (81.5%). Datta et al [25] showed 55.6% sensitivity to Meropenem. Pseudomonas aeruginosa isolates in this study were highly resistant to cephalosporin antibiotics, ampicillin, amoxicillin-clavulanate, trimethoprim-sulfamethoxazole, chloramphenicol, gentamicin, and netilmicin. This follows the antibiotic sensitivity pattern of a study done by Chaudhary et al [20] who reported a very high rate of resistance to almost all antibiotics with cephalosporin antibiotics having the highest resistance (91.1%). Interestingly, a study by Maclean et al. [26] reported isolates of P. aeruginosa being 100% susceptible to trimethoprim-sulfamethoxazole although P. aeruginosa is known to use drug efflux to resist this antibiotic. Acinetobacter species also showed maximum sensitivity (86.66%) to carbapenems-imipenem, and meropenem, followed by aminoglycosides, which displayed moderate sensitivity accounting for 66.66%. This correlates with a study by Asati and Chaudhary [27]. Acinetobacter isolates were highly resistant to tetracyclines, sulfonamides, cephalosporin, penicillin, and quinolones classes of antibiotics. Similar results were reported by Mwanamoonga et al [28], where 21 out of 30 isolates were highly resistant to aminoglycosides, fluoroquinolones, sulphonamides, cephalosporins, carbapenems, and tetracycline classes of antibiotics. Klebsiella isolates showed 100% resistance to ampicillin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, and ceftazidime. Helmy et al [29] reported a similar resistance profile, with complete resistance (100%) to penicillin, ampicillin, cefixime, and sulphamethoxazole/trimethoprim antibiotics. Also, resistance rates over 65% were reported for cephalosporin, fluoroquinolones, and chloramphenicol.

For the detection of biofilm production, the Tube adherence technique and Modified Congo red agar technique were used. This study primarily focused on resource-limited settings, and so both techniques were employed instead of the gold standard tissue culture method due to resource constraints. According to Dhanalakshmi et al [30], Congo red agar and tube adherence methods can be considered as alternatives to detect biofilms in resource constraint conditions. The results for the tube adherence technique were considered because it is the most reliable and common method used to identify biofilm-forming organisms in laboratories compared to the modified Congo red agar method. It is highly sensitive, and specific and correlates well with the standard quantitative assay i.e. tissue culture plate method [31], [32]. In this study, the rate of biofilm production in 44 isolates was higher in the Tube Adherence method by 56.81% than in the Modified Congo red agar method. The variation was statistically insignificant (p-value =0.244). This is in relation to a study by Harman et al [33], who reported that the rate of biofilm production was higher in the tube adherence method (55%) than in the modified Congo red agar method (46.66%). A study by Nabajit [34] reported that the rate of biofilm production was higher by the tube adherence method (57%) than by the modified Congo red agar method (20%). Another study by Shrestha et al. [35] reported that the rate of biofilm production was higher by the tube adherence method (82.35%) with high sensitivity and specificity (82% and 85.9%) similar to that of the tissue culture method. Similarly, Reddy [36] and Khan et al [37] studies reported that the tube method’s sensitivity, as well as its specificity, were 97.3% and 100%; 95.78%, and 99.49% respectively.

Out of the 25 isolates that showed biofilm formation, 12(48%) were found to be biofilm MDR, while out of the 18 isolates that showed non-biofilm formation, 7 (38.8%) were found to be non-biofilm MDR which is similar to the pattern observed by Asati and Chaudhary [27]. Staphylococcus aureus has the potential to attach to a wide range of matrix components to start colonization. This attachment is regularly facilitated by protein adhesions of the family referred to as microbial surface components recognizing adhesive matrix molecules (MSCRAMM). Proteins that have an affinity for collagen and bind to fibronectin belong to this family [38].

Pseudomonas aeruginosa was the predominant pathogen with 9(45%) of the isolates found to be MDR strains. Asati and Chaudhary [27] also observed 70.49% MDR Pseudomonas aeruginosa in their study. To help lower the incidence of infections caused by these antibiotic-resistant organisms, strict measures to prevent infections (such as isolation in a dedicated room, wearing gowns and gloves when interacting with the patient, and practicing hand hygiene before and after each patient encounter) and suitable initial antimicrobial treatment are crucial.

Out of the 5 isolates of the Gram-positive Staphylococcus aureus, 2(40%) were reported as Methicillin-sensitive Staphylococcus aureus (MSSA), and 3(60%) isolates were identified to be Methicillin-resistant Staphylococcus aureus (MRSA). This correlates with a study done by Datta et al [25] where Staphylococcus aureus isolates, 44.44% (16/36) were Methicillin sensitive Staphylococcus aureus (MSSA) and 55.56% (20/36) were Methicillin-resistant Staphylococcus aureus (MRSA). The most predominant gram-positive organism in burn wounds was found to be MRSA. This study reported the MDR rate of Staphylococcus aureus isolates to be 60% which is a high rate corresponding to a study by Asati and Chaudhary [27].

In hospitals, patients with multidrug-resistant (MDR) strains of Pseudomonas aeruginosa and Acinetobacter spp. infections are alarming.  Acinetobacter spp. considered to be a non-pathogenic microbe twenty years ago has appeared as a significant and challenging human pathogen, giving rise to various types of infections. It stands as the second most prevalent nosocomial, aerobic, non-fermentative, gram-negative bacilli pathogen, following closely behind Pseudomonas aeruginosa. These two microorganisms exhibit significant potential for biofilm formation explaining their high antibiotic resistance, survival capabilities, and amplified virulence. Isolates that formed the most biofilms in this study were Pseudomonas aeruginosa. Similarly, Kunwar et al [39] discovered that P. aeruginosa is the most common organism forming biofilm in burn wounds.

Conclusion

The rate of biofilm production in all the isolates obtained from the burn samples was 56.81%. Out of these biofilm-producing bacteria, 48% were multi-drug resistant (MDR).  The study observed a higher rate of multidrug resistance in biofilm-producing isolates. Biofilm decreases antibiotic uptake and further hampers the prognosis. The highest sensitivity was seen for imipenem followed by meropenem and amikacin. Marked resistance was seen for commonly used antibiotics like quinolones, cephalosporins, and cotrimoxazole.

Recommendation

Biofilm production can be detected in routine laboratories by tube adherence method as it is more effective in detecting biofilm-producing organisms. This is easy to interpret and cost-effective. The study therefore recommends that every burn center should regularly identify and monitor precise patterns of burn wound inhabitation and the antimicrobial sensitivity levels of microbes involved. Furthermore, the timely detection of infection resulting from biofilm-producing strains can lead to modification in treatment strategies and improve outcomes in burn patients.

Funding: This research received no external funding

Institutional Review Board Statement: The study was conducted in accordance with the            Declaration of Helsinki, and approved by the Ethics and Protocol Review Committee with Protocol Identification Number: IEC/MMIMSR/1904/15-03-2019

Informed Consent Statement: Written Informed Consent and assent were obtained from all study participants before sampling.

Data Availability Statement: The data presented in this study are available on reasonable request from the corresponding author.

Conflicts of Interest: The authors declare no conflicts of interest.