Association of biofilm production in ESBL and MBL producing clinical isolates of Pseudomonas aeruginosa

Introduction : Pseudomonas aeruginosa is one of the most prevalent nosocomial pathogens that cause a life-threatening infection. One of the important characteristics of P. aeruginosa is biofilm formation and the most studied bacterium related to biofilm formation so far. The biofilm formation and beta-lactamases production synergistically contribute to the extensive dissemination of multi-drug resistant strains. Aim : The present study was conducted to identify, biofilm-producing isolates of P. aeruginosa along with their antibiotic resistance pattern and ESBL and MBL production and to analyze their antibiogram. Materials and methods: Various clinical specimens were collected and totally 82 clinical isolates of P. aeruginosa were included in this study. Biofilm producing isolates were identified by the tube adherence method. Results : Among the total, 22 [26.83%] isolates were biofilm producers and the maximum number was obtained from blood [100%], followed by ETT [75%], and Drain [66.67%]. Biofilm producing isolates were showing more resistance in comparison to non-biofilm producers. All isolates of P. aeruginosa were sensitive to colistin and polymyxin B. Association of ESBL production and biofilm formation found to be statistically significant [p < 0.002], which was a contrast to association of MBL production and biofilm formation. Conclusion : High-level resistance to antimicrobial agents is a characteristic feature of infection caused by biofilm and lead to chronic infections. Knowledge about these biofilm-producing isolates is important in the clinical setting to eradicate these chronic and life-threating infections.


Introduction
Pseudomonas aeruginosa is remarkably considered one of the most adaptive nosocomial pathogens [1]. Infections resulting from P. aeruginosa are frequently life-threatening and hard to treat causing elevated stay in a medical institution or even accelerated morbidity and mortality as it exhibits intrinsically excessive resistance to many antimicrobials and the development of multi-drug resistance in health care settings [2]. Pseudomonas aeruginosa is a gram-negative, non-fermenting, obligately aerobic, the saprophytic bacterium which is widely distributed [3]. It is known for its intrinsic resistance to several antimicrobials, disinfectants, and tolerance to a wide range of physical conditions [4]. One of the important characteristics of P. aeruginosa is biofilm formation [1] and the most studied bacterium related to biofilm formation so far [5]. At present, biofilm is a serious worldwide concern due to its extracellular polymeric substances (EPS) which plays a vital role in antimicrobial resistance [6,7].
Manuscript received: 28 th January 2020 Reviewed: 9 th February 2020 Author Corrected: 14 th February 2020 Accepted for Publication: 19 th February 2020 The biofilm-producing bacteria may show higher Minimal Bactericidal Concentration (MBC) and Minimal Inhibitory Concentration (MIC) of antibiotics up to 100-1000 fold than the planktonic form of bacteria [8].
Biofilm forming bacteria cause chronic persistent infection [8] and life-threatening device-associated infection. The various medical devices are shown to be colonized by biofilm [9] which leads to device-associated nosocomial infection. This biofilm infection affects millions of people each year and many deaths occur as a consequence [10].
NIH publication showed more than 60% of all kinds of infections are related to the formation of biofilm [11]. In the present day, antibiotic resistance is an emerging problem and especially higher antibiotics like carbapenem are under the threat due to the widespread presence of carbapenemase (mainly Metallo Beta-lactamase). The biofilm formation and beta-lactamases (like ESBL and MBL) production synergistically contribute to the wide distribution of multidrug resistant strains [12]. So the present study was conducted to identify, biofilmproducing isolates of P. aeruginosa along with their antibiotic resistance pattern and ESBL and MBL production. Also to find out the association between biofilm formation and drug resistance among P. aeruginosa in the hospital set up.
For ESBL and MBL detection-A Ceftazidime (CAZ 30µg) and Ceftazidime/clavulanic acid (CAZ/CA 30µg/10µg) disk were used to determine ESBL production. If there's an increase of ≥ 5-mm in zone diameter for Ceftazidime/clavulanic acid compared to the diameter of the Ceftazidime, it is considered as ESBL producing isolates [14]. An imipenem (10 µg) and imipenem /EDTA (10 µg /750 µg) disk were used to determine MBL production. If there's an increase of ≥ 7mm in zone diameter for imipenem -EDTA disc compared to zone diameter of imipenem disc, it was considered as MBL producing isolates [15].
Detection of Biofilm formation by Tube Adherence method -The isolated colony of P. aeruginosa was inoculated into a test tube contains trypticase soy broth (TSB) and incubated for 24 h at 35 °C. Next day content of the tube was discarded, and phosphate buffer saline was used to wash the tube and it was dried at room temperature. Then the tube was treated using 0.1% crystal violet for staining and then washed with water and dried. The presence of visible biofilm lining sidewall and the bottom of the tube was considered as biofilm producer [16].
Sample size: A total of 82 P. aeruginosa isolates identified were included in this study.
Data analysis: These study results were analyzed in SPSS version 16 software. Chai Square test was applied and p<0.05 was considered as significant.
Ethical consideration and permission: This study was reviewed and approved by the institutional ethical committee.

Discussion
P. aeruginosa causes the leading and life-threating nosocomial infections, ranking only second among the gram-negative pathogens [4]. As per the CDC statement, the P. aeruginosa infection rate was near about 0.4% in the US hospitals and 4 th common nosocomial pathogen accounts for 10.1% of all hospital-acquired infections [17]. The main problem in treating P. aeruginosa infection is its high-level resistance to various antibiotics.
Studies show that infection by drug-resistant P. aeruginosa leads to increased length of hospital stay, morbidity, and mortality, and chronic infection [18]. The biofilm formation along with beta-lactamase production further complicates the scenario [12]. Production of an extracellular matrix is the hallmarks of a mature biofilm that acts as a barrier for any antibiotics and increases resistance to these antibiotics [19].
In the present study, among a total of 82 isolates of P. aeruginosa, 22(26.3%) were biofilm-producer and this finding is comparable with other studies which show (27.05%) [20], (32.3%) [21] and (33%) [22], but in contrast with others who showed higher rate of biofilm production (73.68%) [12] and (83.33%) [23]. This variation in the rate of isolation also may be due to sample size, type of specimen studied because medical devices were frequently colonized by biofilm-forming organisms and the various methods used for biofilm identification like Congo red agar method or Tissue culture plate which were showing a higher rate of detection.
The present study show, maximum biofilm-producing isolates recovered from specimens received from ICU (63.64%) compared to the ward (36.36%) and similar findings was shown in another study (83.3%) [2]. This could be possibly due to the ICU setup uses multiple medical devices for treatment and intervention of patient care although indwelling devices used widely in hospitals [24] and biofilm is known for colonizing these medical devices.
In the present study, the maximum rate of biofilm positive isolates was identified from the blood (100%) and this finding was similar to another study which showed that 100% sterile fluids isolates were biofilm producers [22].  A catheter might have inserted for several purposes and this can be colonized. After blood samples, the ETT showed biofilm formation in 75% isolates and this could be explained by the fact that more specimens were obtained from patients admitted in ICU who were either intubated or needing ventilator support [24]. It was found that 66.67%, 30.43%, and 23.81% biofilm-producing isolates were from the drain, pus, and sputum respectively. This finding supports the fact that biofilm development is aided by tissue lesions, chronic respiratory disease, implanted medical devices, surgical wounds, etc. [9,25].
The antibiotic susceptibility of biofilm-producing bacteria is reduced because of a restricted antibiotic penetration, adaptive response and the occurrence of persisting cells [2].
In the present study, high resistance was noted among biofilm producers to an antipseudomonal cephalosporin (Ceftazidime and Cefepime), Piperacillin-tazobactam, Ciprofloxacin, and Gentamicin with 77%, 73%, 68%, and 59% respectively. These findings were nearly matching with other studies [21,22]. This may be due to the widespread use of these easily available antibiotics without knowing the infection status. All isolates were susceptible to polymyxin-B and colistin like other studies [1,2,21].
In the present study, resistance to Ceftazidime, Cefepime, Piperacillin-tazobactam, Ciprofloxacin, Gentamicin, and Amikacin was comparatively higher in biofilm producer than a non-biofilm producer. The difference was statistically significant (p < 0.05). It is similar to findings from other studies for most of the antibiotics tested [12,21,22]. So Meropenem, colistin, and polymyxin-B remain the treatment of choice for biofilm-producing isolates. However, due to their high toxicity, polymyxin is used for the treatment of only serious infections.
In the present study, it was found that there is an association between ESBL production and biofilm formation (Statistically significant, p= 0.002). It was similar to another study [26] but the contrast study done by Dumaru et al [12].
No statistically significant association could be established between MBL production and biofilm production (Statistically insignificant, p=0.19) which was in agreement with another study [20]. The resistance to antimicrobials in biofilm-producer may be explained by the fact like, there are an increased plasmid transfer and gene transfer among biofilm bacteria which further intensifies the problem of drug resistance [27] and also by the fact that in the process of biofilm development, drug resistance varies bacterium to bacterium [28].

Conclusion
The present study showed, 26.3% isolates of P. aeruginosa were biofilm producers and also showed that there is an association between biofilm formation and drug resistance. The present study emphasizes the relationship between ESBL production and biofilm formation in P. aeruginosa. Identification of biofilm-producing isolates is important because it leads to treatment failure due to the high drug resistance to multiple antibiotics. Biofilm may be controlled by replacing the device that was colonized and also by taking care of the device or wound that was already existing to prevent biofilm formation.
Limitations-The lack of confirmation of biofilm, ESBL, and MBL production by using molecular technologies are the drawbacks of this study.
What does this study add to the existing knowledge?
The present study highlight's the importance of performing the test for biofilm production in P. aeruginosa isolates. By knowing the resistance pattern of these isolates' clinicians can able to choose the right empirical antibiotic in lifethreatening conditions.