Correlation between biofilm formation and resistance toward different commonly used antibiotics along with extended spectrum beta lactamase production in uropathogenic Escherichia coli isolated from the patients suspected of urinary tract infections visiting Shree Birendra Hospital, Chhauni, Kathmandu, Nepal

A cross sectional study was conducted among the patients suspected of urinary tract infections (having symptoms like burning micturation, frequent or intense urge to urinate, back pain or lower abdominal pain, fever or chills etc.) visiting Shree Birendra Hospital, Chhauni, Kathmandu, Nepal since May to October 2013.

Total 1480 mid stream urine samples collected from the patients (out patients and in patients) suspected of urinary tract infections were cultured by the semi-quantitative culture technique [11]. The patients having laboratory or radiological evidence of other infections as the cause of the symptoms, patients with urinary catheterization and those who already have received antibiotics were excluded from our study. The bacterial isolates from the urine samples were identified by using microbiological techniques as described in the Bergey’s manual which include morphological appearance of the colonies, staining reactions and biochemical properties. The antimicrobial susceptibility testing was done by Kirby-Bauer disk diffusion technique as recommended by clinical and laboratory standards institute (CLSI) [12].

Detection of ESBL producers

Among the uropathogens isolated from suspected cases of UTI, only the strains of E. coli were subjected for detection of ESBL production. The phenotypic confirmation of the ESBL producing strains was done by combined disk method (Fig. 1) [12].

Detection of biofilm producers

Detection of biofilm production was done for both ESBL positive and ESBL negative strains of E. coli by Congo red agar method (CRA) (Fig. 2) [13].

On the basis of the intensity of color change of CRA medium after inoculation of the organisms, which is directly proportional to the amount of biofilm produced by the organisms, the biofilm producing organisms were classified into three categories as strong biofilm producers, moderate biofilm producers and weak biofilm producers [14, 15].

Out of 1480 mid stream urine samples 278, (18.8 %) samples showed significant growth (≥ 10⁵ cfu/ml). E. coli was isolated from 208 (74.82 %) samples. Out of 208 E. coli isolates 69 (33.2 %) were found to be ESBL producers and 139 (66.8 %) were ESBL non producers. Among 208 E. coli isolates 42 (20.192 %) were found to be strong biofilm producers, 42 (20.192 %) were moderate biofilm producers, 24 (11.538 %) were weak biofilm producers and 100 (48.076 %) were biofilm non producers.

Among 208 E. coli isolates, 108 (51.92 %) were biofilm producers. This finding agrees with the findings of different authors from different parts of the world [16, 17]. Biofilm protects bacteria from host defense mechanisms, along with the antibiotics [18].

In this study, the antibiotic resistance of biofilm producing E. coli was found significantly higher than that of biofilm non producing E. coli (p < 0.05). Among biofilm producing E. coli, higher antibiotic resistance was observed in strong and moderate biofilm producers. The association between biofilm production and antibiotic resistance was found to be statistically significant (p < 0.05) except in case of amoxicillin and nitrofurantoin. Microorganisms growing in a biofilm are intrinsically resistant to many antibiotics increasing the antibiotic resistance up to 1000 folds and high antimicrobial concentrations are required to inactivate organisms growing in a biofilm [19, 20]. This may be because of the insufficient concentration of the antibiotics reaching some areas of the biofilms and metabolic inactiveness (along with the presence of active antibiotic degradation mechanisms contributing to halt the accumulation of the drugs up to an effective concentration) of the bacteria located at the base of the biofilms [9].

The biofilm forming ability was found to be significantly higher in ESBL positive strains of uropathogenic E. coli than that of ESBL negative strains [p < 0.05]. The study by Subramanian et al. in India also reported the higher ability of the ESBL producing organisms to form biofilm in comparison to that of ESBL non-producing isolates. It has been postulated that during occurrence of the large numbers of the chromosomal gene rearrangements upon acquisition of the ESBL plasmids the bacteria express several virulence genes [21].

ESBLs are enzymes that are responsible for resistance of bacteria toward third generation cephalosporins and monobactams [22]. Most of the plasmids responsible for ESBL production carry genes encoding resistance to other drugs also [23]. Due to frequent presence of cross-resistance to several other classes of antibiotics (like aminoglycosides and fluoroquinolones), in ESBL-producing organisms, the treatment of the infections by these bacteria are often present as the therapeutic challenges [22]. Further higher ability of the ESBL producing organisms to form biofilm makes the treatment even more difficult, increasing the mortality and severity of the infections [21]. Macrolides (erythromycin, clarithromycin, and azithromycin) are known to have antibiofilm activity against biofilm producing organisms by inhibiting a key component of the biofilm, alginate. And several studies have recommended, the combined therapy (being macrolides one of the first antibiotics chosen) as the treatment of choice in infections caused by biofilm producing organisms [9].

Increasing irrational and haphazard use of antibiotics, sales of substandard antibiotics and transmission of drug resistant bacteria among people may be responsible for the rise in antibiotic resistance among the bacteria [24]. Antimicrobial resistance has become a serious global public health issue. Infections caused by drug resistant bacteria are responsible for increased morbidity and mortality [25]. The selection of the antibiotics for treatment of the bacterial infections should be based on culture and sensitivity reports.

The ability of biofilm formation was found higher among ESBL producing strains of E. coli. There was higher resistance rate among biofilm producing E. coli isolates to almost all the antimicrobial agents except a few. According to our antimicrobial susceptibility pattern for E. coli, to start preliminary treatment for UTI in Nepal, we recommend to use amikacin or nitrofurantoin. Further, for the treatment of the UTI, the antibiotics should be selected on the basis of the urine culture and sensitivity report.