Biofilm eradication studies on uropathogenic E. coli using ciprofloxacin and nitrofurantoin

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1 Available online at Int J Pharm Biomed Res 212, 3(2), Research article International Journal of PHARMACEUTICAL AND BIOMEDICAL RESEARCH ISSN No: Biofilm eradication studies on uropathogenic E. coli using ciprofloxacin and nitrofurantoin N.A. Ghanwate* Department of Microbiology, Sant Gadge Baba Amravati University, Amravati, Maharashtra-444 2, India Received: 16 Jun 212 / Revised: 18 Jun 212 / Accepted: 19 Jun 212 / Online publication: 2 Jun 212 ABSTRACT The chronic nature of some urinary tract infections (UTI) is being attributed to the ability of E. coli to form a biofilm. The armament of therapeutic agents available to treat bacterial infections today is restricted to antibiotics developed specifically to kill (bactericidal) or stop the growth of bacteria (bacteriostatic) in planktonic form and not the biofilm. Bacteria growing within a biofilm lose their sensitivity to antibiotic quickly thus resulting in persistent infections that cannot be resolved with standard antibiotic treatment. The present study was aimed at; in vitro qualitative estimation of biofilm production of E. coli isolated from UTI and correlates it with antibiotic resistance. Minimum inhibitory concentration (MIC) and minimum biofilm eradicating concentration (MBEC) to determine changes in the pattern of antibiotic sensitivity of uropathogenic E. coli (UPEC) from the planktonic to the biofilm phase of growth was evaluated. The uropathogenic E. coli isolated from samples were tested for antimicrobial susceptibility by disc diffusion method. In vitro quantitative estimation of biofilm formation by the isolates was determined by tube method. From 38 isolates of E. coli, 1 were positive for in vitro biofilm production. Among them 44 were classified as strong biofilm producers and 116 as moderate. It was observed that the MIC of ciprofloxacin and nitrofurantoin for the biofilm forming UPEC was more as compared to the non biofilm forming strains and the MBEC of the strong biofilm producers was more than ten folds higher than its MIC. There was a significant correlation between biofilm production and antibiotic resistance. The present study demonstrated that uropathogenic E.coli have high propensity to form biofilm that renders it resistant to conventional antimicrobial therapy. Key words: Biofilm, Minimum inhibitory concentration (MIC), Minimum biofilm eradicating concentration (MBEC), E. coli, Urinary tract infections (UTI), Antibiotics 1. INTRODUCTION Diseases involving bacterial biofilms are generally chronic and difficult to treat because bacteria in biofilm are most resistant to antimicrobial agent than their planktonic (free living) counterparts. Recurrent urinary tract infections caused by uropathogenic E. coli represents classical biofilm disease problem. Such infections may be difficult to treat as they exhibit multidrug resistance. Appropriate antimicrobial therapy is an essential part of recurrent urinary tract infections (UTI) treatments. Usually, floating cells are completely eradicated at the antibiotic levels predicted by *Corresponding Author. Tel: Fax: nirajghanwate@gmail.com 212 PharmSciDirect Publications. All rights reserved. laboratory minimum inhibitory concentration (MIC) studies. However, the concentration of antibiotics required to eradicate cells within the biofilm is uncertain [1]. It is therefore apparent to find out proper concentration of the drug that is capable of inhibiting the formation of biofilms or eradicating existing biofilms. The minimum biofilms eradicating concentration (MBEC) assay could provide a quick and reliable methodology to assess the susceptibility of E.coli cells growing in a biofilm to antibiotics. Some antibiotics are effective at MIC level concentrations both in vitro and in vivo while others are not. This represents the problem faced by the physician dealing with a likely biofilm associated infection. MIC data presents the clinician with a selection of antibiotics that appear efficacious against the patients isolates; however the history of treatment with the current patients have taught the clinician that there may

2 N.A. Ghanwate, Int J Pharm Biomed Res 212, 3(2), be a poor correlation between MIC values and outcome when treating chronic, recurrent or devised related infections. Resolution of the problem regarding choice of antibiotics to treat biofilm infection could be achieved by utilizing an assay system that can select antibiotics and biocides based on activity against biofilms. The MBEC would represent the concentration of antibiotics capable of killing a biofilm. The reliability of in vitro biofilms model and MBEC assay for antimicrobial susceptibility for bacterial biofilms in the anticipation that the MBEC would be more reliable for clinical effective antimicrobials, the present study was aimed at: The investigation of biofilm formation among the clinical isolates of E. coli from urinary tract infection. To examine the correlation between biofilm formation and antibiotic resistance. Evaluation of Minimum inhibitory concentration (MIC) and Minimum biofilm eradicating concentration (MBEC) to determine changes in the pattern of antibiotic sensitivity of uropathogenic E. coli from the planktonic to the biofilm phase of growth. 2. EXPERIMENTAL MBEC was determined by tube method. The tubes with trypticase soya broth were inoculated with loopful culture of biofilm forming E.coli and incubated for 24h. After incubation the tubes were decanted and filled with diluted antimicrobial solutions and the tubes were kept for further incubation for 24h at 37 C. The tubes were rinsed with sterile physiological saline and filled with fresh, sterile 1% peptone water. These tubes were incubated for 24h at 37 C after which the presence of viable bacteria was determined by the pour plate method. Growth of bacteria in a particular tube indicated re-growth of plank tonic bacteria from surviving biofilms. The MBEC value represented the lowest dilution at which bacteria failed to re-grow. 3. RESULTS AND DISCUSSION 3.1. Bacterial isolates from urine sample Out of the total urine samples, 75% were detected of having significant bacteriuria. From the total positive samples, 51% were E. coli, 38% Klebsiella pneumoniae, 6% Enterococcus faecalis followed by 4% Staphylococcus and 1% Micrococcus (Fig.1). A total of midstream urine samples were collected aseptically from patients of IPDs and OPDs of various hospitals from Vidarbha region of Maharashtra state in India. A measured amount of urine i.e..1ml was inoculated by using calibrated loop method on Hi-crome UTI agar (Himedia). The plates were incubated at 37ºC for 24h. After incubation purple colored colonies were counted with the help of colony counter. The plates with the colonies more than 1 5, was considered as significant bacteriuria. From the colonies on Hi-crome UTI agar, E. coli was identified. All the E.coli isolates were assessed for biofilm formation by tube method and tissue culture plate method as described by Christensen et al [2]. The MIC which represents the concentration of antibiotic required to inhibit growth of a plank tonic bacterial population, was determined by using Hicomb MIC strip (Hi media, India) % of Resistant % of sensitive Fig.2. Antibiotics response of uropathogenic E. coli E. coli K. pneumonae E. faecalis S. aureus Micrococcus Fig.1. Bacterial isolates from urine samples 3.2. Antibiotic sensitivity test E. coli isolates were tested for antimicrobial susceptibility by Kirby Bauer Disc Diffusion method, according to norms of Clinical Laboratory Standard Institute (CSLI). Antibiotics used and their concentration in µg included co-trimoxazole (25µg), amoxicillin (1µg), gentamicin (1µg), norfloxacin (1µg), nalidixic acid (3µg), ciprofloxacin (5µg), chloramphenicol (3µg), and nitrofurantoin (3µg) which were obtained from Hi-media, Mumbai. All these isolated E. coli were resistant to commonly used antibiotics such as cotrimoxazole (8%), amoxicillin (78%), gentamicin and norfloxacin (57%), nalidixic acid (52%), ciprofloxacin (43%), chloramphenicol (45%) and nitrofurantoin (14%)

3 N.A. Ghanwate, Int J Pharm Biomed Res 212, 3(2), (Table 1 and Fig.2). The Multiple Antibiotic Resistance Indices (MARI) was calculated as follows: MAR index for isolates N. = N Eqn.1 MAR index for an antibiotics = N. N. N Eqn.2 Table1 Multiple Antibiotic Resistant (MAR) index of antibiotics against isolated uropathogenic E. coli Antibiotics % of Resistant % of Sensitive MAR index Co- trimoxazole Amoxicillin Gentamicin Norfloxacin Nalidixic acid Chloramphenicol Ciprofloxacin Nitrofurantoin Table 2 MIC of different antibiotics for strong, moderate and non biofilm producing UPEC Antibiotics Bacterial strain Strong biofilm producing Moderate biofilm producing Non biofilm forming Ciprofloxacin 5µg/mL 5µg/mL.1µg/mL Nitrofurantoin 1µg/mL 3µg/mL.1µg/mL Resistance of all Isolates Biofilm positive Fig.3. Antibiotic susceptibility results (%) of biofilm producing E. coli Fig.4. Comparative MIC of strong, moderate and non-biofilm producing UPEC 3.3. Biofilm production In vitro quantitative assessment of biofilm formation was determined by tube method. Biofilm formation was considered positive when a visible film lined the wall and bottom of the tube. From the total E. coli isolates 52% were found to be positive for biofilm formation. Out of total biofilm positive E. coli, 28% were strong biofilm producers. There was a significant correlation between biofilm production and resistance to multiple antibiotics such as ampicillin, cotrimoxazole, nalidixic acid and Norfloxacin (Fig.3). In the study conducted by Sharma et al in 29 [1], significant production of biofilm was seen in 54 (67.5%) isolates of E. coli whereas antibiotic sensitivity of the biofilm-producing isolates ranged from 16 to 57% while it was 38-76% for non-biofilm producers in their study. Nonbiofilm-producing E. coli were sensitive to nitrofurantoin, amoxyclav and ceftizoxime. amoxyclav and nitrofurantoin was the most effective antibiotics for E. coli growing as biofilm. The study conducted by Matija et al [3], demonstrated that 53% strains of UPEC were biofilm producing. There was a significant correlation between biofilm production and resistance to multiple antibiotics such as ampicillin, cotrimoxazole, nalidixic acid and norfloxacin as also demonstrated by Suman et al in 25 [4]. Resistance of biofilm forming UPEC to Nalidixic acid was demonstrated by Solo et al [5] MIC and MBEC detection In further study, the MIC and MBEC of ciprofloxacin and nitrofurantoin for the biofilm producing E. coli isolates was determined. It was observed that the MIC for the biofilm forming UPEC (Table 2) was more as compared to the non biofilm forming strains and MBEC was more than ten folds higher than its MIC (Table 3). MIC for the strong biofilm

4 N.A. Ghanwate, Int J Pharm Biomed Res 212, 3(2), A B C D A: Colonies of E. coli on hichrome UTI agar; B: Biofilm production by test tube method 42-week, 73-moderate, 5-strong; C: Biofilm production by TCP method; D: Hi comb method for MIC Table 3 Comparison of MIC and MBEC Antibiotics MIC (µg/ml) MBEC (µg/ml) Ciprofloxacin Nitrofurantoin % 9% 8% 7% % 5% 4% 3% 2% 1% % Ciprofloxacin MBEC MIC Nitrofurantoin Fig.5. Comparative MIC and MBEC of Ciprofloxacin and Nitrofurantoin for strong biofilm forming UPEC producing UPEC was highest than the moderate and weak biofilm producing UPEC (Fig.4). MBEC of ciprofloxacin was increased 95 times and of Nitrofurantoin it increased almost 17 times in case of a strong biofilm producing strain of UPEC (Fig.5). Agrawal et al [6] found that the MBEC of gentamicin in biofilm was 4-fold higher than the MBC of these antibiotics against the free-living forms of P. aeruginosa ATCC CONCLUSIONS Overall the present study demonstrated that, E. coli is the most frequent isolate of urinary tract infection and have high propensity to form biofilm and poor antibiotic sensitivity to conventional antibiotics. Biofilm production in E. coli may promote colonization and lead to increased rate of urinary tract infections. Such infections may be difficult to treat as they exhibit multidrug resistance. There is a significant correlation of biofilm with multiple drug resistance. A greater understanding of the nature of intracellular bacterial communities in chronic or recurrent urinary tract infections will aid in the development of new and more effective treatment for these problematic diseases. In this present study the MBEC of nitrofurantoin was found to be 12µg/mL, which is 17-fold greater than MIC. MBEC of

5 N.A. Ghanwate, Int J Pharm Biomed Res 212, 3(2), ciprofloxacin was found to be 95 times greater than the MIC. From above study it is concluded that the MBEC assay is indeed more predictive than the MIC assay in antibiotic susceptibility testing. Thus it is concluded that MBEC assay could provide a quick and reliable methodology to assess the susceptibility of E. coli cells growing in biofilms to antibiotics. ACKNOWLEDGEMENTS The financial support from the University Grants Commission for minor research project under which this work was carried out is highly acknowledged. REFERENCES [1] Sharma, M., Aparna Yadav, S., Chaudhary, U., Indian J Pathol Microbiol 29, 52, 294. [2] Christensen, G.D., Simpson, W.A., Younger, J.J., Baddor, L.M., Barret, F.F., Meltom, D.M., et al. J Clin Microbiol 1985, 22, [3] Matija Rijavec, Manca Müller-Premru, Breda Zakotnik, Darja Žgur- Bertok, J Med Microbiol 28, 57, [4] Suman, E., Jose, J., Varghese, S., Kotian, M.S., Indian J Med Microbiol 25, 25, 1-3. [5] Solo, S.M., Smithson, J.A., Martinez, J.P., Horcajada, J., Mensa, Vila, J., J Urol 27, 177, [6] Agarwal, G., Kapil, A., Kabra, S.K., Das, B.K., Dwivedi, S.N., The National Medical Journal of India 25, 18, [7] Stamm, W.E., Norrby, S.R., J Infect Dis 21,183, 1-4. [8] Stewart, P.S., Costerton, J.W., Lancet 21, 358, [9] Mathur, T., Singhal, S., Khan, S., Upadhyay, J.P., Fatma, T. Rattan, A., Indian J Med Microbiol 26, 24,