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ORIGINAL ARTICLE
Year : 2015  |  Volume : 4  |  Issue : 4  |  Page : 247-252

Analysis of biofilm formation and antibiotic susceptibility pattern of uropathogens in patients admitted in a tertiary care hospital in India


Department of Microbiology, Lokmanya Tilak Municipal Medical College and General Hospital, Mumbai, Maharashtra, India

Date of Web Publication20-Oct-2015

Correspondence Address:
Ruchi A Tayal
Room No. 405, Department of Microbiology, Lokmanya Tilak Municipal Medical College and General Hospital, Mumbai - 400 022, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-344X.167648

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  Abstract 

Background: Microorganisms attach to surfaces and produce polysaccharides resulting in the formation of biofilms and providing an ideal niche for the exchange of genetic material leading to the emergence of drug-resistant pathogens. Biofilms can develop on anatomical surfaces and implants producing chronic and intractable infections. Aims: Detection of biofilm formation and comparison of antibiotic resistance between biofilm producers and nonproducers. Study Design: Prospective study in which urine specimens from adult patients with urinary tract infection (UTI) during the period of the study were analyzed (1 year). Materials and Methods: Mid-stream clean catch urine from noncatheterized and urine aspirated from in-dwelling urinary catheter in catheterized patients were taken for microbiological processing. Wet mounts, Gram-staining, and urine culture were done. Biofilm formation was detected by tissue culture plate method (TCPM). Statistical Analysis: Chi-square test and mid "P" test were used to analyze the data. A value ofP<0.05 was taken as significant. Results: Gram-negative organisms predominated (89%). Biofilm production was detected in 27% isolates. Maximum biofilm production was seen in Enterococcus spp. (71%), followed by Escherichia coli (26%). Biofilm-producing isolates demonstrated higher antibiotic resistance. All the biofilm-producing Enterococcus spp. showed high-level aminoglycoside resistance. The biofilm-producing isolates of Pseudomonas aeruginosa and Klebsiella pneumoniae demonstrated multi-drug resistance. Conclusions: TCPM is an economical phenotypic method which can be used routinely to detect biofilm formation. Biofilms contribute to an increased resistance to antibiotics used for the treatment of UTIs. Therefore, detection of biofilms is recommended for all patients presenting with chronic or recurrent disease.

Keywords: Biofilm, drug resistance, tissue culture plate, urinary tract infection


How to cite this article:
Tayal RA, Baveja SM, De AS. Analysis of biofilm formation and antibiotic susceptibility pattern of uropathogens in patients admitted in a tertiary care hospital in India. Int J Health Allied Sci 2015;4:247-52

How to cite this URL:
Tayal RA, Baveja SM, De AS. Analysis of biofilm formation and antibiotic susceptibility pattern of uropathogens in patients admitted in a tertiary care hospital in India. Int J Health Allied Sci [serial online] 2015 [cited 2019 Nov 19];4:247-52. Available from: http://www.ijhas.in/text.asp?2015/4/4/247/167648


  Introduction Top


Biofilms are an assembly of microbial cells which can be formed by single or a mixture of bacterial species that are irreversibly associated with a surface and enclosed in a matrix of polysaccharide materials that allow the growth and survival in hostile environments.[1],[2] Biofilms confer advantages to the biofilm-forming bacteria such as protection from antimicrobial agents, exchange of nutrients, metabolites, and/or genetic exchange from proximity to other organisms.[3] Limited penetration of antibiotics into the biofilm and slow rate of cell multiplication of organisms in the biofilm may contribute to the development of chronic infections.[4],[5]

Urinary tract infection (UTI) is considered as the most common bacterial infection worldwide.[6] Although biofilms do form on the anatomical surfaces within the urinary tract, entry into the bladder is met with potent innate defenses, including neutrophil influx and epithelial exfoliation which prevent colonization of the surface by the invading microbes. In contrast, catheter surfaces have no inherent defense mechanisms. Presence of biofilms on the intra-luminal surfaces of in-dwelling catheters and urinary stents is a major cause of concern due to increased duration of hospitalization and increased morbidity for the patient.[7] The most common species present in the mixed population biofilms in UTI are Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis.

Biofilm-producing bacteria, which colonize the urinary tract and in-dwelling catheters, show higher resistance to standard antibiotics used for the treatment of UTIs. This leads to the development of recalcitrant and recurrent episodes of UTI in the affected population. Most studies conducted previously focus on either biofilm production by a single microbe causing UTI or biofilm formation only in catheterized patients. This study was conducted to study the entire spectrum of bacteria causing UTI in catheterized and noncatheterized specimens collected from patients admitted in a tertiary care hospital, detect biofilm formation in these isolates, and compare the pattern of antibiotic resistance between biofilm-producing organisms and the biofilm nonproducers.


  Materials and Methods Top


  • Study design: Prospective study
  • Place of study: Tertiary Care Hospital, Mumbai, India
  • Duration of study: One year (2012–2013)
  • Sample size: 200
  • Inclusion criteria: All adult patients in the age group of 19–60 years admitted in the hospital during the period of study with complaints of UTI were included in the study
  • Exclusion criteria: Patients with underlying anomaly of the genitourinary tract were excluded from the study
  • Ethics clearance: The study was carried out after getting approval from the Institutional Ethics Committee
  • Consent: Informed consent from all the patients included in the study was taken prior to initiation of the study.


Methodology

Sample collection and microbiological processing

Mid-stream, clean catch urine samples from noncatheterized patients were collected after proper urethral toilet. Urine was aspirated aseptically from the in-dwelling urinary catheter using needle and syringe in catheterized patients.

Presence of >5 white blood cells per high-power field in wet mounts of centrifuged urine was taken as an indication of significant pyuria. Presence of even a single bacterium per high power field in Gram-stain of un-centrifuged urine gave a presumptive estimate of 105 CFU/ml of urine which was considered a significant bacteriuria. Culture is considered as the gold standard for diagnosis of UTI. Urine colony count was done semi-quantitatively.[8] The pathogens were identified using routine biochemical tests.

Detection of biofilm formation

Detection of biofilm production was done using the tissue culture plate method (TCPM). Staphylococcus epidermidis ATCC 31484 and S. epidermidis ATCC 12228 were used as standard positive and negative control strains, respectively, for biofilm production.

A loopful of isolated test organisms from overnight cultures were inoculated in 10 ml of Trypticase soy broth with 1% glucose and incubated at 37°C for 24 h. Individual wells of sterile 96 well-flat bottom polystyrene tissue culture treated plate (Sigma-Aldrich, USA) were filled with 200 µl of the bacterial suspension corresponding to 0.5 McFarland after further dilution of 1:100 with fresh medium along with control organisms. Only broth served as a control to check sterility and nonspecific binding of media. The plates were inoculated at 37°C for 24 h. After incubation, contents of each well were removed by gentle tapping and wells were washed 3 times with 300 µl of sterile saline. The remaining attached bacteria were heat-fixed by exposing them to hot air at 60°C for 60 min. Then, 150 µl crystal violet (2%) stain was added to each well. After 15 min, the excess stain was rinsed off by decantation, and the plate was washed. One hundred and fifty microliter of 95% ethanol was added to each well, and after 30 min, the optical densities (ODs) of stained adherent bacterial films were read using a microtiter plate reader at 600 nm. The OD values were calculated for all the tested strains and negative controls, the OD cut-off value (ODc) was established. All the samples were tested in triplicate.

It is defined as three standard deviations (SDs) above the mean OD of the negative control: ODc = average OD of negative control + (3 × SD of negative control). Final OD value of a tested strain was expressed as average OD value of the strain reduced by ODc value (OD = average OD of a strain − ODc); ODc value was calculated for each microtiter plate separately. When a negative value was obtained, it was presented as zero, while any positive value indicated biofilm production. For interpretation of the results, strains were divided into the following categories:[9]

  • Nonbiofilm producer (0) OD ≤ ODc,
  • Weak biofilm producer (+ or 1) = ODc < OD ≤ 2× ODc,
  • Moderate biofilm producer (++ or 2) = 2 × ODc < OD ≤ 4× ODc,
  • Strong biofilm producer (+++or 3), 4 × ODc < OD.


Antibiotic susceptibility testing

Bacterial susceptibility to anti-microbial agents was determined by Kirby–Bauer disk diffusion method on Mueller-Hinton agar as per CLSI 2012 guidelines. Antibiotic discs were procured from HiMedia Laboratories Pvt. Ltd., India, and were used after quality control testing. Staphylococcus aureus ATCC 25923, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 were used as controls. Anti-microbial susceptibility was done using the following antibiotic discs.[10]

Penicillin (10 units), nitrofurantoin (300 µg), norfloxacin (10 µg), erythromycin (15 μg), co-trimoxazole (1.25/23.75 μg), gentamicin (10 μg), clindamycin (2 μg), nalidixic acid (30 µg) for S. aureus; amoxicillin/clavulanic acid (20/10 µg), amikacin (30 µg), nalidixic acid (30 µg), norfloxacin (10 µg), nitrofurantoin (300 µg), co-trimoxazole (1.25/23.75 µg), cefotaxime (30 µg), piperacillin (100 µg), ceftazidime (30 µg), ciprofloxacin (5 µg), imipenem (30 µg), netilmicin (30 µg), cefepime (30 µg), piperacillin-tazobactam (100/10 µg) for Gram-negative isolates; and Penicillin (10 units), ampicillin (10 µg), vancomycin (30 µg), linezolid (30 µg), tetracycline (30 µg), nitrofurantoin (300 µg), norfloxacin (10 µg), nalidixic acid (30 µg), high level gentamycin (120 µg), and streptomycin (300 µg) for Enterococcus spp.

Statistical analysis

The comparative statistical analysis for all methods was done by 2 × 2 tables where applicable. Parameters such as sensitivity and specificity were determined from these tables. Chi-square test, mid "P" test, and Fischer's exact test were used to analyze the data wherever applicable. P < 0.05 was taken as significant.


  Results Top


A total of 200 urine specimens were analyzed for biofilm formation and antibiotic susceptibility. Of these, 30.5% (61) urine specimens were catheterized samples and 69.5% (139) were mid-stream urine specimens. 64% (128) of the participants in the study were females and 36% (72) were males. Maximum patients belonged to the age group between 51 and 60 years (35%) followed by the age group between 20 and 30 years (25%). 92.5% of the patients presented with signs and symptoms suggestive of UTIs, while 7.5% had no symptomatology indicative of UTI. A history of UTI, catheterization, and a prolonged duration of catheterization (≥7 days) correlated significantly with increased propensity of microorganisms to form biofilms in the urinary tract [Table 1].
Table 1: Risk factors and their correlation with growth (n = 200)

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Growth was seen in 66.5% samples (137/200). Gram-negative organisms were the predominant isolates from the urine specimens accounting for 89% (122), while Gram-positive organisms were 11% (15). E. coli was isolated from more than half the urine specimens (54.01%) followed by Klebsiella pneumoniae (11.66%) and Enterococcus spp. (10.22%). There was only one isolate of methicillin-sensitive Staphylococcus aureus (MSSA) [Table 2].{Table 2}

Biofilm formation was seen in 37 (27%) isolates. The maximum biofilm production was seen in Enterococcus spp. (71%). 26% of E. coli isolates showed biofilm formation followed by 18% of K. pneumoniae isolates. There were no biofilm-producing isolates of Acinetobacter spp.and P. mirabilis found in the present study [Table 2].

Biofilm-producing urinary isolates demonstrated a slightly higher antibiotic resistance pattern as compared to their nonbiofilm-producing counterparts. Biofilm-producing and nonbiofilm-producing E. coli isolates did not demonstrate a significant difference in the antibiotic susceptibility patterns. All biofilm-producing isolates of K. pneumoniae showed poor susceptibility to the tested antibiotics. The organism showed the highest sensitivity to amikacin at 65% followed by norfloxacin and nitrofurantoin at 33% each. All isolates were resistant to the remaining tested antibiotics. Nonbiofilm producers showed better susceptibility to both amikacin (77%) and nitrofurantoin (46%). Some isolates were also found to be susceptible to cefotaxime and co-trimoxazole (8% and 15%, respectively). Biofilm-producing isolate of P. aeruginosa demonstrated multi-drug resistance and was susceptible only to imipenem and piperacillin-tazobactam combination as compared to the nonbiofilm producers which showed 78% susceptibility to amikacin and 56% susceptibility to norfloxacin and piperacillin. However, only one biofilm-producing isolate of P. aeruginosa was found in the present study. This number is too small (11%) to draw any significant conclusions from the above result. There was no significant difference in the antibiotic patterns for both groups of Enterobacter spp.isolates. Only one isolate of Morganella morganii was found in the present study and it showed biofilm production and susceptibility to amikacin, nitrofurantoin, and co-trimoxazole. Biofilm-producing isolate of Klebsiella oxytoca was multi-drug resistant and showed susceptibility to only imipenem [Table 3]. Biofilm-producing isolates of Enterococcus spp. showed 100% susceptibility to vancomycin and linezolid. However, susceptibility to nitrofurantoin was only 50% as compared to 75% in the nonbiofilm producers. There was absolute resistance to norfloxacin and tetracycline among the biofilm producers as compared to 50% and 25% susceptibility, respectively, seen in the nonbiofilm producers. All biofilm producers showed high-level aminoglycoside resistance as compared to only 25% of the nonbiofilm-producing isolates. MSSA was isolated from one urine specimen. It showed biofilm production and was susceptible to nitrofurantoin and gentamycin. It did not show inducible clindamycin resistance [Table 4].
Table 3: Antibiotic susceptibility patterns for Gram-negative isolates

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Table 4: Antibiotic susceptibility patterns for Gram-positive isolates

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  Discussion Top


History of UTI, catheterization, and a prolonged duration of catheterization (≥7 days) increased the propensity of microorganisms to form biofilms in the urinary tract. Soto et al. in 2006 suggested that a significant history of UTI is a major indicator for the recurrence of UTI due to biofilm formation.[11] Trautner et al. have demonstrated a positive correlation among catheterization, duration of catheterization, and biofilm formation [Table 1].[12],[13]

Gram-negative organisms were the predominant isolates from the urine specimens accounting for 122 (89%) of the total growth, while Gram-positive isolates were 15 (11%). E. coli was isolated from 54.01% specimens followed by K. pneumoniae (11.68%) and Enterococcus spp. (10.22%). These findings correlate well with the findings reported in studies conducted by Behzadi et al. in 2010, Jain et al. in 2011, Subramanian et al. in 2012, and Noor et al. in 2013 [Table 2].[14],[15],[16],[17]

Biofilm production on in-dwelling urinary catheters has been reported in a large number of studies conducted all over the world and is a major cause of nosocomial and recalcitrant UTI. TCPM was selected because of its ease of performance and economy. Moreover, this method is considered the gold standard phenotypic test for the detection of biofilms owing to its higher sensitivity. One hundred and thirty-seven clinical isolates from urine were tested for their ability to form biofilms using this in vitro method. A total of 37 (27%) isolates showed biofilm formation. The maximum biofilm production was seen in Enterococcus spp.where ten out of the 14 isolates (71.43%) showed biofilm production. This number is greater than that reported by Ira et al. in 2013[18] who found 53% of Enterococcus spp.isolates to be biofilm producers. 25.68% isolates of E. coli showed biofilm production. This number is significantly lower than that reported by Sevanan et al. in 2011 (90%)[19] and Subramanian et al. in 2012 (60%).[16],[20] No biofilm-producing isolates of Acinetobacter spp. and P. mirabilis were found in the present study [Table 2].

Biofilm producers demonstrated a higher degree of antibiotic resistance than the nonbiofilm-producing isolates. E. coli isolates did not demonstrate a significant difference in the antibiotic susceptibility patterns observed in biofilm-producing and nonbiofilm-producing isolates. Both groups showed about 85% susceptibility to amikacin and nitrofurantoin. However, these findings are significantly higher than the 30% susceptibility to amikacin detected by Sevanan et al.[19] All biofilm-producing isolates of K. pneumoniae and P. aeruginosa showed poor susceptibility to the tested antibiotics. There was no significant difference in the antibiotic patterns for both groups of Enterobacter spp.isolates.

The present study showed that the most effective antibiotics against biofilm-producing Gram-negative isolates from UTIs were found to be imipenem and amikacin and for Gram-positive isolates was vancomycin. This is in agreement with Abdallah et al. who, in 2011, reported maximum efficacy of imipenem and amikacin for Gram-negative isolates and vancomycin and ciprofloxacin for Gram-positive isolates.[9] Savas et al. in 2006 also found that the most effective antibiotics against Gram-negative bacteria were imipenem and meropenem.[21] Similar results were reported by EL-Banoby et al. stated in 2007 that aminoglycosides were the most common antibiotics to which the organisms were sensitive for nosocomial UTI (46.2%) followed by monobactam (34.6%), quinolones (30.8%), and vancomycin (3.8%).[22]


  Conclusions Top


Biofilms are a major cause of recurrent and recalcitrant UTI, leading to increased morbidity in the patient, increased duration of hospital stay, and increased economic burden and drain on resources. Biofilm-associated antibiotic resistance is a common phenomenon in chronic and nosocomially acquired infections. Detection of biofilms is, therefore, recommended for all patients presenting with chronic or recurrent UTI before the institution of empirical antibiotic therapy. In the present study also, a greater degree of resistance has been detected in the biofilm-producing isolates. However, a larger study is needed to understand the true impact of biofilms on antibiotic susceptibility.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Behzadi P, Behzadi E, Yazdanbod H, Aghapour R, Akbari Cheshmeh M, Salehian Omran D. A survey on urinary tract infections associated with the three most common uropathogenic bacteria. Maedica (Buchar) 2010;5:111-5.  Back to cited text no. 14
    
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    Tables

  [Table 1], [Table 1], [Table 3], [Table 4]


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