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Australas Med J. 2012; 5(7): 344–348.
Published online Jul 31, 2012. doi:  10.4066/AMJ.2012.1193
PMCID: PMC3412999
Antiobiotic resistance pattern of biofilm-forming uropathogens isolated from catheterised patients in Pondicherry, India
Pramodhini Subramanian, 1 Niveditha Shanmugam, 2 Umadevi Sivaraman, 3 Shailesh Kumar, 3 and Stephen Selvaraj 3
1. Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pondicherry, India.
2. MBBS Student, Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pondicherry, India.
3. Professor, Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pondicherry, India.
Corresponding Author: Dr. Pramodhini S, Assistant Professor, Department of Microbiology, Mahatma Gandhi Medical College and Research Institute, Pillaiyarkuppam, Pondicherry-607402 Email: pramo4/at/yahoo.co.in
Background
Microbial biofilms pose a public health problem for persons requiring indwelling medical devices, as micro-organisms in biofilms are difficult to treat with antimicrobial agents. Thus the present study includes biofilm formation and antibiotic resistance pattern of uropathogens in hospitalised patients with catheter associated urinary tract infections (UTI).
Method
This prospective analysis included 100 urine samples from catheterised patients with symptoms of UTI over a period of six months. Following identification, all isolates were subjected to antibiotic sensitivity using modified Kirby- Bauer disc diffusion method. Detection of biofilms was done by tube adherence method and Congo red agar method.
Results
E.coli was found to be the most frequently isolated uropathogen 70%, followed by Klebsiella pneumoniae 16%, Pseudomonas aeruginosa 4%, Acinetobacter spp 2%, coagulase negative Staphylococci 6% and Enterococci Spp 2%. In the current study 60% of strains were in vitro positive for biofilm production. Biofilm positive isolates showed 93.3%, 83.3%, 73.3% and 80% resistance to nalidixic acid, ampicillin, cephotaxime and cotrimoxazole, respectively, compared to 70%, 60%, 35%, 60% resistance showed by biofilm non-producers for the respective antibiotics. Approximately 80% of the biofilm producing strains showed multidrug resistant phenotype
Conclusion
To conclude E.coli was the most frequent isolate, of which 63% were biofilm producers. The antibiotic susceptibility pattern in the present study showed quinolones were the least active drug against uropathogens. The uropathogens showed the highest sensitivity to carbapenems. The next best alternatives were aminoglycosides. Significant correlation between biofilm production and multi-drug resistance was observed in our study.
Keywords: Biofilm, Uropathogens, Tube adherence method, Congo red agar method
Biofilms are microbial communities of surface-attached cells embedded in a self-produced extracellular polymeric matrix.1 They can cause significant problems in many areas, both in medical settings (e.g. persistent and recurrent infections, device-related infections) and in non-medical (industrial) settings (e.g. biofouling in drinking water distribution systems and food processing environments).
Biofilms have major medical significance as they decrease susceptibility to antimicrobial agents. The decreased susceptibility to microbial agents within a biofilm arises from multiple factors, including physical impairment of diffusion of antimicrobial agents, reduced bacterial growth rates, and local alterations of the microenvironment that may impair activity of the antimicrobial agent. Furthermore, the proximity of cells within a biofilm can facilitate plasmid exchange and hence enhance the spread of antimicrobial resistance.2
Biofilm is the predominant mode of growth in aquatic ecosystems and, as such, plays a central role in the pathogenesis of Catheter Associated Urinary Tract Infections (CAUTI). Most aspects of the diagnosis, treatment, and prevention of CAUTI are influenced by the tenacity of biofilm-associated uropathogens. The biofilm mode of living is a highly advantageous response of the micro-organisms to the environmental stresses of the urinary tract environment.
Thus the present study was done to study the antibiotic resistance pattern of biofilm forming uropathogens in patients with catheter associated urinary tract infections.
This prospective, analytic study was done for a period of six months. A total of 100 urine samples were collected from patients of all age groups and both the sexes with a urinary catheter for at least two days suffering from symptoms of UTIs. The study was approved by the Institutional Human Ethical Committee (IHEC) and informed written consent was taken from the patients before collection of samples. Samples were collected under complete aseptic conditions with a sterile syringe from the distal end of the urinary catheter and transferred to a sterile urine container and transported immediately to the laboratory without any delay.
Urine samples were inoculated on Blood Agar, MacConkey’s agar and Cystine lactose electrolyte deficient (CLED) medium with a calibrated loop to determine colony-forming units (CFU). All specimens with bacteriuria of >103 colony- forming units (cfu)/mL urine (which defines CAUTI) of one or two organisms were analysed to determine their causative uropathogens and their antibiotic susceptibilities.3
Identification of isolates was done by colony morphology, gram staining and standard biochemical tests. Bacterial susceptibility to antimicrobial agents was determined by the Kirby Bauer disk diffusion method on Muller-Hinton agar. Isolates were categorised as susceptible, moderately susceptible, and resistant, based upon interpretive criteria developed by the Clinical and Laboratory Standards Institute (CLSI).4 Antibiotic discs (Hi-Media) ampicillin (10μg), cefuroxime (30μg), cefotaxime (30μg), amikacin (30μg), gentamicin (10μg), cotrimoxazole (1.25/23.75μg), norfloxacin (10μg), netilmycin (30μg), nitrofurantoin (300μg), nalidixicacid (30μg), ciprofloxacin (5μg), erythromycin (15μg), oxacillin (1μg), linezolide (30μg), imipenem (10μg) and vancomycin (30 μg) were used for antimicrobial susceptibility tests.
Detection of biofilms was done by tube adherence method and Congo red agar method.
Tube adherence method by Christensen et al 5
Suspension of tested strains was incubated in the glass tubes containing Brain Heart Infusion Broth (broth) aerobically at the temperature of 35°C for a period of two days. Then the supernatant discarded and the glass tube was stained by 0.1% safranin solution, washed with distilled water three times and dried. A positive result is defined as the presence of a layer of stained material adhered to the inner wall of the tubes. The exclusive observation of a stained ring at the liquid-air interface should be considered negative
Congo red agar method by Freeman et al 6
Suspension of tested strains was inoculated onto a specially prepared solid medium - brain heart infusion broth (BHI) supplemented with 5% sucrose and Congo red. The medium was composed of BHI (37 gms/L), sucrose (50 gms/L), agar no.1 (10 gms/L) and Congo red stain (0.8 gms/L). Congo red was prepared as concentrated aqueous solution and autoclaved at 121°C for 15 minutes, separately from other medium constituents and was then added when the agar had cooled to 55°C. Plates were inoculated and incubated aerobically for 24 to 48 hours at 37°C.
A positive result was indicated by black colonies with a dry crystalline consistency. Weak slime producers usually remained pink; though occasional darkening at the centres of colonies was observed. A darkening of the colonies with the absence of a dry crystalline colonial morphology indicated an indeterminate result. The experiment was performed in triplicate and repeated three times.
Biofilm positive by any one these two methods were taken as positive for biofilm production.
Quality control
The following international reference strains were used as controls: the biofilm producers S. epidermidis ATCC 35984 (positive control) and the non-biofilm producers S. epidermidis ATCC 12228 (negative control).
Statistical analysis
The statistical analysis was done by taking percentage and simple ratios.
The number and percentage of each uropathogen isolated from catheterised urine samples are shown in Table 1.
Table 1:
Table 1:
Percentage distribution of uropathogens from catheter urine samples
Out of these 100 strains, E.coli was found to be the most frequently isolated pathogen 70%, followed by Klebsiella spp 16%, Pseudomonas aeruginosa 4%, Acinetobacter spp 2% ,coagulase negative Staphylococci 6% and Enterococci 2%.
In the current study 60% of strains were in vitro positive for biofilm production and 40% were negative for biofilm production (Table 2).
Table 2:
Table 2:
Screening of 100 urinary isolates for biofilm formation
A comparison of slime production by both methods showed that there was complete agreement between two methods in 84 of the total 100 isolates (Table 3).
Table 3:
Table 3:
Results of methods for detecting biofilm production
A total of 54 (90%) isolates were biofilm positive by Congo red agar method and 50 (83.3%) isolates were biofilm producers by Christensen's method.
The overall percentage of resistance observed among all the isolates including biofilm producers and biofilm non producers are shown in Table 4.
Table 4:
Table 4:
Antibiotic resistance pattern of biofilm and non biofilm producers
There was 80% resistance to nalidixic acid, ampicillin, cephotaxime and cotrimoxazole, respectively, compared to 70%, 60%, 35%, 60% resistance showed by biofilm non- producers for the respective antibiotics. Multidrug resistance pattern of the biofilm producing isolates is shown in Table 5.
Table 5:
Table 5:
Multiple drug resistant pattern of biofilm producers
There was a significant correlation between biofilm production and resistance to multiple antibiotics such as ampicillin, co-trimoxazole, cefotaxime, nalidixic acid and norfloxacin. Out of the total 60 strains isolated, 48 (80%) strains were multidrug resistance phenotype.
CAUTI is the most common nosocomial infection in hospitals and nursing homes, comprising >40% of all institutionally acquired infections.7-9 The relevance of biofilm to CAUTI is that a foreign body, such as an indwelling urethral catheter, connecting a normally sterile, hydrated body site to the outside world will inevitably become colonised with microorganisms. 10
The present study showed out of these 100 strains, E.coli was the most frequently isolated pathogen 70%, followed by Klebsiella spp 16%, Pseudomonas aeruginosa 4%, Acinetobacter spp 2% , coagulase negative Staphylococci 6% and Enterococci 2% which was similar to the findings of Hassin11 showing E.coli (74%) as the predominant organism followed by Klebsiella spp 17.7% & Pseudomonas spp 2.5%. Ronald in his study found that E. coli remains the predominant uropathogen (80%) in community acquired infections followed by S. saprophyticus (10-15%), Klebsiella, Enterobacter, Proteus spp.12
The present study also showed significant correlation between biofilm production and multidrug resistance, where 80% of strains producing biofilm were multidrug resistant phenotypes.21 Therapy against UTI should be guided by antimicrobial susceptibilities as increasing numbers of urinary isolates are developing resistance to commonly used antibiotics. Increasing Antimicrobial resistance of uropathogens has led to reconsideration of traditional treatment of recommendations in many areas.
Microbial biofilms have been associated with a variety of persistent infections which respond poorly to conventional antibiotic therapy. This also helps in the spread of antibiotic resistant traits in nosocomial pathogens by increasing mutation rates and by the exchange of genes which are responsible for antibiotic resistance. Antibiotic therapy against device associated biofilm organisms often fails without the removal of the infected implant. An elevated expression of the efflux pump is another mechanism for the development of antibiotic resistance in biofilm bacteria. Physiological heterogeneity is another important characteristic which is observed in biofilm bacteria. This phenomenon affects the rate of growth and metabolism of the bacteria and is reflected by interbacterial quorum signals, the accumulation of toxic products and the change in the local micro environment. These so-called persister cells are not resistant to antibiotics per se, but become resistant when associated with the biofilm.22
Conclusion
In conclusion, E.coli was the most frequent isolate, of which 63% were biofilm producers. The antibiotic susceptibility pattern in the present study showed quinolones were the least active drug and the uropathogens showed the highest sensitivity to carbapenems and aminoglycosides. Significant correlation between biofilm production and multidrug resistance was observed in our study. As biofilm production was detected in many of our isolates, it is necessary to establish standard guidelines on the care of catheter for all units in the hospital with a view to preventing nosocomial infections associated with the device in patients.
Footnotes
PEER REVIEW
Not commissioned. Externally peer reviewed.
CONFLICTS OF INTEREST
The authors declare that they have no competing interests
FUNDING
Nil
Please cite this paper as: Pramodhini S, Niveditha S, Umadevi S, Kumar S, Stephen S. Antiobiotic resistance pattern of biofilm-forming uropathogens isolated from catheterised patients in Pondicherry, India. AMJ 2012, 5, 7, 344-348. http//dx.doi.org/10.4066/AMJ.2012.1193.
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