Purpose: Bacterial biofilm develop on the surfaces of urinary catheter and proceed to cause full blown bacterial infections and sepsis. Urinary catheters, infection rates increase with the duration of catheterization at rates of per day with virtually all of those who undergo long-term catheterization becoming infected. Also antibiotics results in the adaptation and development of resistance leading to treatment failure, prolonged hospitalization, increased costs of care, and increased mortality. Methods: In the present study total 200 used urinary catheters were studied from the different hospitals of Amravati city in 2015-2016. Different bacterial uropathogens were isolated by conventional method and biofilm formation was studied by tissue culture plate (TCP). Antibiotic sensitivity was performed by disc diffusion method. Minimum inhibitory concentration (MIC) and Minimum biofilm eradicating concentration (MBEC) of triclosan was determined by TCP. Results: Out of total samples 93% are contaminated. Around 59% urinary catheters contain mixed consortia. Pseudomonas aeruginosa was found to be the strong biofilm forming and multidrug resistant organism. The most effective drug over seven bacteria isolates were chloramphenicol. Triclosan was used to test against the strong and moderate biofilm forming isolates the MIC of triclosan ranged between 1.5 and 1000 μg/ml and MBEC was between 800 and 3200 μg/ml Conclusions: From the study it was concluded that female are more prone to be infected with catheter associated infection. Pseudomonas aeruginosa was found to be deadly caused of infection, as it is highly resistant to antibiotics. Also triclosan showed effective result on the bacterial uropathogens.
Urinary tract infection (UTI) is one of the most common bacterial infections in humans, with an estimated annual incidence rate of nearly 13% in women [
The risk of UTI can be related to the length of time the catheter in place. Most patients catheterized for a week or less would escape from infection, but for the many elderly and disabled patients who are catheterized for several months or years, bacteriuria is inevitable [
Patients with chronic indwelling catheters may have >105 viable bacteria per milliliter of urine [
1) Introduction at the time of catheter insertion,
2) Migration of bacteria present in the urethra around the catheter,
3) Ascent of bacteria through the lumen of the catheter from a contaminated drainage system.
The development and increased use of closed methods of catheter drainage have contributed to reduction of the latter problem and markedly improved the management of UTI. However, a closed catheter system merely postpones the inevitable; a recent study by [
The catheter helps to connect the heavily colonized perineum with the sterile bladder, thus providing a route for bacterial entry into the bladder. For example P. mirabilis, Providenncia stuartii, Morganella morganii and K. pneumonia produce urease and form a unique type of crystalline biofilm on catheters [
The biofilm is a complex unit. It can be up to 500 cells deep, but may only be composed of a small percentage of bacterial cells with the remainder being comprised of extrapolymeric substances. Within the urinary tract, the matrix may also contain large quantities of encrusting minerals [
Triclosan is a broad-spectrum hydrophobic antimicrobial agent. Because of its favorable safety profile, Triclosan has been used for the past 2 decades in several dermatological preparations and oral hygiene products. Results of toxicology studies show that triclosan and its metabolites are well tolerated by a variety of species, including human beings [
Triclosan is a synthetic, broad-spectrum antimicrobial agent. It has been available in the market for about four decades. Medical uses of it include: eradication of methicillin-resistant Staphylococcus aureus (MRSA) in patients by reducing skin colonization [
Triclosan acts by blocking the active site of the enoyl-acyl carrier protein reductase enzyme (ENR), which is an essential enzyme in fatty acid synthesis in bacteria [
The present study was carried out to know the incidence of catheter associated nosocomial infections in the Hospitals of Amravati, Maharashtra State, India in 2015-2016. The study focuses on isolation and identification of nosocomial bacterial uropathogens from urinary catheters and detection of biofilm formation of the isolated uropathogens by Tissue culture plate (TCP) method. Furthermore, the antibiotic susceptibility of the uropathogens was studied against the different class of antibiotics and effect of triclosan on strong and moderate biofilm forming isolates by three different methods disc diffusion, MIC and MBEC by TCP.
A retrospective analysis of culture results of used urinary catheter was performed at Department of Microbiology at Sant Gadge Baba Amravati University. The sex, age and duration of catheterization of patients, the organism isolated, biofilm forming ability, antimicrobial susceptibility profiles and effect of triclosan were studied. All experiment assays were performed independently and in triplicate.
A total of 200 used urinary catheters from different hospitals were collected from Amravati city in sterilized HiDispoTM bag [HiMedia Laboratories Pvt. Ltd.] in aseptic condition. The urinary catheter was immediately brought to the laboratory and was processed in aseptic conditions. The collected urinary catheters were washed with sterile distilled water and flushed with saline solution than swabbed with 70% alcohol swab from outer side of the catheter to remove planktonic bacteria. and were then sectioned into five appropriate parts A, B, C, D, E from tip and each part were suspended into test tubes containing sterile Ringer’s solution (10ml) separately. Sonication for 5 minutes at 35 kHz in water bath and vortex mixing for 2 minutes were performed to remove and disrupt the colonizing biofilm.
Resulting cell suspensions were cultured by taking a loopful of suspension on to Hi-chrome UTI agar plates [
Antibiotic susceptibility testing against isolates was performed according to Kirby Bauer’s method and interpreted as per Clinical and Laboratory Standards Institute (CLSI) recommendations (CLSI, 2005). The antibiotic discs (Himedia, India) used were Amikacin (30 μg), Amoxicillin (30 μg), Cefaclor (30 μg), Cefixime (30 μg), Ceftriaxone (30 μg), Chloramphenicol (30 μg), Ciprofloxacin (5 μg), Gatifloxacin (5 μg), Nalidixic acid (30 μg), Nitrofurantoin (300 μg), Piperacillin (100 μg) and Vancomycin (30 μg). Antibiotics were stored as per the manufacturer’s recommendations. Each experiment was performed in triplicates.
Organisms isolated from fresh agar plates were inoculated in 10 ml of trypticase soy broth with 1% glucose. Broths were incubated at 37˚C for 24 h. The cultures were then diluted 1:100 with fresh medium. Individual wells of sterile 96 well flat bottom polystyrene tissue culture treated plates were filled with 200 μl of the diluted cultures. The control organisms were also incubated, diluted and added to tissue culture plate. Negative control wells contained inoculated sterile broth. The plates were incubated at 37˚C for 24 h after incubation; contents of each well were removed by gentle tapping. The wells were washed with 0.2 ml of phosphate buffer saline (pH 7.2) four times. This removed free floating bacteria. Biofilm formed by bacteria adherent to the wells were fixed by 2% sodium acetate and stained by crystal violet (0.1%). Excess stain was removed by using deionised water and plates were kept for drying. Optical density (OD) of stained adherent biofilm was obtained by using micro ELISA auto reader at wavelength 570 nm. Experiment was performed in triplicate [
Mean OD values | Adherence | Biofilm formation |
---|---|---|
<0.120 | Non | Non/weak |
0.120 - 0.240 | Moderately | Moderate |
>0.240 | Strong | High |
Disk diffusion AST (Antimicrobial susceptibility test) is also referred to as the Kirby-Bauer method, the top portion of three to five, 18 to 24 h old, well isolated, colonies transferred to a tube containing broth medium. The broth culture is incubated at 35˚C until the turbidity of the culture meets the turbidity of a 0.5 McFarland turbidity standard. Inoculation of the MH agar plate is accomplished as described by the CLSI. In essence, it involves swabbing the entire surface of the plate three times, in three different directions, to ensure that the organism grows over the entire surface of the plate. Small filter paper disks, each containing a triclosan an antimicrobial agent, are then placed on the agar surface, making sure that the entire lower surface of each disk is in contact with the agar surface. The plate is then inverted and incubated for 16 to 18 h at 35˚C in an incubator. During the incubation period, the drug diffuses into the agar After 16 to 18 h of incubation, the plate is removed from the incubator, and the diameter of each zone of no growth is carefully measured in millimeters.
The Triclosan to be tested is added to Muller Hinton (MH) agar, which is then placed in dilution plates and diluted with varying levels of water. After this, the pathogen to be tested is added to each plate, plus a control plate that does not receive Triclosan. The dilution plates are then incubated at 37˚C. The plates are then incubated for 24 h, although incubation time may be less for bacteria populations that divide quickly. After incubation, the plates are examined to determine if bacterial expansion has occurred. The lowest concentration of Triclosan that stopped the spread of the bacteria is considered to be the minimum inhibitory concentration of that bacterium.
The MBEC (Minimum biofilm eradicating concentration) Assay can be used to determine the efficacy of an antimicrobial agent against biofilm. The MBEC assay uses a 96-well plate with lid that allow for the adherence and growth of biofilm. MBEC assay is a very time efficient and accurate method of testing antimicrobial agent efficacy against biofilm. In this method take a sterile 96 well Tissue culture plate. In which add fresh trypticase soya broth (100 μL) and blank and control well also set up. 1 µL bacterial suspension is added into each well except blank and control therefore the mixture is diluted as 1:100. Incubated at 37˚C for 48 h. After incubation biofilm is formed in tissue culture plate. Then wash the biofilm plate with phosphate buffer saline for four times. Then add different concentration of Triclosan into each well. Incubated at 37˚C for 48 h. Again the plate is wash with phosphate buffer saline solution. Add 1% peptone water in each well and again incubated at 37˚C for 24 h. Therefore the resistant bacteria can regrow in tissue culture plate. Presence of viable bacteria is determined by streak plate method on Muller-Hinton agar plates. Lowest concentration of Triclosan at which bacteria failed to regrow within a biofilm considered as MBEC.
A total of 200 used urinary catheters were collected during the period of study. The urinary catheters were collected from the patients at different hospitals of Amravati region mentioned in
It was observed that the female catheterized patients are more prone to infection as the catheter get contaminated with in less period of time as compare to male patients though the number of male catheter samples was more but the more frequently contamination were observed in the female catheterized patients. The maximum contaminations were observed in urinary catheters of males and females with duration of 6 - 10 days and 26 - 30 (
In present study total 368 bacterial uropathogens were isolated from 200 urinary catheters. Out of which strains of different bacterial species were identified by using conventional methods and through VITEK2 automated identification.
TCP the standard method is used to detect biofilm formation of the isolates, Pseudomonas aeruginosa (41.84%) Enterococcus faecalis (19.02%) and Staphylococcus aureus (16.84%) were strong biofilm forming. No species of Escherichia coli were found to be of strong biofilm forming explained in
Bacterial species | Number (%) of catheters colonized by each species | ||
---|---|---|---|
All catheter bacterial species | Mixed-bacterial species in (118 (59%) urinary catheters) | Single-bacterial species in (82 (41%) urinary catheters) | |
Pseudomonas aeruginosa | 154 (77) | 94 (79.6) | 60 (73.1) |
Klebsiella pneumonia | 28 (14) | 28 (23.7) | 0 (0.0) |
Escherichia coli | 32 (16) | 28 (23.7) | 4 (4.8) |
Stenotrophomonas maltophilia | 10 (5) | 9 (7.6) | 1 (1.2) |
Proteus vulgaris | 6 (3) | 6 (5.0) | 0 (0.0) |
Dermacoccus nishinomiyanensis | 6 (3) | 5 (4.2) | 1 (1.2) |
Enterococcus faecalis | 70 (35) | 58 (49.1) | 12 (14.6) |
Staphylococcus aureus | 62 (31) | 57 (48.0) | 5 (6.0) |
the similar manner, major bacterial colony species were same to the urinary catheter colonized by single bacterial species but the least species was observed that of stenotrophomonas maltophilia and Dermacoccus nishmaneyasis.
The predominant growth of single bacteria was seen in 41% used urinary catheters. More than 60% of the isolates were sensitive to amikacin around 45% of the isolates were sensitive to ciprofloxacin and Gatifloxacin (
Escherichia coli, P aeruginosa, Enterococcus fecalis, and Staphylococcus aureus susceptibile to ciprofloxacin, amikacin, Gatifloxacin ceftriaxone followed by Gatifloxacin which showed high sensitivity against Klebsiella further Stenotrophomonas maltophilia was found to be equally susceptible to only four antibiotics i.e. amikacin, chloramphenicol, gatifloxacin, Nitrofurantoin. Proteus vulgaris completely sensitive to all the antibiotics equally and no resistant were observed. Dermacoccus nishmaneyasi rare species were highly susceptible to chloramphenicol, ciprofloxacin, gatifloxacin, nalidixic acid and resistant to all other antibiotics. Enterococcus faecalis resistant to Cefixime (
As per our primary study Triclosan has shown lots of promising effects over eradication and prevention of biofilm in urinary catheter. The MIC of triclosan
Number (%) susceptible and resistance of each species | ||||||||
---|---|---|---|---|---|---|---|---|
Antimicrobial agent | Pseudomonas aeruginosa (n = 154) | Klebsiella pneumonia (n = 28) | Escherichia coli (n = 32) | Stenotrophomonas maltophilia (n = 10) | ||||
Susceptible | Resistant | Susceptible | Resistant | Susceptible | Resistant | Susceptible | Resistant | |
Amikacin | 84 (54.5) | 70 (45.5) | 18 (64.2) | 10 (35.7) | 24 (75) | 8 (25) | 4 (40) | 6 (60) |
Amoxycillin | 0 (0.0) | 154 (100) | 0 (0.0) | 28 (100) | 0 (0.0) | 32 (100) | 0 (0.0) | 10 (100) |
Cefaclor | 22 (14.2) | 132 (85.7) | 6 (21.4) | 22 (78.5) | 0 (0.0) | 32 (100) | 0 (0.0) | 10 (100) |
Cefixime | 16 (10.3) | 138 (89.6) | 4 (14.2) | 24 (85.7) | 0 (0.0) | 32 (100) | 0 (0.0) | 10 (100) |
Ceftriaxone | 36 (23.3) | 118 (76.6) | 4 (14.2) | 24 (85.7) | 20 (62.5) | 12 (37.5) | 0 (0.0) | 10 (100) |
Chloramphenicol | 30 (19.4) | 126 (81.8) | 24 (85.7) | 4 (14.2) | 14 (43.7) | 18 (56.2) | 4 (40) | 6 (60) |
Ciprofloxacin | 56 (36.3) | 98 (63.6) | 4 (14.2) | 24 (85.7) | 26 (81.2) | 6 (18.7) | 0 (0.0) | 10 (100) |
Gatifloxacin | 50 (32.46) | 104 (67.5) | 28 (100) | 0 (0.0) | 24 (75) | 8 (25) | 4 (40) | 6 (60) |
Nalidixic acid | 6 (3.8) | 148 (96.1) | 4 (14.2) | 24 (85.7) | 6 (18.7) | 26 (81.2) | 0 (0.0) | 10 (100) |
Nitrofurantoin | 4 (2.5) | 150 (97.4) | 14 (50) | 14 (50) | 24 (75) | 8 (25) | 4 (40) | 6 (60) |
Piperacillin | 22 (14.2) | 132 (85.7) | 4 (14.2) | 24 (85.7) | 0 (0.0) | 32 (100) | 0 (0.0) | 10 (100) |
Number (%) susceptible and resistance of each species | ||||||||
---|---|---|---|---|---|---|---|---|
Antimicrobial agent | Proteus vulgaris (n = 6) | Dermacoccus nishinomiyanensis (n = 6) | Enterococcus faecalis (n = 70) | Staphylococcus aureus (n = 62) | ||||
Susceptible | Resistant | Susceptible | Resistant | Susceptible | Resistant | Susceptible | Resistant | |
Amikacin | 6 (100) | 0 (0.0) | 0 (0.0) | 6 (100) | 48 (68.5) | 22 (31.4) | 40 (64.5) | 22 (35.4) |
Amoxycillin | 6 (100) | 0 (0.0) | 0 (0.0) | 6 (100) | 18 (25.7) | 52 (74.2) | 12 (19.3) | 50 (80.6) |
Cefaclor | 6 (100) | 0 (0.0) | 0 (0.0) | 6 (100) | 20 (28.5) | 50 (71.4) | 0 (0.0) | 62 (100) |
Cefixime | 6 (100) | 0 (0.0) | 0 (0.0) | 6 (100) | 0 (0.0) | 70 (100) | 6 (9.6) | 56 (90.3) |
Ceftriaxone | 6 (100) | 0 (0.0) | 0 (0.0) | 6 (100) | 31 (44.2) | 39 (55.7) | 27 (43.5) | 35 (56.4) |
Chloramphenicol | 6 (100) | 0 (0.0) | 6 (100) | 0 (0.0) | 40 (57.1) | 30 (42.8) | 22 (35.4) | 40 (64.5) |
Ciprofloxacin | 6 (100) | 0 (0.0) | 6 (100) | 0 (0.0) | 34 (48.5) | 36 (51.4) | 35 (56.4) | 27 (43.5) |
Gatifloxacin | 6 (100) | 0 (0.0) | 6 (100) | 0 (0.0) | 24 (34.2) | 46 (65.7) | 28 (45.1) | 34 (54.8) |
Nalidixic acid | 6 (100) | 0 (0.0) | 6 (100) | 0 (0.0) | 32 (45.7) | 38 (54.2) | 28 (45.1) | 34 (54.8) |
Nitrofurantoin | 6 (100) | 0 (0.0) | 0 (0.0) | 6 (100) | 24 (34.2) | 46 (65.7) | 24 (38.7) | 38 (61.2) |
Piperacillin | 6 (100) | 0 (0.0) | 0 (0.0) | 6 (100) | 26 (37.1) | 44 (62.8) | 12 (19.3) | 50 (80.6) |
ranged between 1.5 to 1000 /ml and MBEC was between 800 to 3200 µ/ml i.e. BEC values were 3 to 1800 folds than MIC values. This in vitro antimicrobial activity of triclosan is mentioned in
Present study revealed that, indwelling urinary catheter gets contaminated by various nosocomial pathogens during the course of catheterization. Contamination also depends on the gender of patient. Female catheter are more prone to get contaminated around 95.83% female patients urinary catheter was contaminated. Duration of catheterization also influences of contamination. Maximum catheters are of 26 - 30 days contamination of urinary catheter is also related to biofilm forming ability of the contaminants. If the bacteria are biofilm producer, there is more risk of its persistence and subsequent infection. Thus it was concluded that 93% of catheter gets contaminated during course of catheterization. 52.7% contaminants are biofilm forming
Similarly, Abdallah and Balasubramanian, [
Also Taiwo and Aderounmu, found that Klebsiella spp were the commonest pathogen isolated with 46 (36.6%), followed by Pseudomonas spp 34 (27.0%), Escherichia coli 26 (20.6%), Staphylococcus aureus 12 (9.5%), Proteus mirabilis 4 (3.2%), Candida albicans 4 (3.2%) and coagulase negative staphylococci 2 (1.6%). But in present study Pseudomonas aeruginosa was the commonest contaminant organism were found and two rare species of bacteria were also obtained Stenotrophomonas maltophilia and Dermacoccus nishinomiyaensis. Out of total 59% urinary catheters contains mixed consortia of bacteria in the present investigation.
In present study it was found that susceptibility profile shows multiple drug resistance among most of the biofilm forming isolates. Pseudomonas aeruginosa was found to be the most multidrug resistant organism. The most effective drug which were effective over seven bacterial isolates were chloramphenicol and gatifloxacin while amikacin, ciprofloxacin, nalidixic acid and nitofurantoin were effective over 6 isolated microorganisms which was followed by Ceftriaxone and Piperacillin which were found to be effective over five bacterial isolates. The least effective drug was amoxycillin, cefaclor and cefixime (
Reiter and Rajamoha, found that frequency of UTI was greater in women as compared to men as 66% of the patients were females and 34% were male it was observed that as the no. of male samples are more but the contaminated samples are more of female catheterized patient . Duration of catheterization is one of the leading reasons for the catheterized infection during the sampling it was observed that as longer the duration there is more chances of infection and blockage of the urinary catheter. Abdallah, Taiwo and Aderounmu observed that period ranging from 48 hours to over 28 days before change or removal and all were routinely placed on prophylactic systemic antibiotic following catheterization Pseudomonas aeruginosa among the three aminoglycosides, amikacin showed 72% susceptibility and in the present work it was observed that only 54.5% susceptible.
Enterococcus isolates were studied by Sivraj et al. total 68 species where checked for antibiotic resistance and found that high rates of resistance to ciprofloxacin, rifampicin and erythromycin were as in present study Enterococcus were completely resistant to cefixime. Also Koshariya were found that isolated Escherichia coli from urinary catheter were resistant to amoxicillin 58.7%, cefixime 83.6% but in the present study, it shows 100% resistance to amoxicillin, cefixime, piperacillin and cefaclor. During the present investigation two rare bacterial species was identified Dermacoccus nishmaneyasis and Stenotrophomonas maltophilia isolated from the urinary catheter sample. Similarly Mengegloglu et al. isolated Stenotrophomonas maltophilia from the three different sources respiratory tract followed by blood and wounds.
In the present study the number of catheter sample is more of male than female, but the bacterial contaminations were more observed in the female catheterized patients. Also the duration of catheterization is one of the important factors which is the main cause of contamination observed that as the patient get catheterized for longer duration the more is the encrustation and formation of slime layer inside the lumen of catheter. The main contaminant found was strong biofilm forming Pseudomonas aeruginosa highest in number among all single and multi species contaminated catheters sample of catheterized patients. During the study, two rare species Dermacoccus nishinomiyaensis and Stenotrophomonas maltophilia both show resistance to all other antibiotics except chloramphenicol and gatifloxacin. Triclosan used against strong and moderate biofilm forming isolates are very good. Minimum concentration is required for the inhibition of planktonic bacterial growth as performed by disc diffusion and MIC by TCP method and the concentration required for MBEC is higher than that of MIC values. The MIC of triclosan ranged between 1.5 and 1000 µ/ml and MBEC was between 800 and 3200 µ/ml i.e. MBEC values were 3 to 1800 folds than MIC values.
This work was supported by funds from the University Grants Commission (UGC) [F.No 43-472/2014 (SR)], New Delhi, India, under the Major research project.
Tiwari, A.A. and Ghnawate, N. (2017) Detection of Biofilm Forming Bacterial Communities from Urinary Catheter of Patients with Change in Its Antibiotic Susceptibility Pattern and Triclosan Effect from Different Hospitals of Amravati City Maharashtra, India. Open Journal of Medical Microbiology, 7, 51-66. https://doi.org/10.4236/ojmm.2017.73005