Catheter-Associated Urinary Tract Infection and Obstinate Biofilm Producers

Background Biofilms, or colonies of uropathogen growing on the surface of indwelling medical devices, can inflict obstinate or recurring infection, thought-provoking antimicrobial therapy. Methods This prospective analysis included 105 urine samples from catheterized patients receiving intensive care. Ensuing phenotypic identification, antibiotic sensitivity test was performed by modified Kirby–Bauer disc diffusion method following CLSI guidelines; MDR isolates were identified according to the combined guidelines of the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC). Biofilm-forming uropathogens were detected by the tissue culture plate (TCA) method. Results The predominant uropathogen in catheter-associated UTIs (CAUTIs) was Escherichia coli 57%, followed by Klebsiella pneumonia 15%, Pseudomonas aeruginosa 12%, Staphylococcus aureus 8%, Enterobacter spp. 3%, Enterococcus faecalis, Acinetobacter spp., and Proteus mirabilis 1.5%, of which 46% isolates were biofilm producers. Prime biofilm producers were Escherichia coli 33%, followed by Klebsiella pneumoniae 30%, Pseudomonas aeruginosa 20%, Staphylococcus aureus 10%, Acinetobacter, and Enterobacter 3.33%. Multidrug resistance associated with biofilm producers were greater than biofilm nonproducers. The Gram-negative biofilm producers found 96.15%, 80.76%, 73.07%, 53.84%, 53.84%, 46.15%, 19.23%, and 11.5% resistant to amoxyclave, ceftazidime, tetracycline, gentamicin, meropenem, nitrofurantoin, amikacin, imipenem, and fosfomycin, respectively. Gram-positive biofilm producers, however, were found 100% resistant to tetracycline, cloxacillin, and amoxyclave: 66.67% resistant to ampicillin while 33.33% resistant to gentamicin, ciprofloxacin, and nitrofurantoin. Conclusion High antimicrobial resistance was observed in biofilm producers than non-biofilm producers. Of recommended antimicrobial therapies for CAUTIs, ampicillin and amoxicillin-clavulanate were the least active antibiotics, whereas piperacillin/tazobactam and imipenem were found as the most effectual for gram-negative biofilm producer. Likewise, amoxicillin-clavulanate and tetracycline were the least active antibiotics, whereas vancomycin, fosfomycin, piperacillin-tazobactam, and meropenem were found as the most effective antibiotic for Gram-positive biofilm producer. In the limelight, the activity fosfomycin was commendable against both Gram-positive and Gram-negative biofilm producers.


Background
Of nearly 40 percentile of all health care-associated infections, urinary tract infections (UTIs) are the foremost cause of infections; out of these, a bulky proportion, 80%, involve catheter-associated urinary tract infections (CAU-TIs) [1]. e urinary catheters are routinely used in urology practice; albeit, advances in design and materials used, UTIs persist as the major snags, owing to the contamination of such indwelling devices [2]. Approximately, prior admission, 12 to 16% of adult hospital inpatients have an indwelling urinary catheter, however, known to be associated with high morbidity, high mortality, increased the length of hospital stay, and increased the cost of treatment [1][2][3].
Furthermore, the catheter-associated biofilm producers, preceding drug resistivity, and their thought-provoking infection control procedures have been reported in aforementioned studies, which raises our concern on CAUTIs and biofilm producers in our settings [4,5].
Biofilms are the sessile polymicrobial communities attached to the substratum of biotic and abiotic surfaces and are sheathed within a self-produced extracellular polymeric matrix, that is, polysaccharides intercellular adhesin [2,5,6]. e extracellular matrix facilitates communications among the cells through biochemical signals-acyl-homoserine lactone in Gram-negative bacteria and oligopeptides in Gram-positive bacteria-in a phenomenon called as quorum sensing [7]. Not only the matrix precludes the pathogen against host defense but also attributes antimicrobial resistance, by subordinating antibiotic penetration, horizontal transmission of plasmid-associated drug-resistant gene, and altered microenvironment [6,7]. In this standpoint, early detection of biofilm producers is crucial, to reduce the irrational antimicrobial burden proceeding antimicrobial resistance in the patient; hence, it would be an auxiliary in controlling device-associated infections in medical centers." e rationale of our study was to explicate bacterial etiologies, illuminate biofilm-associated resistivity patterns, and to endorse suitable antimicrobial therapy against biofilm producers in CAUTIs.

Study Design and Setup.
e cross-sectional study was conducted at the Department of Microbiology, Janamaitri Foundation Institute of Health Science (JFIHS), Nepal, over a period of six months. e study hospital is a referral centre with medical, surgical, gynecological, pediatric, geriatric, and other specialties.

Inclusion and Exclusion Criteria.
e urine sample of all catheterized patients irrespective of gender and age between 12 and 70 years who met the criteria of CAUTI were included in the study. Nevertheless, noncatheterized patients, either nursed in ward or formerly under antimicrobial therapy at least 48 h prior catheter insertion and no more than two types of organism grown from the clinical sample, were considered as contaminated and consequently, excluded from the study.

Laboratory
Methods. CAUTI was defined using a combination of clinical signs and symptoms and laboratory criteria as described by Stamm [2]. A total of 105 urine sample from the catheterized patient, admitted in intensive care units, were processed semiquantitatively by inoculating 0.001 ml of the specimen (by using a calibrated wire loop) onto the Cystine Lactose Electrolyte Deficient (CLED) agar for the isolation and identification of significant uropathogens [8]. Following the inoculation, the plates were incubated for 24 hours at 37°C in an aerobic atmosphere. e growth of a single organism with a count of ≥10 2 colonyforming units (CFU)/ml was considered to represent as CAUTIs and was identified using appropriate routine identification methods including colony morphology, Gram stain, and an in-house set of biochemical tests [8].

Detection of Biofilm Production.
e detection of biofilm was done by tissue culture method/microtiter plate method (TCA), the gold standard method, as described as Christensen et al. [11]. In brief, the bacterial isolates from fresh agar plates were inoculated in 2 ml of BHI broth and incubated for 24 h at 37°C. e cultures were then diluted 1 : 40 with fresh medium (BHI broth supplemented with 1% glucose); 200 µl of the sample was dispensed in the individual microtitration plate (AD Touch, apDianv) and incubated further 24 h at 37°C. With a gentle tapping, the content was removed further with a subsequent washing with phosphate buffer saline (pH 7.2) three times to remove free floating sessile bacteria. e adherent bacteria, biofilm producer, were fixed with sodium acetate (2%) and stained with crystal violet (0.1% w/v) for 10-15 min. e unbound crystal violet solution was removed with a triplicate washing with PBS, and the plate, then, was kept for drying. Finally, all wells were filled with 200 µl ethanol (95%) to release dye from the well and Optical Density (OD) was taken at the wavelength of 630 nm. For a precision, the experiment was performed in triplicate two times. Average OD values of each test strain and negative control were calculated, and OD cutoff values (ODc) were assessed as described by Stepanovie et al. [12].

Data Analysis.
e information regarding patients' profile and the results were entered into a computer program. Data analysis was carried out using the Statistical Package for Social Sciences (SPSS ™ ) version 20.0 (IBM, Armonk, NY, USA) and presented in percentage base distribution.

Ethical Consideration.
Written approval was taken from the Institutional Review Committee (IRC) of Janamaitri Foundation Institute of Health Science (JFIHS) after submitting and presenting the research proposal. Written informed consent was taken from every patient or their guardians before enrollment in the study.
Microtitration plate shows the biofilm production by TCA method as shown in Figure 1.

e Antimicrobial Resistant Pattern in Biofilm Producers
and Nonproducers. Gram-negative biofilm producers, more than 90%, were resistant to ampicillin and amoxicillinclavulanate, and nearly 94% of biofilm producer and nonproducer were found to be sensitive to fosfomycin. Besides, the greater percentile of antimicrobial resistivity was found to be associated with biofilm producers than biofilm nonproducers.

Discussion
Most aspects of the diagnosis, treatment, and prevention of CAUTI are influenced by the tenacity of biofilm-associated uropathogens [13]. Meanwhile, in patients with underlying diseases or under intensive care, the relevance of detection of biofilm producers is crucial since CAUTIs is a common nosocomial infection [1]. e prevalence of catheterassociated urinary tract infection was found to be 61.9%, with predominance in female sufferers (43, 66.2%). Similar prevalence was reported in a review of Nicolle from 15 developing countries like ours [14]. is might be due to the anatomical differences of urogenital organs-anal proximity and shorter urethra in female [15].
Antibiotic resistance patterns of biofilm producer and nonproducer were observed; sequentially, high resistance against tested antibiotics was attributed in biofilm producers. In Gram-negative bacterial isolates, the resistance against four groups of antibiotics, ampicillin, nitrofurantoin, tetracycline, and meropenem, was equated in biofilm producer versus non-biofilm producer isolates; the consecutive antibiotics were 92. 3% [18]. Furthermore, fosfomycin has shown promising in vitro activity against both Gram-positive and Gram-negative biofilm producers, as earlier experienced by Neuner et al., Mihailescu et al. and Marquès et al. in treating urinary tract infections [26][27][28].
Hence, the antimicrobial resistivity in the isolate possibly attributed with the biofilm productions. In Gram-positive isolates, the resistance against 3 groups of antibiotics amoxicillin-clavulanate, tetracyclin, and cloxacillin was equated in biofilm producer versus non-biofilm producer isolates; the consecutive antibiotics were 100% resistant in biofilm producers while 33.3%, 66.7%, and 66.77% were   Canadian Journal of Infectious Diseases and Medical Microbiology found to be resistant in non-biofilm producers. Similarly, MRSA, resistant pattern linked with the tenacity of biofilm producers, was observed by Ando et al. [29]. In biofilms, poor antibiotic penetration, nutrient limitation and slow growth, adaptive stress responses, and formation of persister cells are hypothesized to constitute a multilayered defense [30]. e backdrop of antimicrobial resistant in biofilm producers urges seeking of naive therapeutic alternatives despite conventional antibiotic therapies. In the meantime,  Canadian Journal of Infectious Diseases and Medical Microbiology to prevent or to combat these obstinate biofilm producers, small molecules (N-acetylcysteine, Ca2+ and Mg2+ chelators), and matrix-targeting enzymes (dispersin B, DNase I, proteinase K and trypsin), bactericidal and antiadhesion coatings (trimethylsilane, carboxybetaine methacrylate, organoselenium, heparin, and hyaluronic acid; polymer brush coatings; and furanones) were commenced successfully in developed nations [31]. Ironically, the indorsed therapeutic solutions are farther than reach in the developing countries like ours.

Conclusion
Biofilm producing bacteria are responsible for many recalcitrant infections and are notoriously hard to eradicate, owing to the possible acquisition of multidrug status. High antimicrobial resistance was observed in biofilm producing pathogens than nonproducers. Of recommended antimicrobial therapies for CAUTIs, ampicillin and amoxicillinclavulanate were the least active antibiotics, whereas piperacillin/tazobactam, fosfomycin, and imipenem were found as the most effective antibiotics among Gram-negative biofilm producers. Likewise, among Gram-positive biofilm producer isolates; amoxicillin-clavulanate and tetracycline were the least active, whereas vancomycin, fosfomycin, piperacillin/tazobactam, and meropenem were found as the most effective antibiotic. In the limelight, the activity of fosfomycin was laudable against both Gram-positive and Gram-negative biofilm producers. Hence, an early identification of biofilm producers with subsequent detection of antibiotic resistivity pattern is mandatory, to improve the clinical management of CAUTIs when the patient requires an intensive care.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
Written informed consent was obtained from every patient for granting participation in an interview and to extract pertinent sociodemographic and clinical data from their respective clinical files, respecting confidentiality.

Consent
Written informed consent was obtained from the patients for relevant investigations, and publication of the findings was taken from every patient and coauthors.