International Journal of Medical Laboratory

Urinary tract infection (UTI) is a broad term that encompasses asymptomatic microbial colonization of urine and symptomatic infection with microbial invasion and inflammation of the human urinary tract [1]. A major cause of morbidity and mortality, UTI has an enormous disease burden and represents the most commonly acquired bacterial infections in human accounting for an estimated 25.0-40.0% of nosocomial infections [2]. About 150 million cases are reported globally per annum with significant incidences also being reported in Nigeria, costing the world economy billions of dollars in treatment and work loss [3].
Although every individual is susceptible to UTIs, certain specific subpopulations are more predisposed to UTIs. These include infants, pregnant women, the elderly, patients with spinal cord injuries and/or on urinary catheters, patients with diabetes, multiple sclerosis, impaired immunity and patients with underlying urologic abnormalities. The risk of developing UTI increases significantly with the use of indwelling devices such as catheters and urethral stents or sphincters common with patients suffering from obstructive uropathy [4]. This risk is further increased when any breach in the closed system is encountered such as during emptying the catheter drainage bag or taking urine sample [5].
The presence of a urinary catheter is the most important risk factor for bacteriuria with a daily incidence rate of 3.0-10.0% [6]. Typically, in the absence of antimicrobial therapy, catheterized patients will become colonized with low level bacteriuria which usually progresses to >105 colony forming unit (CFU) within 24 to 48 hours and biofilm begins to form within 72 hours of catheterization [7].
Several published articles have documented various virulence factors that contribute to the ability of uropathogens to cause disease, which includes; fimbrial adhesins, toxins, flagella, auto transporter proteins and iron-acquisition systems [8]. Biofilm formation amongst bacteria cells have been described as an important virulence factor currently estimated to be responsible for over 65.0% of nosocomial infections and 80.0% of all microbial infections [9]. As reported by Donlan, majority of biofilm producing bacteria are from catheterized patients. Urinary catheters are tubular latex or silicone devices, which when inserted, may readily acquire biofilm on the inner or outer surfaces [10]. Uropathogenic E.coli strains an important member of the Enterobacteriaceae family present a suitable model for this study as they are consistently reported to be the most common extra-intestinal pathotypes identified in patients with UTIs thereby accounting for about 90.0% of reported cases. These uropathogenic bacteria refer to certain strains that are selected from faecal flora, not by chance or based on prevalence but because of specific virulence factors which aid their persistent, colonization and circumventing host defense to allow invasion of the normally sterile urinary tract [9, 10]. Biofilm formation described as a transition from planktonic living, presents itself as sticky accumulation of small colonies of bacteria surrounded by an extracellular polysaccharide matrix in which cells aggregate and adhere in an irreversible manner to various surfaces, including medical devices and injured tissues [11].
Antimicrobial resistance development associated with biofilm producing uropathogens have been linked to upsurge in treatment failures which pose a threat for patients confined to the use of indwelling catheter or undergoing urological surgery in general, and men subjected to prostate biopsy in particular [12]. The emergence of antibiotic resistance in the management of urinary tract infection is a serious public health issue, particularly in the developing world where apart from high level of poverty, ignorance and poor hygienic practices, there is also high prevalence of fake or adulterated drugs of questionable quality in circulation [12, 13].
In a recent study, biofilm producing bacteria had higher antimicrobial resistance than that of nonbiofilm producers to amoxicillin-clavulanate and ciprofloxacin, respectively Moreover, multidrug resistance was observed more among biofilm producing isolates than their counterparts to amoxicillin and cephalexin. This higher antimicrobial resistance among biofilm producing bacteria may emerge from increased properties of efflux mechanism. In addition, it may be also associated with higher plasmid transfer, modified target genes, and metabolic pathway that allow for resistance to antimicrobial agents. Furthermore, it was also reported that about two-third of the biofilm producing bacteria are multidrug resistant to at least three or more antimicrobial agents [13]. Global data shows increasing antibiotic resistance among the uropathogens. The resistance is mostly encountered with the antibiotics frequently used for the treatment of these infections and it varies among communities depending on their prescription pattern [14]. This study is therefore designed to determine the prevalence of biofilm producing UPEC in catheter urine at University of Ilorin Teaching Hospital and to establish association between antibiotic susceptibility pattern and biofilm production.
Materials and Methods
This study was conducted at the Department of Medical Microbiology and Parasitology of the University of Ilorin Teaching hospital (UITH), Ilorin. UITH belongs to the second generation of Teaching Hospitals in Nigeria. It is a tertiary health care centre and the only referral centre in Kwara State with a capacity of over 450 beds and an average of 10,000 to 12,000 annual admissions of in-patient and out-patient’s visits respectively in the last five years as captured by the Department of Health Information Management, UITH, in Ilorin, 2015. It is located in the North central region of Nigeria. The Hospital provides quality health care services to the neighboring states like Oyo, Kogi, Niger, Osun, and Ekiti states.
This was is a hospital based prospective study.
The study population comprises catheterized patients, both outpatient and inpatients of all age groups and gender at clinics /units of the hospital such as General Outpatient Department (GOPD), Urology clinic, Male and Female Surgery wards, Accident and Emergency unit.
Random sampling technique was employed for the selection of patients who had met the inclusion criteria including: patients with indwelling catheter ≥48 hrs calendar days; and not on any antibiotics within the last 72 hrs before recruitments. Patients with indwelling catheter < 24 hrs and on antibiotics within 72 hrs of recruitments were excluded from this research. Ethical approval was sought from the University of Ilorin Teaching Hospital Ethical Review Committee.
Analytical laboratory procedures
catheter urine sample collection and transportation
Urine sampling from patient with an indwelling urinary catheter was obtained from a sampling port using aseptic technique. Where there was no sampling port, the drain tubing was detached from the catheter bag and about 10-15 ml urine was allowed to drain aseptically into a sterile receptacle. Collected urine in the catheter bag was not considered. All sample collection was carried out with the assistance of an assigned doctor. Samples collected were promptly transported to the medical microbiology and Parasitology department for analysis. However the samples that were unavoidably delayed were refrigerated at 2-8˚C for not more than 4 hrs.
Culture and isolation
A modified semi-quantitative culture technique was used. Standard calibrated bacteriological loop (to determine CFU) was used to aseptically transfer 0.001 ml of a well-mixed urine sample on appropriately well labeled culture of Cysteine Lactose Electrolyte Deficient (CLED) agar (Biomarker) and 5.0% sheep blood agar (Biomarker) media. All culture media were prepared according to the manufacturer’s specifications. Incubation was at 37˚C for 18-24 hrs aerobically.
Characteristic of isolates
E.coli appears on CLED as Large, elevated opaque yellow lactose fermenting colonies with a slightly deeper yellow center. E.coli appears on blood agar as large, opaque, sticky and colorless, ± narrow clear hemolysis zone.
Bacterial biochemical testing
A Commercially available phenotypic qualitative miniaturized systems API 20E (Bio Merieux, France) for Enterobacteriaceae identification was used for the physiological       or biochemical confirmatory identification of UPEC isolates.
Biofilm assay using tissue culture plate (TCP) method
A phenotypic quantitative TCP Method first developed and described by Christensen et al [13]. and considered the gold standard for biofilm detection was used for the detection of biofilm production in all UPEC isolates.
Antibiotic susceptibility testing
This was performed according to the Clinical and Laboratory Standards Institute (CLSI), 2007 guidelines. In vitro antibiotic susceptibility testing of established biofilm producing UPEC isolated was carried out by modified Kirby-Bauer disc diffusion method on Muller-Hinton agar plates. The following antibiotics were used; Amoxicillin-clavulanate (10 μg) Cefriazone (30 μg), Ceftazidime (30 μg), Ciprofloxacin (5 μg), Gentamicin (10 μg), Nitrofurantoin (300 mg), and Imipenem (10 μg).
Extended spectrum beta lactamase (ESBL) detection
The double disk synergy test (DDST) using third generation cephalosporins (3 GS) which includes ceftazidime (30 μg) and ceftriaxone (30 μg) antibiotic discs alongside amoxicillin-clavulanate (10 μg) disc were used for the detection of ESBL production amongst UPEC isolates. This was done by placing the antibiotic disk at distance of 20 mm from each other (center to center). Following incubation at 37˚C aerobically for 18-24 hrs, a positive synergistic effect showed inhibition zone between disks. A >5 mm increase in a zone diameter for either antimicrobial agent was tested in combination with clavulanic acid versus its zone which when tested alone is taken as a positive ESBL production. 
Statistical analysis
Data were analysed using statistical apcakage for social sciences (SPSS) version 21, California, USA. Redukts were presented as frequencies and percentages. Chi-square test was used to determine association between ESBL, biofilm production and antibacterial resistance. A p value less than 0.05 at 95% confidence interval was considered significant.
Results
Catheter urine samples from one hundred and thirteen patients comprising both inpatients and outpatients who has met the inclusion criteria set out for this study were collected within the month of April-June, 2016. Catheter associated urinary tract infection in this study was 70.8% of samples analysed. Of this, Escherichia coli, 44 (55.0%) proved to be the most predominant pathogen. This study revealed UPEC, biofilm and ESBL production being 38.9%, 81.8% (Fig. 1) and 27.2%, respectively. ESBL production was significantly associated with degree levels of biofilm formation (p<0.005) (Fig. 2). Both strong and moderate biofilm producers showed the same level of resistance to ceftazidime of 31.6%. Moderate biofilm producers were 46.7% resistant to cefriaxone. Resistance to Amoxillin-clauvanate significantly occurred in all grades of biofilm producers (p>0.05). Imipenem however was the most sensitive with no resistance (Table 1). There was a statistical significant association between ESBL production and degree of biofilm formation. Highest incidence was observed in strong biofilm producers (100.0%) (Fig. 2). Resistance patterns and degree of biofilm formation is shown in figure 3.
Discussion
This study involved the enrollment and evaluation of 113 patients with indwelling urinary catheter, within the period of April and June, 2016. These patients comprised both outpatients and inpatients. The prevalence of CA-UTI in the study area was (70.8%), 55.0% of which was caused by UPEC isolates. Prevalence of biofilm production potential by UPEC isolates was 31.8%. UPEC accounted for 44 (55.0%) of the total uropathogens isolated in this study. In consonance with this study, Yakubu [15]; Ahmed et al. [16] and Niveditha et al. [17] reported similar findings where UPEC was observed as the most prevalent uropathogen despite variation in the prevalence rates of 24.14%, 31.7% and 71.0% respectively.




 

Table 1. Antibiotic susceptibility pattern of Uropathogenic E. coli isolates