Prevalence, antibiotic susceptibility patterns, including ESBL production and associated risk factors of Klebsiella species among UTI suspected patients at Bahir-dar town, Northwest Ethiopia.

Background Klebsiella species are opportunistic bacteria responsible for different infections such as pneumonia, urinary tract infections (UTI) and septicemia in nosocomial and community environments. Beta-lactam drugs are used extensively to treat patients with Klebsiella infections. But today, most Klebsiella species are resistance to third- and fourth-generation cephalosporins and monobactams. Objectives To assess the prevalence, antibiotic susceptibility patterns including ESBL production and associated risk factors of Klebsiella species among UTI suspected patients attended at Bahir Dar town health institutions, Northwest Ethiopia. Methods Institutional based cross-sectional study was conducted at Bahir Dar town health institutions from January to May 2019. A simple random sampling technique was used to enroll 384 study participants. Midstream urine samples were collected and inoculated onto Cystine–lactose–electrolyte-decient and MacConkey agars. Identication was carried out by using biochemical tests. Antimicrobial susceptibility patterns of the isolates were determined by Disc-diffusion method. ESBL-producing Klebsiella species was detected by using combined and single antibiotic disks on Mueller Hinton agar. A statistical analysis was done using SPSS version 20 statistical package. P-value < 0.05 was considered as statistically signicant with 95% condence interval. Result was them were Klebsiella pneumonia, K. K. Moreover; regression hospitalization statistically biochemical Ltd. England) and Muller Hinton agar Ltd. England) a positive control for ESBL production. Escherichia coli (ATCC 25922) strain was used as ESBL negative control. Laboratory identication procedures like inoculation of culture media, colony characterization and measuring of antibiotic susceptibility testing were checked by an experienced microbiologist.


Background
The Genus Klebsiella, names after Edwin Klebs a 19 th century German scientist, is a member of the family Enterobacteriaceae. Non-motile, aerobic, facultative anaerobic, rod shaped, gram negative and capsulated is a unique feature to Klebsiella species. The capsule covers the entire cell outer layers, accounts for the large appearance of the organism on gram stain, and provides resistance against many host defense mechanisms (1). Of the genus Klebsiella, commonly isolated species are K. pneumoniae and Klebsiella oxytoca. Klebsiella species cause urinary tract infection mostly caused by K. pneumoniae (1,2). The pathogenic success of K. pneumoniae in causing urinary tract infections can be attributed to its ability to form bio lms, particularly on indwelling medical devices (3).
Urinary tract infection is the microbial invasion of any tissues of the urinary system and is a common medical problem with an unpredictable natural history. Even though many urinary infections resolve spontaneously, some others can progress to destroy the kidney (4). The incidence of UTI is greatly in uenced by age, sex and predisposing factors that impair the normal defense mechanism, maintaining the sterility of the normal urinary tract. Females are more prone to suffer from UTI because of short urethra and have in close proximity of the anus and urethral trauma during intercourse (5, 6 &8).
Moreover; anatomic and hormonal changes during pregnancy can cause UTI that may lead to serious complications in both the mother and fetus (8, 10, &11).
UTIs remain common among outdoor and indoor patients and pose a serious challenge to antimicrobial therapy. Antibiotics are usually given empirically before the laboratory results of urine culture are available. Globally; it has been estimated that more than 6 million out patient's visits and 300,000 hospital stays every year are due to UTI. Approximately 10% will have urinary tract infections at some time during their lives (7). UTI is one of the most common bacterial infections of which Klebsiella pneumoniae is one cause of epidemic and endemic hospital associated UTI (9 &10).
Antibiotic resistance is an increasing global problem that leads to signi cant challenges and costs in the health care system. The resistance level of pathogens against commonly used antibiotics of uncomplicated UTI has signi cantly increased in recent years. Recognition of this fact has led to an international re-evaluation of therapeutic recommendations for uncomplicated UTIs (11). Due to the rapidly evolving adaptive strategies of bacteria, antibiotic resistance pro le of bacterial uropathogens have changed considerably over the past years, both in community and nosocomial infections. Apparent shift in the etiological agents of UTI and associated problem of antibiotic resistance among bacterial uropathogens from time to time and from one institution to another have initiated health institution to carry out continuous evaluation of UTI from the view point of their spectrum and drug susceptibility testing (12). Since Klebsiella. Spp are able to develop cross resistance to many of the commonly used antibiotics including cephalosporin and aminoglycosides, patients suffering from such infections are enforced to visit health institutions frequently may be due to treatment failure (10).
Accurate identi cation of bacterial uropathogens and determining their drug susceptibility pattern are critical for e cient management of patients with UTI. They are also associated with signi cant clinical and nancial bene ts, via the reduction of mortality rates and overall hospitalization costs (12). Antibiotic resistance in urinary pathogens is increasing worldwide and varies according to geographic locates and is directly proportional to misuse of antibiotics. Understanding the rate of drug resistance to the commonly cause of UTI is critically important to determine the empirical therapy (5).
Antimicrobial resistance (AMR) is a signi cant concern in developing nations due to mutations, gaining new genetic material, contact to cells with new genetic material and use of antimicrobial agents as growth supporters in animal feeds destined for human feeding contribute to increase multidrug resistance. But, misuse, over-use of antimicrobial agents, widespread availability of substandard drugs, and poor infection control measures are a current situation worldwide (14).
The scarcity of reliable and timely information, particularly in sub-Saharan Africa, may further limit epidemiological surveillance and effective stewardship efforts. Antimicrobial resistance with clinical isolates of K. pneumoniae has become progressively more serious problem over the past 20 years and there are several examples of rapid global spreading of novel mechanisms of resistance, such as ESBL producing K .pneumoniae, K. pneumoniae carbapenemase (KPC) and the New Delhimetallo-betalactamase (NDM) (13). So, this study was intended to determine the prevalence, antibiogram, ESBL production patterns and its associated risk factors of Klebsiella species isolated from urinary tract infection suspected patients.

Study area, design and period
An institutional based cross-sectional study was conducted to determine the prevalence, antibiogram, ESBL production patterns and its associated risk factors of Klebsiella species isolated from UTI suspected patients attended at FelegeHiwot referral Hospital, Gammby private general Hospital and Bahir dar health center from January to March 2019.

Source population
The Source population was all population who were attended in Bair-dar town health institution during the study period. The study population was all UTI suspected patients seeking medical service in the selected health institutions during the study period.

Sample size determination and sampling techniques
The sample size was calculated using a single population proportion formula based on the assumption of 5% expected margins of error, and considering 95% con dence. The nal sample size became 385.
A simple random sampling method was employed to select two hospitals and one health center. All UTI patients who were attended before one month of the study period in the selected health institutions were found to be 395 and the estimated number of patients during the data collection period, from January to March 2019, was calculate as 1185. Therefore the 385 study participants were allocated proportionally as 278 for Felege-Hiwot referral Hospital, 83 for Gammby private general Hospital, and 24 for Bahir-dar health center.
Data collection and Laboratory processing Structured questionnaires were administered to collect the socio-demographic variables (age, sex, marital status, education level, occupation, and residence), potential risk factors for infection and other relevant clinical data (history of catheterization, history of UTI, history of previous hospitalization and history of antibiotic use). To ensure the reliability of information, all the questionnaires were administered by native Amharic speakers and were checked for completeness and consistency every day.
After giving an orientation on sample collection processes, sterilized urine cap was given to the patient or patient guardian to collect urine sample and was transported to the laboratory unit for examination.
The collected midstream urine specimens were inoculated onto Cystine-lactose-electrolyte-de cient and MacConkey agars. Identi cation of the species were carried out by using biochemical tests and their antimicrobial susceptibility patterns were determined by disc-diffusion method. Also, ESBL-producing Klebsiella species were detected by using combined and single antibiotic disks on Mueller Hinton agar.

Culture and Identi cation
Each urine sample was inoculated onto Cystine-lactose-electrolyte-de cient (CLUDE) agar and incubated at 37 0 C for 24 hrs. The counted colonies yielded bacterial growth of 10 5 /ml urine was considered as signi cant for positive bacteriuria. Pure colony was transferred to MacConkey agar and incubated at 37 0 C for further 24 hrs. The sample that form pink colony in MacConkey agar showed positive for lactose fermenter and was considered as Gram negative bacteria. Finally, a series of biochemical tests namely; triple sugar iron agar, indole, Simon's citrate, urea, and motility were performed on pink colonies from MacConkey agar for nal identi cation of the isolates.

Antimicrobial Susceptibility Testing (DST)
A suspension of pure colony from each con rmed culture isolate was inoculated in sterile normal saline and incubated at 37 0 C for 30 minutes. The suspension was adjusted at 0.5% McFarland standard (32&33). A sterile cotton applicator stick was used for uniform distribution of the suspension on Muller Hinton agar (Oxoid Ltd. England). Then Modi ed Kirby-Bauer disk diffusion technique was implemented for antibiotic susceptibility pattern using different antibiotics such as ampicillin (10µg), amoxicillin/clavulanic acid (20µg), cipro oxacin (5µg), gentamycin (10µg), cotrimoxazole (25µg), chloramphenicol (30µg), nitrofurantoin (300µg), meropenem (10µg), ceftazidime (30µg), cefotaxime (30µg) and cefoxitin (30µg). After incubation at 37 0 C for 24hrs, the zones of inhibition were measured by a calliper. Finally the results were measured, recorded and classi ed as susceptible, intermediate and resistant using clinical and laboratory standards institute (CLSI) 2018 performance standards for antimicrobial susceptibility testing interpretation (26).

Extended Spectrum β-Lactamase Detection
The screening for ESBL were done by using ceftazidime (≤22 mm) or cefotaxime (≤27 mm). The organisms that showed zone of inhibition lower than the minimum for any of the above antibiotic disc were considered as ESBL positive. The phenotypic con rmations were done by testing the isolated bacterial species against ceftazidime (30μg) and ceftazidime-clavulanic acid combination (30/10μg/discs). In these tests, an overnight culture suspension of the bacterial isolate was adjusted to 0.5% McFarland's standard. Lawn culture were done on the Mueller-Hinton Agar (MHA) plate. The ceftazidime and ceftazidime-clavulanic acid discs were placed at 20mm apart on the agar surface. After incubating at 37 0 C for 24 hours, a ≥5mm increase in the zone diameter in comparison to ceftazidime were taken as indicative for ESBL positive/producer. Escherichia coli ATCC 25922 was used as an ESBLnegative and Klebsiella pneumoniae 700603 was used as an ESBL-positive reference strain (28).
Data management and quality assurance The questionnaire was pre-tested before the actual study undertook to make sure that whether the questionnaire was appropriate and understandable on UTI suspected patients for the actual study area. The questionnaire was prepared in English and translated to Amharic then translated back to English to check the accuracy of the translation.
Sterility of cotton swabs, test tubes and petri-dishes were maintained by autoclaving at 121 0 C for 15 minutes. Moreover, contaminations were avoided by using proper aseptic techniques such as; wearing clean laboratory coats, gloves and washing hands. MacConkey agar (Oxoid Ltd. England), Muller Hinton agar (Oxoid Ltd. England) and different biochemical test (Oxoid Ltd. England) were incubated aerobically at 35 0 C for 24hrs to cheek the sterility of the prepared culture media. Moreover, known strains of Klebsiella pneumonia (700603) was inoculated in to MacConkey agar (Oxoid Ltd. England) biochemical test (Oxoid Ltd. England) and Muller Hinton agar (Oxoid Ltd. England) as a positive control for ESBL production. Escherichia coli (ATCC 25922) strain was used as ESBL negative control. Laboratory identi cation procedures like inoculation of culture media, colony characterization and measuring of antibiotic susceptibility testing were checked by an experienced microbiologist.

Data entry and analysis
Data was entered and coded in Epi data and was exported and analyzed using SPSS version 20 statistical package. The characteristics of the study population were summarized using frequencies, mean and standard deviation. Binary logistic regression was done to determine the association of variables with Klebsiella species and Crude odds ratio was calculated. Moreover, adjusted odds ratio was computed using multivariate logistic regression for variables with p value < 0.2. P-value < 0.05 was considered as statistically signi cant at 95% con dence interval. Finally, the ndings of the study were presented in tables and gures.

Ethical considerations
This study was ethically approved by Ethical Review Committee of School of biomedical and laboratory sciences, University of Gondar. Moreover; legal permission was obtained from Felege-Hiwot referral Hospital, Gammby private general Hospital and Bahir-dar health center of higher management prior to data collection. Written assent and consent were taken from each participant after they understood the purpose of the study. All the subject's data were kept in full con dentiality and was not be disclosed to an unauthorized person.

Socio-demographic characteristics
A total of 385 clinically UTI suspected patients were included in the study; of these, 205 (53.2%) were females and majority of the study participants (37.9%) were in the age groups of 16-30 with participants median age of 32 years. Moreover; 60.8% of the participants were from urban and 24.4% were illiterate ( Table 1).
The current nding also showed that K. pneumoniae 25(65.7%) was higher than a study done in Saudi Arabia (21.5% and 23.5%) ( these difference might be due to type and quality of specimen collection, processing procedure and population difference.
Klebsiella species harboring ESBLs have caused numerous outbreaks of infections and now are signi cant problem in hospitalized patients throughout the world leading to an increasing therapeutic problem.
The current study showed high level of amoxicillin, amoxicillin/clavulanic acid, and cotrimoxazole resistance. One of the reasons for development of these resistance might be the occurrence of ESBL in these species 10/25(40%).
This nding was in agreement with the study reported from Ilam 39.4%, Tehran 50.7% and Tabriz 45.8%. However lower than the previous study reported from India (58.7%) (13).
The current nding pointed out that meropenem was the most effective antibiotics against ESBLproducing and non ESBL producing Klebsiella species as it showed sensitivity of 80 and 100% in ESBL producers and non ESBL producers Klebsiella isolates respectively. This was in agreement with a study conducted in India, meropenem (65.7%) sensitivity (28).
Among the predictor variables for Klebsiella species prevalence for UTI's, pervious antibiotic use, history of hospitalization and tight closing habit were signi cant risk factors for UTI. On the present study the participant's previous history of antibiotic use was signi cant risk factor to develop UTI by Klebsiella species with odds ratio (2.618), con dence interval at 95% (1.18-6.129) and p-value of 0.027. Additionally, the predictor variables of previous hospitalization were showed signi cant statistical association with odds ratio (5.873), con dence intervals at 95% (2.355-14.6) and p-value of (0.000) the present ndings were supported by previous study reported from India where previous antibiotics usage was noted as a risk factor for acquisition of an ESBL-producing organism (34).

Conclusion
Klebsiella species were prevalent in varying magnitude among UTI suspected patients in bahir-dar town health institutions. The prevalence of ESBL producing Klebsiella species was higher as compared to previous studies leading to development of multidrug resistance. ESBL producing patterns of Klebsiella pneumoniae was higher than other Klebsiella species. Moreover; empirically use of broad-spectrum antibiotic, history of hospitalization and tight dressing had signi cant association for infection. To