Prevalence and Antibiotic Resistance Pattern of Escherichia coli and Klebsiella pneumoniae in Urine Tract Infections at the La Paz Medical Center, Malabo, Equatorial Guinea

The study was conducted to isolate and determine the antibiotic resistance in Escherichia coli and Klebsiella pneumonia from urine samples over a 2-year period (August 2013-September 2015) at the La Paz Medical Center, Malabo. A retrospective analysis of 785 urine culture samples over a 2-year period August 2013-September 2015 was carried out according to the routine protocol of urinalysis. Bacterial etiological agents were isolated from 155 (19.7%) samples with highest prevalence of Escherichia coli (55.5%) followed by Klebsiella pneumonia (23.2%), Proteus mirabilis (4.5%), Pseudomonas species (3.2%), Enterobacter species (2.6%), Enterococcus faecalis (2.6%) and others species (8.4%). The E. coli and K. pneumonia represent 78.7% of all isolated bacterial strains. The E. coli and K. pneumoniae isolates possess highly resistant to ampicillin, Trimethoprim/Sulfamethoxazole, Doxycycline, Amoxicicline/Clavulanic acid. Whereas K. pneumonia demonstrated also to be highly resistant to Gentamycin, Cefuroxime and Ceftriaxon, low level of resistance to Piperacilin/Tazobactam, Amikacin and the lowest to Imipenem. The alarming level of MDR strains to the first choice antibiotics treatment was observed.

pneumonia, P. mirabilis, P. aeruginosa, S. aureus, E. faecalis, S. saprophyticus are most common bacteria causing UTIs in human beings [4] [5]. Among the uropathogenic bacteria, Escherichia coli is predominant in both community and nosocomial UTI [2] [3] [6]- [15]. Infections are gradually becoming more and more difficult to treat and may lead to therapeutic dead ends. These resistance patterns have shown large inter-regional variability. Understanding the spectrum and resistance patterns may help guide effective empirical antibiotic therapies, decrease treatment failure and costs. Resistance patterns of microorganisms vary from country to country, state to state, large hospital to small hospital and hospital to community [1] [6]- [14]. In Equatorial Guinea, the problem of antibiotic resistance is compounding because of overuse and misuse of antibiotics. It is a first study of antibiotic resistance of uropathogenic bacteria in Equatorial Guinea.
The aim of this study was to assess the diversity and state of resistance of dominant uropathogenic bacteria such as E. coli and K. pneumoniae to antibiotics in Equatorial Guinea.

Subjects and Methods
The observational and prospective study was conducted at La Paz Medical Center, Malabo, Equatorial Guinea. We cultured and analysed 785 urine samples from patients of different departments at La Paz Medical Center. These samples were quickly recorded and processed according to the routine protocol of urinalysis.

Samples Processing
Urine sample (0.01 mL) was inoculated on Triptic Soy Agar with 5% Blood sheep (TSAB), MacConkey's agar and CHROME Orientation agar (HyLabs Ltd.) by spread plate technique. The isolated bacteria were then identified by using Gram Stain and their biochemical characteristics using Remelrap ID system kits.

Antimicrobial Susceptibility Testing
Antibiotic susceptibility was determined using the disc diffusion method on Mueller Hinton agar according to the Guidelines of the Clinical Laboratory Standards Institute (GCLSI) [16]- [18].
Different families of antibiotics (discs obtained from OXOID) were studied such as: Cephems (cefuroxime, ceftriaxone); Penicillins (Ampicillin); Beta-lactam + inhibitor (Amoxicillin/Clavulanic acid, Piperacilin/Tazobactam); fluo quinolones (Ciprofloxacin); Aminoglycosides (Gentamicin, Amikacin); Tetracyclins (Doxycyclin); Trimethoprim/Sulfamethoxazole; Carbapenemes (Imipenem) After incubation, the diameter of the inhibition zone formed around the disc was measured and compared to the critical values "d" and "D" of each antibiotic disc (according to CLSI) to qualify the target bacteria as sensitive (diameter of the inhibition > D) or resistant(diameter of the inhibition < d) or again intermediate (d < diameter of the inhibition < D). Multi Resistant strains were divided into MDR (Multiple Drug-Resistant), XDR (Extensively Drug-Resistant) and PDR (Pandrug-resistant) according to the Center for Disease prevention and Control [19]. MDR bacteria are defined as resistant to at least three different classes of antibiotics. XDR bacteria are characterized by their sensitivity to only one class of antibiotics and the PDR bacteria are resistant to all classes of antibiotics recommended for treatment [20].

Epidemiology of Urinary Tract Infections
The total of 785 urine samples of both outpatients and in-patients of La Paz Medical Center was determined. 443 (55%) were from males while 342 (45%) were from females. A total of 155 bacteria strains (19.7%) were successfully isolated from urine samples. 76 (17.3%) positive sample were belong to males and 79 (23.1%) were belong to females. Out of 155 positive samples the E. coli (56%) isolation was the highest followed by Klebsiella pneumonia (24%), Proteus mirabilis (5.3%), and Pseudomonas aeruginosa (2.7%) and others ( Table 1). Both E. coli and Klebsiella pneumonia isolates represent 78.7% of all isolated bacterial strains. Therefore, antibiotic resistance rates were performed specifically these two strains.

Antibiotic Resistance
The comparison of antibiotic resistance rates was performed specifically for E. coli and K. pneumonia (

Multi-Resistant Strains
Based on their phenotypes, multi-resistant strains were classified as MDR, possible Extensively Drug-Resistant

Discussion
Urine tract infection caused by microorganisms is one of the most common infections in the world [2]- [4]. Increasing the high resistance to broad spectrum antibiotics of urine pathogens especially E. coli and K. pneumoniae as the prevalence UTIs pathogens is alarming [2] [5] [21]- [23]. The rapid and correct choice of the antibiotic enables rapid cure of the patient, and sometimes even to save the patient's life. So it is very important to determine the antibiotic resistance patterns of UTIs pathogens. According to the Antimicrobial Resistance Global Report of WHO the information and data about antibiotic resistance situation obtained from the most African countries are still not enough or clear [2]. Our study is the first one about an antibiotic resistance pattern of prevalence UTI bacteria in Equatorial Guinea. In this study we compared the diversity of uropathogens isolated at La Paz Medical Center (Malabo, Equatorial Guinea) from 2013 to 2015 to that reported in similar studies from other African countries. According to this study out of 155 (19.7%) cultures were positive. 76 (17.2%) and 79 (23.1%) were isolated from male and female respectively. The finding that positive urine samples were more frequent in women than men is in agreement with previous studies [7] [11] [14]. The rate of positive urine samples from this study was 19.7%, which is lower than figures from previous studies [5] [7] [10] [14] in Ivory Coast (25.1%), Ethiopia (27.4%), South Africa (51%), and Cameroon (58.3%). It is however, higher than in Nigeria (13.1%) and Ghana (9.5%) [11] [22]. The difference in rates of positive samples can be explained by the differences in location and health situation in the region, capacity of hospital and the status of the patient (patient has the acute UTI or after antibiotic treatment).Our study showed that E. coli and by Klebsiella pneumonia were implicated in 78.7% of all Urine Tract Infection pathogens. As expected, the rate of E. coli (55.5%) isolation was the highest followed by Klebsiella pneumonia (23.2%). This is consistent with other studies in different African countries ( Table 5). The prevalence of K. pneumoniae (23.2%) is similar to those reported from studies in Maroco (22%) [6] Nigeria (25%) [11], Ghana (26%) [22], and more highly from studies in other countries of African continent ( Table 5).

Antibiotic Resistance
The antimicrobial resistance profile of isolates in our study revealed a generally higher resistance rate than reported in African studies [1]. The almost 100% resistance of E. coli and K. pneumoniae to Ampicillin and Trimethoprim/Sulfamethoxazole is the highest rate in African continent, except Nigeria and Moroco where were  [11] and Maroco [6].
Our study showed inefficiency to the first choice line drugs for the treatment of urine tract infection due to E. coli and K. pneumoniae isolates and this information is alarming. Just Imipenem was broadly the most sensitive drug, followed by Piperacilin/Tazobactam and Amikacin. The explanation is probably the fact that these are very powerful drugs used only in hospital settings and not as first-line therapy.

Multi Drug Resistance
The present study showed that the prevalence of MDR among E. coli (74.4%) and K. pneumonia (91.7%) isolates was higher compared to the study carried out in Ivory Coast [5] where the prevalence of MDR E. coli and K. pneumoniae was 14.4% and 23.1% respectively. Our ratio of MDR E. coli isolates was relatively lower than those reported in other African countries such as Sudan (92.2%) [21], Ethiopia (87.4%) [23] and Nigeria (83.9%) [11]. Like other studies elsewhere, our MDR E. coli and K. pneumonia isolates were found sensitive to Imipinem, Piperacilin/Tazobactamand Amikacin.

Conclusion
Our study has shown an overview of the common uropathogens bacteria founded in Equatorial Guinea compared to others African countries. Escherichia coli and Klebsiella pneumoniae were the most predominant strains in urine tract infection (UTI). The high level of Multi Drug Resistant (MDR) strains and the emergence level of Extensively Drug-Resistant (XDR) strains to the first choice antibiotics were observed.