Detection of Extended-spectrum β-lactamases’ (ESBLs) Resistance among Urinary Tract Pathogens in Khartoum State

Aims: The aim of this study is to isolate and identify the extended spectrum beta lactamase (ESBLs), the causative agents of urinary tract infection and detection of their resistance against β lactam drugs. Study Design: Descriptive cross sectional studies in which 100 patients with UTI. Methodology: One hundred urine samples were collected from Khartoum state Hospitals and identified on the basis of their culture characteristics and morphological appearance using Gram stain technique and biochemical tests. The isolates were subjected to antimicrobial susceptibility against the third generation cephalosporins (Cefotaxime, Ceftazidime and Ceftriaxone) using Disk-Original Diffusion method. The bacterial isolates were inoculated to show their ability to produce ESBL using Combination Disk Method (Calvulanic acid + Third generation cephalosporins). The ESBL producers were evaluated among non-ESBL producers. Results: The isolates were identified and enumeratetd as E. coli (49), Proteus species (15), Klebsiella pneumoniae (18), Pseudomonas aeruginosa (9), Enterococcus faecalis (7) and 2 Gram positive bacteria (Staphylococcus species). The isolates subjected were found to show their antibacterial susceptibility against third generation cephalosporins (Cefotaxime, Ceftazidime, and Ceftriaxone) and the results observed were that sixty out of one hundred isolates depicted resistance to third generation cephalosporins and it included E. coli (29), Klebsiella pneumoniae (18), Proteus species (7), Pseudomonas aeruginosa (4) and Enterococcus faecalis (2). Among sixty bacterial species that showed resistance to the third generation cephalosporins, E. coli 20(68%), Klebsiella pneumoniae 7(38%), Proteus species 3(42%) and Enterococcus faecalis 1(50%) respectively were found to be ESBL producer. Pseudomonas aeruginosa showed non-ESBL producers. Conclusion: We conclude that the ESBL producers were found in large proportion which may be due to the misuse of antibiotics or inadequate treatment.


INTRODUCTION
The urinary tract is a system in the body for removing waste and extra water. It consists of bladder, kidneys, ureters, and urethra. The kidneys filter the blood and remove waste and extra water which becomes components of urine. Urinary tract infections are the second most common infection in the body. Over 50% of the women develop UTI. The majority of UTIs is caused by E. coli, the common symptoms include frequent urination & painful burning sensation while urination [1].
UTIs diagnosis usually is made by observing symptoms and examining urine samples of infected patients. UTI infections are commonly treated with antibiotics e.g. Gentamicine, ciprofloxacin, cefetriaxone, cefixime, cefuroxime, nalidixic acid and amoxicillin-clavulanate, but observed that bacterial causing UTI exhibits resistance against wide range of beta lactam antibiotics mentioned above. Beta-lactamase is enzymes produced by bacteria that inactivate either penicillin or cephalosporine, but some can inactivate both classes of drugs. Most Gram positive bacteria secrete beta-lactamases so that beta-lactam drugs are inactivated extracellularly, i.e., in the surrounding medium. By contrast, the beta-lactamase of Gram negative remains inside the cell and inactivate beta lactam drugs in periplasmic space, i.e., the space between the outer membrane and cytoplasmic membrane [2].
ESBLs are not the only β-lactamases to confer resistance to 2 nd and 3 rd generation cephalosporins while sparing carbapenems, but are the most important. Moreover, as plasmidmediated enzymes, they have great potential to spread. They occur mostly in Enterobacteriaceae (eg E. coli, Klebsiella species and Enterococcus species) and rarely in non-fermenters (eg. P. aeruginosa). They should be distinguished from other important modes of resistance to 2 nd and 3 rd generation cephalosporins [3].
Between February 2008 and January 2009 in India, a total of 213 isolates were tested for ESBLs production by using both the double disk approximation and the combination disk methods [4].
In 2011, study was done in a Chennai suburban tertiary care hospital (India) and its antibiogram pattern a total of 131 urine sample were collected from out patients clinically suspected to have UTIs Out of 131 urine samples obtained, 126 samples were considered positive for UTI; the positive samples included E. coli (58), Klebsiella species (39), Proteus mirabilis (13), Pseudomonas areuginosa (9), Staphylococcus species (6) and Citrobacter species (1). Only E. coli and Klebsiella species were included to detect ESBLs production, and the result was positive for both [5].
The resistance against ᵝ -lactam has become one of the major problems in therapy of urinary tract infections in our country as well as world. [6] The aim of this study is to isolate and identify the extended spectrum beta lactamase (ESBLs), the causative agents of urinary tract infection and detection of their resistance against β lactam drugs.

Experimental Work
This descriptive cross sectional studies included 100 individual of UTI induced. Approval was obtained from Al-Neelain Research Ethical Board.

Collection of Specimen
The mid-stream urine was collected in clean, dry, wide neck, screw capped container [7].

Isolation of Pathogens
Urine sample was inoculated into Citrate Lysine Electrolyte Deficiency (CLED) media and incubated at 37°C over night. The isolates were identified base on of their cultural characteristics, Gram stain technique and their reaction of biochemical tests [7].

Antibiotic Susceptibility Test
An overnight culture suspension of the test isolates which was adjusted to 0.5 McFarland's standard was inoculated by using a sterile cotton swab on the surface of a Mueller Hinton Agar plate. Susceptibility of different isolates against 30-μg disc of Cefotaxime, Ceftazidime, and Ceftriaxone was performed using Kirby-Bauer disc diffusion technique placed 15 mm apart. After incubating overnight at 37°C, the presence of zones of inhibition was interpreted as positive for 3 rd -generation cephalosporins susceptibility [2].

Combination disk method
The combination-disk test using Cefotaxime, Ceftazidime, Ceftriaxone, and Amoxyl alone and in combination with clavulanic acid, were performed for the detection of ESBL according to the Clinical Laboratory Standards Institute (CLSI) guidelines [8]. In this test, an overnight culture suspension of the test isolates which was adjusted to 0.5 McFarland's standard was inoculated by using a sterile cotton swab on the surface of a Mueller-Hinton Agar plate. The Cefotaxime (30 μg) and cefotaxime-clavulanic acid (30 μg / 10 μg) disks were placed 20 mm apart on the agar. Similarly, the ceftazidime (30 μg) and ceftazidime-clavulanic acid (30 μg / 10 μg) disks were placed 20 mm apart. This step was repeated with ceftriaxone, ceftriaxonecalvulanic acid and amoxyl-calvulanic acid. After incubating overnight at 37°C, a diameter ≥ 5-mm increase in the zone diameter for either antimicrobial agent which was tested in combination with clavulanic acid vs. its zone when tested alone was interpreted as positive for ESBL production [8].

RESULTS AND DISCUSSION
As can be seen in Table 1, total of 100 urine samples were collected from patients of UTI from hospitals of Khartoum State -Sudan during September-December 2014.  (7), Pseudomonas aeruginosa (9) and (2) Gram positive bacteria (Staphylococcus species) were isolated from these specimens but only 60 (60%) microorganisms showed resistance against the third-generation cephalosporins and they were tested for the ESBL production. These findings were in agreement with Yuksel et al., [9] who reported that the UTI most causative agent was E. coli (87% of cases) followed by Klebsiella pneumoniae (10%).
The findings of this study are supported by Umadevi et al. [4] who reported that the overall ESBL producers were 74.32%and the overall isolates which exhibit to 3 rd -generation cephalosporin were 67% which is disagree with our findings which appeared as 60% for 3 rd generation cephalosporins resistance and 51.67% for ESBL production.
Bradford [10] states that the resistance to extended spectrum cephalosporins is mainly mediated by the production of ESBLs and it is also agreed with this study.
In this study we found that the common ESBL producer is E. coli (68.00%), followed by K. pneumoniae (38.00%) and this is in disagreement with the findings of Jarlier et al. [11], Walsh et al. [12], and Nayal et al. [13] who stated that K.pneumoniae was observed high resistance to ESBL more than E. coli.
Also Walsh et al. [12], Nayal et al. [13], and Singhai et al. [14] reported Pseudomonas specie ESBL production is less as compared to Enterococcus faecalis, because their resistance is mediated by various other mechanisms such as the production of metallo-beta-lactamases, lack of drug penetration due to mutations in the porins and the loss of certain outer membrane proteins and efflux pumps which is in agreement with the present findings. Also the present study results reveal that none of Pseudomonas was ESBL producer.  In a similar study by Babypadmini and Appalaraju [14], 62% of the E. coli and 73% of the K. pneumoniae isolates were reported to be ESBL producers and less than our study 68.00% in E. coli.

CONCLUSION
In conclusion, the ESBL-producing organisms were multidrug-resistant pathogens that are increasing rapidly and have become a major problem in the area of infectious diseases. It is essential to report ESBL production along with the routine sensitivity reporting, which will help the clinicians in prescribing proper antibiotics.