Antibiotic Resistance Study and Detection of Virulence Gene among Uropathogenic E.coli

A total of two hundred and fourteen outpatient and inpatient children their ages ranged from 1day to 14 years, whether symptomatic or asymptomatic were studied for urinary tract infections (UTIs) during the period (February to May 2012) at Rapareen Teaching Hospital for Children in Erbil City. Urine samples were collected and examined using microscopic, dipstick test for detection of leukocyte esterase and culture techniques. Isolated organisms were identified using microscopical, morphological and biochemical tests (including recent Vitek 2 system) and the results showed that positive urine cultures were detected in 134 (62.6 %) children, among females were 98 (73 %), while among males were 36 (27 %). Microorganisms that had been isolated from urine culture were mainly Escherichia coli 70 (52.2 %) followed by others. The isolates appeared to be varied in their resistance to antibiotics, and the highest resistance were for ampicillin 67 (95.7 %), while the most effective antibiotic used was imipenem .Total E.coli isolates also tested for their ability to produce hemolysin and the results showed that 27 (38.5%) were α-hemolysin, 9 (12.8%) β-hemolysin and 34(48.5%) were γ-hemolysin producers.. Out of 70 Escherichia coli isolates, 41 (58.5%) were found to be ESBLs producers, while 29 (41.4%) were non ESBLs producers. The plasmid profile of studied E.coli isolates revealed that plasmids were found in 64 (91.6%) of isolates with different molecular weights ranged from 1more than10 kbp. Curing of plasmid DNA content were conducted using sodium dodecyl sulfate (SDS) and elevated temperature at 46C o . The results revealed that the curing percentage for isolate E3 was (50%) while for isolate E25 Kirkuk University Journal /Scientific Studies (KUJSS) Volume 10, Issue 3, September 2015 , p.p(205-229) ISSN 1992 0849 Web Site: www.kujss.com Email: kirkukjoursci@yahoo.com, kirkukjoursci@gmail.com 206 was (41.6%) for antibiotics understudy. On the other hand these two isolates when incubated at 46 C o the curing percentage were (66.6%) and (58.3%) respectively. Also the cured colonies tested for their ability to produce ESBLs and the results demonstrated that the cured colonies cannot produce ESBL indicating that the genes responsible for this trait are plasmid situated. Among 70 UPEC isolates, 61(87.1%) UPEC were positive for the existance of aerobactin virulence gene using PCR.


1.INTRODUCTION
Urinary tract infection, or UTI, is an infection that can happen anywhere along the urinary tract. Urinary tract infections have different names, depending on what part of the urinary tract is infected [1]. Uropathogenic Escherichia coli (UPEC) is the most frequent agent causing UTI in adults and children [2]. At least 80% of urinary tract infections in children are caused by Escherichia coli followed by Klebsiella spp., Proteus mirabilis, Pseudomonas aeruginosa and Enterobacter spp. [3]. While 10% of UTIs are caused by gram positive bacteria include Staphylococci, Enterococci and Streptococci [4]. Pathogenic potential of E. coli strains is thought to be dependent on the presence of virulence factors (VFs), which are located either on bacterial plasmid or on chromosome. The virulence genes most commonly associated with UPEC include P fimbriae (pap), afimbrial adhesin I (afaI), aerobactin (aer), type 1 fimbriae, hemolysin (hly), S fimbriae (sfa), cytotoxic necrotizing factor 1 (cnf1), adhesins and fimbriae [5]. Virulence factors are encoded by genes located on the chromosome or on the plasmids. The location of virulence factors on such genetic mobile elements may facilitate the spread of virulence properties within bacterial communities [6]. Increasing rates of resistance to antibiotics among uropathogens have caused growing concern in both developed and developing countries [7]. Understanding antibiotic resistance patterns and molecular characterization of plasmids and other genetic elements is epidemiologically useful [8]. Plasmids allow the movement of genetic material, including antimicrobial resistance genes between bacterial species and genera. Controlling of antibiotic resistance at the molecular levels such as using curing agents may limit or decrease the resistance of pathogenic bacteria [9]. Curing may occur naturally through cell division or by treating the cells with chemical and physical agents [10]. Therefore the aim of this study are isolation and identification of uropathogenic E. coli (UPEC), study the antibiotic resistance patterns of these isolates to different antimicrobials, elimination the bacterial resistance to antibiotics for some isolates by curing the plasmid using SDS and elevated temperature, and finally detection of virulence genes (Aerobactin gene) of E.coli isolates using Polymerase Chain Reaction ( PCR) assay and gel electrophoresis.

Collection of samples:
This study was carried out during the period 25 th Feb.2012 to 17 th June 2012. A total of two hundred fourteen (214) urine specimens were collected from infants and children, aged one day-14 years attending (Rapareen Teaching Hospital for Children in Erbil City). Provisional diagnosis of urinary tract infections made by pediatrician. All urine specimens were obtained by mid stream cleancatch, catheterization or from urine bags. The specimens were transferred to the laboratory and processed within half an hour of collection.

Microscopic examination:
For microscopic examination, about 5ml of urine was centrifuged (2000 rpm for 5 min.), the supernatant was discarded and a drop (o.1ml) of the sediment was placed on a clean microscopic slide. The preparation was examined under high power (x40) lens. The common finding expected to be seen are: Pus cell (white blood cell), red blood cell (RBC), casts, crystals, epithelial cells, bacteria and parasites.

Leukocyte esterase test (LE):
This test was used to detect leukocyte esterase enzyme released from leukocytes in urine using urine test strip. The positive result depend on changing the color of strip from yellow to deep brown [11].

Urine culture (isolation of E.coli):
A loop full of undiluted urine samples was spreaded on appropriate culture media (blood, MacConkey agar and CLED agar) plates. The plates were incubated overnight at 37C°. Well isolated single colonies were sub-cultured on the same media to check for the purity of the isolated bacteria. Purified isolates were identified using microscopical, morphological, biochemical and Vitek 2 system as a more accurate method for identification.

Bacterial count and significant bacteriuria:
Urine culture is based on colony forming units per ml (CFU/ ml). The diagnosis of urinary tract infection is based on a positive urine culture of ≥ 10 5 colony-forming units/ml [12].The  [11].

Antimicrobial Susceptibility Testing:
The antibiotic resistance pattern of seventy (70) E.coli isolates from urine specimens and laboratory strain E.coli DH5oe were screened for their resistance to thirteen widely used antibiotics. Disc diffusion method, also known as the Kirby-Bauer-method were used [ 13].The

Detection of extended spectrum β-lactamases (ESBLs):
A double disc diffusion test was performed with amoxicillin-clavulanic acid surrounded by aztreonam and third generation cephalosporin discs cefotaxime and ceftazidime [14,15].

Isolation of plasmid DNA content from bacterial isolates understudy:
DNA spin™ Plasmid DNA Purification Kits (Intron/ Korea) procedure is based on alkaline lysis method of bacterial cells followed by adsorption of DNA onto silca in the presence of high salt.

Study of plasmid profile purified from E.coli isolates agarose gel electrophoresis technique:
The method described by [16] with few modifications was followed and include the following steps: The edges of a clean, dry glass plate sealed with tape to form a mold. Samples of DNA were mixed with appropriate volume of loading buffer (bromophenol blue).
The sample mixture slowly loaded into the slotes of submerged gels using a disposable micropipette. Size standards was loaded into slotes on both the right and left sides on the gels.
The DNA will migrate toward the positive anode at 10 volt / cm for 1 hour, the gel run until the samples have migrated an appropriate distance through the gel. The electric current turned-off and the lid was removed. Finally we examined the gel by UV-transilluminator, and then photographed .

3.RESULTS AND DISCUSSION
Prevalence of microorganisms isolated from urine samples in relation to gender :  These results do agree with those reported by [37] who found that amikacin and nitrofurnatoin showed good activity against uropathogen isolates. (38) showed that the resistance to nitrofurantoin which is one of the oldest urinary anti-infective drugs in use remains minima.

Antibiotic resistance pattern
[39] stated that the amikacin have good anti-Pseudomonas activity and other bacteria that are resistant to other antibiotics, and is usually recommended for serious UTI. Our results showed that most isolates were highly sensitive to ciprofloxacin. These results parallel with those reported by [40[ and [41]. This is due to that the ciprofloxacin is newly used in treatment in   resistance to other classes of antibiotics may reside within the same plasmid and therefore be spread together. Plasmid profiling is also an important tool for epidemiological typing and has got diagnostic value as well.

Plasmid profile of E.coli isolates
The results revealed that among 70 E. coli isolates, 64 (91.6 )% isolates harbored from one to seven bands and the size of the bands ranged from 1Kbp to more than 10Kbp . Figure (1    Many researchers demonstrated the mechanism by which the elevated temperature create curing of plasmid DNA, of these, the effect of elevated temperature on plasmid curing may be due to decreasing the amount of synthesized DNA. This may be due to the fact that the enzymes which contribute in the DNA replication processes are more affected by this high temperature.