Escherichia coli (E. coli)is the prototype of the large bacterial
family Enterobacteriaceae. It is facultatively anaerobic with both fermentative
and respiratory type of metabolism. E. coli is one of the most frequent
causes of some of the many common bacterial infections of man such as urinary
tract infection, neonatal meningitis, choleycystitis, bacteremia, cholangitis,
travelers diarrhea and pneumonia (Ochoa and Cleary,
2003). In the past the isolation of E. coli was done by simple methods.
Clinical specimens may be stained by Grams method for microscopical examination
or cultured on MacConkeys agar or other suitable media. In the case of
suspected urinary tract infection, cultures are semi quantitative (Chart,
Much of the past practice and thinking about E. coli were based on the
classical views and behavior of the organism. These views are changing rapidly
under the influence of accumulating fundamental molecular knowledge. Currently,
molecular techniques are finding an increasing use in the diagnoses of E.
coli. The most widely used method is the Polymerase Chain Reaction (PCR).
Not only does this technique provide tools for highly sensitive and specific
detection of the organism in clinical specimens, but certain characteristics
including virulence, toxins and antimicrobial resistant genes may also be determined
(Ram et al., 2008). Compared with the classical
urine culture methods, PCR is more rapid and can detect smaller number or fragments
of bacteria; which would otherwise undetectable (Yoshimasa,
A number of modifications have been made to the standard PCR reaction the most
important of which is the real-time PCR which has expanded the use of the technique
and broadened the spectrum of the microorganism that may be detected. The technique,
in addition to being a closed system, is highly sensitive, rapid and accurate
The present study was designed, essentially, to establish real-time PCR for the detection of E. coli directly in clinical specimens in the Sudan.
MATERIALS AND METHODS
This study was conducted during the period May 2007 to March 2008. A total of 46 patients (24 males and 22 females) attending Khartoum, Omdurman and Khartoum North Teaching Hospitals and the National Health Laboratory suffering from urinary tract infection were enrolled in this study. Urine specimens (n = 46) were collected from each patient. The age of patients ranged between five years to eighty years. Urine specimens were collected in sterile containers, without preservatives, transported to the laboratory and immediately processed.
Bacterial DNA was extracted directly from each urine specimen using the
Phenol-Chloroform method as described by Snounou et al.
(1993) with some modifications. Five milliliter of urine and 10 mL of red
cell lysis buffer (RCLB) were transferred under aseptic conditions and centrifuged
at 6000 rpm for 5 min. The supernatant was discarded and the deposit was re-suspended
in 800 μL of white blood cell lysis buffer (WCLB) containing 10 μL
of Proteinase k (10 mg mL-1) and incubated overnight at 37°C.
Equal volume of phenol/chloroform/isoamyl alcohol (PCI) was added. The suspension,
mixed thoroughly on a vortex shaker, was centrifuged at 6000 rpm for 5 min.
The upper layer was transferred to a clean eppendorf tube and an equal volume
of chloroform/isoamyl alcohol (CI) was added, mixed on a vortex shaker and centrifuged
for 5 min at 6000 rpm. The upper layer was transferred to a clean eppendorf
tube, 2 volumes of 95% cold ethanol and 1:10 of sample volume of 3M Na acetate
were added and the mixture was incubated at -20°C over night prior to centrifugation
for 10 min at 12000 rpm. The supernatant was discarded and the pellet was re-suspended
in 8.2 mL of 70% ethanol. The suspension was centrifuged for 7 min at 12000
rpm and the supernatant was discarded. The last step was repeated, the supernatant
was discarded and the pellet was air dried for 15 min, dissolved in sterilized
distilled water (100 μL) and stored at -20°C till used.
The DNA amplification and analysis were carried out using Thermocycler (Techne-Quantica).
One set of primer (Left Primer 5AGGCAGCAAATGAATTACGC 3 and Right
Primer 5AGCCTGTTGACGCTCTTCAT 3) was used. 2X sensimix NORef DNA
Kit comprising 2Xsensimix NORef that contains reaction buffer, heat- activated
Taq DNA polymerase, dNTPs, 6 Mm MgCl2, stabilizers and SYBER Green
dye was utilized in this study. For a 100 reaction/plate, sterilized distilled
water, 2X sensimix, 100 μL forward primer, reverse primer and SYBER Green
dye were mixed in sterile eppndorf tube under sterile condition (Clean BenchD Lab Tech). The reagents were added according to manufacturers recommendation
with some modifications as follows: 500 μL H2O, 1250 μL
2X sensimix, 3-100 μL forward primer, 100 μL reverse primer and 5-50
μL SYBER Green dye. The plate was prepared as follows: 20 μL of master
mix was placed on the wall of each well using automatic pipette, and a 5 μL
aliquot of sample was placed on the other wall of the same well. Samples were
made in duplicate. E. coli genomic DNA and distilled water (5 μL
each) were added in two wells as a positive and negative control respectively
for comparison. Finally, the plate was sealed by a sealing machine (Thermosealer-AB
Gene-Combi Ltd.). For DNA amplification, the Thermocycler was programmed to
denaturation at 95°C for 600 sec, amplification at 95°C for 30 sec,
annealing at 58°C for 30 sec and extension step at 72°C for 30 sec).
RESULTS AND DISCUSSION
Forty six specimens were collected from patients attending Khartoum Teaching
Hospital, Omdurman Teaching Hospital, Khartoum North Teaching Hospital and National
Health Laboratory (NHL). The majority of specimens 20 (43.5%) were obtained
from NHL and so the positive specimens 13(61.8%) (Table 1).
||Distribution of positive results according to the hospital
||Real-time PCR curve for positive control
||Real-time PCR curve for negative control
Real-time PCR sigmoid curves for specimens showing positive
infection by E. coli
||Distribution of positive results according to sex
||Distribution of positive results according to the age groups
Using real time PCR the overall results showed that 45.7% of the cases examined
were positive for E. coli with relative fluorescence >12,000 (Fig.
3) and the rests were negative with no relative fluorescence detected (Fig.
1, 2). The number of positive samples for females and
males were 13 (61.9%) and 8 (38.1%), respectively (Table 2).
The study clearly indicate the prevalence of E. coli among urinary tract
infected patients. This finding is in line with that of Alizadeh
et al. (2007) who reported that 39% of the urinary tract infections
were caused by E. coli. Similar results were reported by Hinata
et al. (2004), who ascertained the high prevalence (84%) of E.
coli among urinary tract infected patients. Furthermore, our findings are
in conformity with those of Hinata et al. (2004),
who indicated a higher prevalence of E. coli in urine collected from
female urinary tract infected patients.
Our results taken in conjunction with those of Alizadeh
et al. (2007) and Hinata et al.
(2004) suggested that E. coli is a major causative agent of urinary
tract infection at least in many parts of the world.
The study of Hinata et al. (2004), examined
200 urinary tract infected patients, who showed a close similarity in E.
coli prevalence levels using real- time PCR and the conventional culture
technique. However, real- time PCR is advantageous as it is simple, more rapid,
highly sensitive and more quantitative than the conventional culture techniques
in the diagnosis of E. coli UTI.
Based on the results of this study it could be concluded that urine samples
collected from patients attending the National Health Laboratory had the highest
frequency of E. coli infection (61.8%). The age group of adult is more
exposed to E. coli infection (56.5%), while the age group of children
is less exposed (13.1% E. coli infection) (Table 3).
The real-time PCR technique is more sensitive, specific, rapid and it can easily
be adopted as a routine work in hospitals settings in the Sudan.