Genetic basis of multiple drug resistant Escherichia coli from urine samples in Ikpoba Okha Local Government Area of Edo State , Nigeria

This study was aimed at characterizing bacteria at the molecular level and determining the genetic basis of the multidrug resistant bacteria isolates. Bacteria were isolated from eighty urine samples from urinary tract infection patients. Phenotypically identified isolates of Escherichia coli were selected. Multiple drug resistant (MDR) bacteria were generated from the isolates by carrying out antibiotic susceptibility tests using the Kirby-Bauer disc diffusion technique. MDR bacteria were selected and molecular characterization using polymerase chain reaction technique with species-specific primers was performed for confirming microbial identity. Plasmid DNA profiling was carried out to detect MDR Escherichia coli with plasmid and subsequent plasmid curing of isolates. The polymerase chain reaction result showed that all twenty isolates were Escherichia coli, fourteen out of the twenty isolates were multiple drug resistant Escherichia coli. The gel electrophoresis indicated that eleven out of the fourteen multiple drug resistant Escherichia coli contains plasmids with a molecular weight of 48.5kb. The E. coli isolates that habors plasmid were cured of the plasmids and the results of the antibiotics sensitivity tests showed that E. coli that showed resistance before curing became sensitive after curing. This study has shown that multidrug resistant E. coli are plasmid mediated in this locality.


Introduction
Urinary tract infection is one of the significant illnesses that cause burden of national concern.Due to widespread and careless use of antibiotics at community level, we encountered more and more resistance pattern of micro-organisms to common antibiotics [1].Urinary tract infections (UTIs) are among the most widespread infectious diseases of humans and a chief cause of morbidity and mortality [2].It is estimated that 40-50 % of healthy adult women have experienced at least one UTI episode [3,4].UTI has become the most common hospital acquired infection, accounting for as many as 35.0% of nosocomial infections, and it is the second most common cause of bacteraemia in hospitalized patients [5].Previous reports have also suggested that UTI can occur in both males and females of any age [6,7].The leading causes of acute and uncomplicated UTI in patients have been reported to be due to Escherichia coli, Staphylococcus aureus, Proteus spp., Klebsiella spp.and Pseudomonas aeruginosa [8].Escherichia coli are the most common organism associated with asymptomatic bacteriuria [9].Escherichia coli are responsible for more than 80.0% of all UTIs and causes both asymptomatic and symptomatic UTI [9].The main cause predisposing an individual to urinary tract infection has been attributed to poor personal hygiene and culture habit imposition [8].
Multiple drug resistance isolates causing UTI has serious implications regarding therapy against pathogenic isolates and for probable co-selection of antimicrobial resistance mediated by multiple drug resistant plasmids [10].E. coli from clinical isolates are known to harbor plasmids of different molecular sizes.It has been widely reported that bacteria harbor antibiotic resistant genes which can be horizontally transferred to other bacteria [11].The widespread occurrence of drug resistant E. coli and other pathogens in our environment has necessitated the need for regular monitoring of antibiotics susceptibility trends to provide the basis for developing rational prescription programs, making policy decisions and assessing the effectiveness of both [12].Therefore, this study was carried out to molecularly identify, determine antibiotic sensitivity pattern and plasmid profile of multidrug resistant Escherichia coli isolated from Urinary tract infection patients in Ikpoba Okha Local Governmen Area, Nigeria.

Sample collection
A total of 80 clean-voided, mid-stream urine samples of about 20ml were collected from both inpatient and outpatient attending Lahor Medical Centre in Benin City with their respective bio-data.Urine samples were collected in sterile universal bottles.

Ethical clearance
Approval was obtained from the Medical Director of the hospital whose patients participated in this study and the patients gave their consent after being informed of the objectives of study.

Bacteriological procedures/identification of isolates
Specimens were aseptically inoculated onto MacConkey, Blood and Nutrient agar and incubated aerobically at 37C for 24 hours and observed for colonial growth.Isolates were screened for Escherichia coli.The specimens were processed at Lahor research Laboratories, Benin City, Nigeria using standard microbiological methods.All isolates were identified using conventional techniques [13] and confirmed with polymerase chain reaction technique.

Antibiotic susceptibility testing
The susceptibility of isolates to commonly used antibiotic was determined by the Kirby-Bauer disk diffusion method for in vitro antibiotic susceptibility as described by NCCL (2002), against the following antibiotics for Gram negative bacteria include: Augmentin (AUG, 30µg), Ofloxacin (OFL 5µg), Cefixime (CXM 5µg), Gentamycin (GEN 30µg), Cefuroxime (CRX 30µg), Ceftazidime (CAZ 30µg), Ciprofloxacin (CPR 5µg), Nitrofurantion (NIT 300µg).The concentrations of antimicrobial sensitivity and interpretation of zones of inhibition were in accordance to Performance Standards for antimicrobial disk susceptibility tests of Clinical and Laboratory Standards Institute [14].

Bacteria genomic DNA extraction
Multiple drug resistant bacteria isolates were subcultured overnight in Luria-Bertani broth (Merck, Germany) and genomic DNA was extracted from typical colonies of Escherichia coli using Zymo research fungi/bacterial DNA MiniPrep DNA extraction kit (Irvine, CA, USA), according to manufacturer's instructions.

Identification of Escherichia coli using polymerase chain reaction technique
Polymerase Chain Reaction (PCR) was used for the amplification of Escherichia coli species-specific genes.Forward and reverse primers (URF-301-TGTTACGTCCTGTAGAAAGCCC; URR-432-AAAACTGCCTGGCACAGCAATT) were used in a Peltier thermal cycler PCR machine at Lahor Research Laboratories, Benin City, Nigeria.Quick load OneTaq one-step PCR master 2x (New England Biolab, USA) was purchased from lnqaba Biotech, Hartfield, South Africa incorporated and used according to the manufacturer's instruction.The PCR was performed in 25 µl reaction mixture containing 12.5 µl Quick load OneTaq one-step PCR master mix (2x), 1.25 µl of each species-specific forward primer (20 µM), 1.25 µl of each species-specific reverse primer (20 µM), 5.0 µl of nuclease free water and 5 µl of DNA template was added last.The PCR was started immediately as follows: Initial denaturation at 94 C for 3 minutes, denaturation at 94 C for 30 seconds, annealing at 55 C for 30 seconds, extension at 72 C for 1min, for 35 cycles, final extension at 72 C for 15 minutes and final holding at 4 C forever.Ten microliters (10 µl) of the amplified PCR products were fractionated on a 1.0% agarose gel containing ethidium bromide in Tris/Borate/EDTA (TBE) Buffer.Electrophoresis was performed at 90 volts for 60 minutes.Products were visualized by Wealth Dolphin Doc UV transilluminator and photographed.Molecular weights were calculated using molecular weight standard maker.

Plasmid DNA extraction and gel electrophoresis
Plasmid isolation was carried out using a commercial plasmid isolation kit (ZR Plasmid Miniprep Classic, USA) and the process was carried out according to the manufacturer instructions as described by D'Anteo [15].Isolated plasmids were thereafter electrophoresed in a horizontal tank at a constant voltage of 90V for 60 minutes.After electrophoresis, plasmid DNA bands were viewed under UV transilluminator and photographed.Molecular weights were calculated using molecular weight standard maker which ranged in size from 0.5kb to 48.5kb, and results recorded.

Plasmid curing procedure
Multidrug resistant bacteria isolates were subjected to plasmid curing experiment using procedure as described by Ehiaghe et al. [16].Freshly prepared nutrient broth (9 ml) was inoculated with 1 ml overnight culture that was grown in Luria broth (LB) containing antibiotics for 24 hours at 37 C.The resultant mixture was incubated for 4 hours to allow for minimal growth of the microorganisms.Aliquot of 1 ml of the 10 % sodium dodecyl sulphate curing agent was added to 9 ml nutrient broth culture, and was incubated at 37 C for 24 hours.The cured culture of 1 ml was inoculated unto 9 ml freshly prepared nutrient broth and incubated at 37 C for 24 hours.The overnight broth culture was then used to carry out post susceptibility test on Muller-Hinton agar plate with the necessary antibiotic discs placed and incubated for 24 hours at 37 C.

Results and discussion
The prevalence of antimicrobial resistance among microorganisms that cause UTI is increasing worldwide and is a major factor in selecting antibiotics for treatment.There are local variations in the antimicrobial susceptibility among urinary pathogens in different hospitals.Diagnosis of UTI is a good example of the need for close cooperation between the clinician and the microbiologist.The present study gives an insight about the common trend of increased resistance of uropathogens in this region which may be due to geographical variation or indiscriminate or sub lethal use of antibiotics.In this study, a total of 80 urine samples were collected.Of these, 20 (25%) isolates were found to be E. coli.All the 20 isolates revealed characteristic features of E. coli, which were Gram negative bacilli, produced pink lactose fermenting colonies on MacConkey agar and characteristic greenish metallic sheen on Eosin methylene blue agar.All the isolates showed typical IMViC pattern of E. coli viz., Indole and Methyl Red tests positive, Voges Proskauer and Citrate utilization tests negative.Malonate utilization was negative for all the isolates.These findings were in hormony with that of Arya [17].All isolates produce yellow colour colony on Triple Sugar Iron slant (TSI) as also reported by Lehman [18].Glucose fermentation test positive for all the isolates and all isolates were motile which was in line with the report by Mittal et al. [19], they were positive for nitrate reduction test as also shown by Tiso and Schechter [20]. .Thus, all the isolates were confirmed as E. coli by PCR.This corroborate with the result reported by Bejet al. [21] in the detection of low levels of microorganisms in environmental samples by using polymerase chain reaction.Antibiotic sensitivity testing of the isolates revealed that 14(70%) isolates out of the twenty Escherichia coli were found to be multidrug resistant isolates (Table 1).All isolates in this study were resistant to between 3 and 8 antimicrobial compounds, with different resistance profiles recognized.The resistance rate of E. coli in decreasing order are as follows: ceftazidime (75%), cefixime and ciprofloxacin (65%), gentamycin (60%), cefuroxime (55%) augmentin (50%), ofloxacin (45%), nitrofurantion (40%) as shown in table 2. Primarily, antimicrobial therapy is one of the primary control measures for reducing morbidity and mortality due to UTI.However, majority of E. coli isolates tested in this study demonstrated resistance to ceftazidime (75%), cefixime and ciprofloxacin (65%), gentamycin (60%) and displayed multiple antimicrobial resistances as typified by resistance to as many as four different antimicrobial classes.These resistance levels are comparable to those previously reported for clinical E. coli isolates [22] and for both fecal and clinical E. coli isolates recovered from turkeys in West Virginia and Virginia [23].Similar resistances have been reported in other countries as well for E. coli isolated from avian species [24,25,26], with the majority of isolates exhibiting resistance to tetracyclines, sulfa drugs, and aminoglycosides.On the contrary, Patel et al. [27] reported moderate percent of isolates (33.33%) were found to be resistant to ciprofloxacin which was also similar to the findings of Paul et al. [28] who observed less than 30 percent of E. coli isolates resistant to ciprofloxacin in their study while moderately high percentage (65%) of isolates were found to be resistant to antibiotic ciprofloxacin in our study.It has been reported that pathogenic isolates of E. coli have relatively high potential for developing resistance [29].Escherichia coli isolates obtained from this research were more susceptible to nitrofurantion (60%) and ofloxacin (55%).It has been observed that antibiotic susceptibility of bacterial isolates is not constant, but dynamic and varies with time and environment [30].Bacteria resistance to specific antimicrobials is sometimes encoded by plasmids, which may distribute resistance in susceptible bacteria through horizontal gene transfer [31].Figure 3 and 4 showed the Agarose gel electrophoretic analysis of the plasmids DNA from the multiple drug resistant Escherichia coli isolates.Lane M, is the standard molecular marker used (0.5kb -48.5kbDNA ladder).Plasmid DNA analyses revealed that there were detectable plasmids in 10(71.4%) of the 14 multidrug resistant E. coli isolates tested.Four of the isolates possessed no plasmid.The ten isolates possessed single sized plasmids of the same molecular weight (48.5kb).E. coli isolates which harbors plasmid are: E1, E2, E4, E5, E6, E10, E11, E12, E13 and E15 while isolates E8, E16, E17 and E19 do not harbor plasmid.Resistance to antibiotics has been ascribed in most instances to the presence of plasmids [32].In the study carried out in Benin City, Nigeria, 11.4% of the Pseudomonas isolates was plasmid mediated, and were highly transferable with a frequency range of 2x10 -2 to 6x10 -4 [33].The emergence of R-plasmids in this study could be ascribed to the indiscriminate and widespread use of antibiotics caused by over the counter availability of antibiotics as well as the higher exposure of people to enteric flora in places with poor sanitation [34,35].Smith et al. [36] reported that 47 of the E. coli isolated from animals in Lagos harbors detectable plasmids which ranged in sizes from 0.564kb to >23kb.Danbara et al. [37] reported plasmids of sizes between 3.9kb and 50kb in E. coli strains isolated from traveller's diarrhoea.

Conclusion
This study showed that Escherichia coli were most susceptible to nitrofurantion and ofloxacin.It was observed that all Escherichia coli used in this study were resistant to cefixime, gentamycin, cefuroxine, ceftazidime and ciprofloxacin.This study has also highlighted the emergence of multidrug resistant R-plasmids among Escherichia coli causing urinary tract infections in Ikpoba Okha Local Government Area, Edo State, South-south Nigeria.Concerted strategies at monitoring and prescribing habits of clinicians, the diagnostic efficiency of laboratory scientist, the dispensing habits of pharmacists, the inappropriate use of antibiotics, as well as encouraging good hygienic measures could help curtail possible transmission of MDR E. coli infections within and outside the hospital environment.Our findings also indicate that the E. coli recovered in this study expressed high levels of resistance to antimicrobials that are commonly used in clinical medicine.This could contribute to the spread and persistence of antimicrobial resistant bacteria and resistance determinants in humans and the environment.

Figure 1
Figure 1 Polymerase chain reaction results for bacteria isolates analyzed with 1.0% agarose gel electrophoresis stained with ethidium bromide.L is 100 -1517 bp DNA ladder (molecular marker).Samples E1 -E10 are positive for Escherichia coli with band at 154 bp, NC: negative control Polymerase chain rection (PCR) was performed for identification of bacterial isolates.Escherichia coli species-specific set of primers (URF-301 and URR-432) gave amplicon of 154 bp in all isolates screened (figure 1, 2).Thus, all the isolates were confirmed as E. coli by PCR.This corroborate with the result reported by Bejet al.[21] in the detection of low levels of microorganisms in environmental samples by using polymerase chain reaction.

Figure 2
Figure 2 Polymerase chain reaction results for bacteria isolates analyzed with 1.0% agarose gel electrophoresis stained with ethidium bromide.L is 100-1517bp DNA ladder (molecular marker).Samples E11 -E20 are positive for Escherichia coli with band at 154 bp, NC: negative control

Figure 3
Figure 3 Agarose gel electrophoresis showing profiles of plasmid DNA of clinical isolates of Escherichia coli.Lane L is the molecular weight marker of size ranges from 0.5kb to 48.5kb.Isolates E1, E2, E4, E5, E6, E10, E11, E12 and E13shows plasmid bands at 48.5kb while isolate E8 do not show plasmid band.NC: negative control.

Figure 4
Figure 4 Agarose gel electrophoresis showing profiles of plasmid DNA of clinical isolates of Escherichia coli.Lane L is the molecular weight marker of sizes ranging from 0.5kb to 48.5kb.Isolate E15 shows plasmid band at 48.5kb while isolate E16, E17 and E19 do not show plasmid band.NC: negative control.

Table 1
Antibiotic sensitivity testing of Escherichia coli

Table 2
Antibiotic susceptibility pattern of Escherichia coli