Extended Spectrum Beta-Lactamase-Producing Uropathogenic Escherichia coli in Pregnant Women Diagnosed With Urinary Tract Infections in South-Western Nigeria

Extended spectrum beta-lactamase (ESBL)-producing uropathogenic Escherichia coli (UPEC) in symptomatic pregnant women with confirmed urinary tract infections in Southwest. Nigeria was reported. Susceptibility of UPEC isolates to β-lactam and other classes of antibiotics was determined by the Kirby-Bauer’s disc diffusion method on Mueller-Hinton agar plate. Detection of plasmid DNA in multiple antibiotic resistant isolates was carried out by alkali lysis (TENS) method. Extended-spectrum-β-lactamase (ESBL) production was determined by double disk synergy test (DDST). Isolates that were positive to ESBL were mated with non-ESBLproducing E. coli and other enterics in a conjugation experiment. Transfer of ESBL-enzyme and other resistance phenotypes in the transconjugants was investigated by DDST. Data obtained were statistically analyzed using SPSS 17. Greater percentage of the isolates were multiple antibiotic resistant (MAR). Sixty-nine (26.1 %) of UPEC were ESBL producers. Some of the ESBL producers transferred ESBLenzyme and other resistance determinants to the recipients. Large size plasmid DNA of molecular weight (23.13-33.04 kb) was detected in some representative MAR isolates.


Introduction
Escherichia coli have been reported to be common causes of hospital acquired infections which can have severe clinical implications with corresponding multiple antibiotic resistance (Aibinu et al., 2002).Extended spectrum β-lactamases (ESBLs) are plasmid mediated enzymes that confer resistance to penicillin, third generation cephalosporins and aztreonam but are inhibited by clavulanic acid (Paterson and Bonomo, 2005).In Africa, ESBLs-producing bacteria have been reported in Egypt, Morocco, Tunisia, Senegal and South Africa (Bloomberg et al., 2005).
Extended-spectrum β-lactamase (ESBL)-producing enterobacteriaceae have become widespread in hospitals and are increasing in community settings where they cause a variety of infections.In addition to hydrolyzing most β-lactam agents, bacteria harboring these enzymes display resistance to other unrelated antimicrobial agents and thus often pose a therapeutic dilemma (Maina et al., 2013).Increase in ESBL-producing enteric Gram-negative bacteria has led to the choice of inappropriate therapy; as a result, the rate of resistance has increased.Antibiotic therapy of infections due to ESBL-producing pathogens is still a clinical challenge.In most cases, carbapenems and fluoroquinolones have been used (Ramphal & Ambrose, 2006).
Horizontal gene transfer by plasmid exchange between E. coli strains is a recognized source of rapid spread of antimicrobial resistance phenotypes (Fang et al., 2006).The significance of plasmids in disseminating antimicrobial resistance genes is further enhanced by the association of plasmids with mobile genetic elements, such as transposons, integrons and insertion elements (Pitout et al., 2007).Resistance to third generation cephalosporins, ciprofloxacin, trimethoprim-sulfamethoxazole, gentamicin and amikacin was detected in the ESBL-producer group.Selective pressure of the antimicrobials selects those strains that are resistant to the applied antimicrobials causing the resistant strains to multiply and spread.In the last decade, CTX-M enzymes have replaced TEM and SHV mutants as the most prevalent ESBLs worldwide, with E. coli being the major host (Livermore et al., 2007;Paterson & Bonomo, 2005).Extended spectrum beta-lactamases production have been reported among E. coli in both hospital and community settings ( (Pitout et al., 2007)).They have also been detected in pets and farm animals, products of the food chain and sewage (Warren et al., 2008).The resistance rate of E. coli to third generation cephalosporins (3GCs) is a broad indicator of the occurrence of ESBLs.It has been discovered that, travellers to countries with high rates of ESBLPCs (e.g.Egypt, India, etc) readily acquire asymptomatic faecal carriage.
Extended spectrum β-lactamase-producing E. coli and other enterobacteriaceae, particularly those producing CTX-M, have spread rapidly among humans and there is evidence of spread among animal populations.Factors that influence the spread of resistance genes as well as resistant bacteria include, antimicrobial usage, co-selection for resistance genes A number of risk factors of acquiring ESBL-producing bacteria have been identified in hospitalized patients, most of which also apply to other multi-resistant Gram-negative bacilli.These risk factors include: prolonged hospital stay; prior hospitalization; previous use of 3GCs, aminoglycosides and quinolones; presence of medical devices such as urinary catheters and mechanical ventilation (Rodriguez-Bano et al., 2006).In the case of community acquired ESBL infections, older age, female gender, recurrent UTIs/prior invasive procedures (e.g.catheterisation), known faecal carriage, contact with healthcare facilities/residents in care homes and previous antimicrobial treatment are the risk factors (Moor et al., 2008).
Travellers to areas of the world such as India where very high rates of ESBL are present, have been noted to become readily colonized, asymptomatically, with CTX-M-producing ESBL strains (Tham et al., 2010).The present study reports the prevalence of ESBL-producing UPEC in pregnant women with confirmed UTIs in Ondo and Ekiti States, south-western Nigeria.

Study Design and Sample Collection
The study population include symptomatic UTIs pregnant women in Ekiti and Ondo States, South-western Nigeria.Ekiti State is located between longitudes 40°51′ and 50°451′ east of the Greenwich meridian and latitudes 70°151′ and 80°51′ north of the Equator while Ondo State lies between longitudes 4"30" and 6" East of the Greenwich Meridian, 5" 45" and 8" 15" North of the equators The preliminary identification of E. coli recovered from 400 voided mid-stream urine samples of symptomatic pregnant women with confirmed cases of UTIs was based on colonial morphology by a characteristic green metallic sheen on EMB agar (Oxoid Ltd., Hampshire, England) plate.The identity of isolate was confirmed by various conventional biochemical tests with reference to Bergey's Manual of Determinative Bacteriology (Holt et al., 1994).

Detection of β-lactamase Enzymes Producing E. coli
Extended spectrum β-lactamase production among the isolates was detected following the double-disk synergy technique (DDST) (Clinical and Laboratory Standards Institute (CLSI), 2012).The standardized isolates were subjected to double-disk synergy tests (DDST) on sterile Mueller Hinton agar plates.Escherichia coli ATCC 25922 was used as reference.The test was performed by placing ceftazidime (30 µg) and cefotaxime (30 µg) at 20 mm (center to center) from a centre disk containing augmentin (30 µg) (amoxicillin (20 µg) plus clavulanate (10 µg).Enhancement or potentiation in the zone of inhibition of any of the antibiotic disks toward the center disk containing clavulanic acid indicates the presence of ESBLs (Clinical and Laboratory Standards Institute (CLSI), 2012).

In Vitro Conjugation Experiment
Minimum inhibitory concentrations of the antibiotics (augmentin, amoxicillin, cefadroxil, Cefotaxime, cefepime, ceftazidime, cefuroxime, alidixicAcid, ciprofloxacin, ofloxacin, pefloxacin, gentamicin, tetracycline, cotrimoxazole) used in the conjugation experiments were determined by agar dilution method as prescribed by CLSI.[6]Conjugation experiment was performed by the mating assay using the method of Aibinu et al. (2003) in tryptic soy broth for six of the ESBL-producing E. coli isolates as donors and non-ESBL-producing E. coli ATCC 25922 and other enterobacteriaceae (Klebsiella sp, Proteus sp., Salmonella sp. and Shigella sp) as recipients.The donors were tested and confirmed to be sensitive to the antibiotic resistance markers on MHA plates.A suspension of each organism was made in the sterile double strength nutrient broth at 37°C and adjusted to 0.5 McFarland Standard.The donor and the recipient were then mixed in a ratio 1:9 (50 μl of the donor to 450 μl of the recipient) and incubated at room temperature for 3 h for conjugation to take place.
One milliliter of each conjugated samples was serially diluted (10-folds) and 0.1 ml from 10 -3 and 10 -4 dilution fractions was spread inoculated onto the surface MacConkey, eosin methylene blue agar and Salmonella-Shigella agar plates supplemented with appropriate minimum inhibitory concentration of antibiotics used as the recipients' markers.Transfer of resistance was read by observation of recovery of the recipient colonies on the agar plates containing the corresponding antibiotics.The transconjugants were subjected to DDST to confirm the transfer of ESBL enzymes and co-transfer of other resistance determinants present in the donor isolates.

Plasmid Profiling
Plasmid DNA extraction of selected multiple antibiotic resistant ESBL-producing isolates was performed using the alkaline lysis 'TENS' (Tris 25 mM, EDTA 10 mM, 0.1 N NaOH and 0.5% SDS-all Sigma products) method of Kraft et al. (1988) and Lech et al. (1987).The extracted plasmid DNA was separated on 0.8% agarose gel (Oxoid, Basingstoke, England) in a 20-40 µl of TE (Tris-EDTA) buffer and a 100 bp ladder (Promega, Madison, USA) was used as standard.The electrophoretic products were viewed using ultraviolet trans-illuminator and the plasmid size was compared to the reference marker.

Statistical Analysis
Significant differences and relationship between the prevalence of ESBL-producing UPEC strains in pregnant women in the study areas were compared using analysis of variance (SPSS 17 version).A value of p < 0.05 was set as significant   1).

Results
Each of the ESBL producers was resistant to more than one class of antibiotic.The ESBL-producing UPEC (52.2%) were resistant to all the six classes of antibiotics tested, 26.1% to five, 15.9% to four, and 5.8% to three classes (Table2).
Figure 1 shows the percentage rate of transfer of resistance phenotypes to the transconjugants.Extended spectrum β-lactamase enzyme and other antibiotic resistance phenotypes were transferred to non-ESBL-producing E. coli, Salmonella sp., Shigella sp., Proteus vulgaris and Klebsiella sp.recipients.Consequently, resistance to other antibiotics was also transferred in the same trend, augmentin, cefotaxime and ceftazidime were 100% transferred to the transconjugants, followed by amoxicillin (98.0%), tetracycline (94.0%) and cotrimoxazole (90.0%).However, pefloxacin, ciprofloxacin, ofloxacin and nalidixic acid resistance traits were not transferred (Figure 1).Molecular weights of plasmid DNA in the representative MAR -UPEC isolates are presented in table 4 and the gel electrophoresis of the amplified plasmid DNA is shown in Figure 2. Large plasmid size of molecular weights ranging from 23.13-33.04kb were harboured by the selected MAR isolates, and 62.5% of the isolates were ESBL-producing strains.Some of the non-β-lactam antibiotic resistant isolates had same plasmid DNA size as those of ESBL-producing UPEC (Table 3).
In Japan, Korea and Hong Kong, the percentage of ESBL production among E. coli was also low (Ho et al., 2000).
In South and Eastern Nigeria, prevalence of ESBL among E. coli isolated from pregnant patients from 2003-2007 was very high (52.4%)(Aibinu et al., 2003;Iroha et al., 2008) and is at variance with the findings of the present study.This may likely due to geographical location and antimicrobial usage.The implication of ESBL-producing UPEC in pregnant women may include prolonged stay in the hospital, cost, treatment failure and relapsed cases.A number of risk factors of acquiring ESBL-producing bacteria have been identified in hospitalized patients, most of which also apply to other multi-resistant Gram-negative bacilli.These risk factors include: prolonged hospital stay; prior hospitalization; previous use of 3GCs, aminoglycosides and quinolones; presence of medical devices such as urinary catheters and mechanical ventilation (Rooney et al., 2009).In the case of community acquired ESBL infections, older age, female gender, recurrent UTIs/prior invasive procedures (e.g.catheterization), known faecal carriage, contact with healthcare facilities/residents in care homes and previous antimicrobial treatment are the risk factors (Moor et al., 2008).Transfer of ESBL enzyme and other antibiotic resistance phenotypes in some of the isolates is plasmid-linked.This may be due to the fact that the ESBL genes were located on transposable element or integron thus resulting in transfer function (Akortha et al., 2011).Inter-generic transfer rate in this study is lower than intra species transfer rate.This has been linked to fertility inhibition, incompatibility, inability to synthesize adhesion or narrow host range (Akortha, 2009).Transmission in the community is probably quite complicated.Individuals in long-term care homes where high carriage rates of CTX-M producing enterobacteriaceae have been observed may spread strains (Rooney et al., 2009) to other noncare-home residents.The evidence of a significant spread amongst household contacts has been presented in a Spanish study which showed that 70% of index cases of patients with ESBL-producing strains causing UTI in the community had positive contacts with 16.7% of their household members.The finding of this study explains co-selection of resistance as resistance to other non-β-lactam antibiotics is located on the same plasmid as ESBL factor.
The presence of plasmid-mediated ESBL resistance among the isolates in the present study is an evidence of its transfer capability of ESBL and other resistance phenotypes between its species and other genera.This implies that under favourable condition, horizontal gene transfer of resistance plasmid by conjugation could occur in vivo (Yah et al., 2008).This indicates that plasmid carrying ESBL gene in one bacterium can spread rapidly to members of the same or organisms of different species in the same or different individual.Greater percentage of UPEC in the study area MAR capable of transferring ESBL enzymes and other resistance phenotypes.

Conclusion
In conclusion, antibiotic resistance remains one of the nature's never ending process whereby organisms develop tolerance to new environmental condition.The development and spread of ESBL-producing UPEC and horizontal transfer of resistance in the study is of great concern especially in therapeutic management of UPEC-induced urinary tract infections.

Figure 2 .
Figure 2. Gel electrophoresis of the amplified plasmid DNA of selected multiple antibiotic resistant uropathogenic E. coli in Ondo and Ekiti Sates Lane M: DNA marker, Lanes 1-24: the test isolates.

Table 1 .
Prevalence of antibiotic resistance among ESBL-producing uropathogenic E. coli in pregnant women with UTI in Ondo and Ekiti States Key: MAR= Multiple antibiotic resistant; ESBL= Extended spectrum β-lactamase.

Table 2 .
Multiple antibiotic resistance (MAR) among ESBL-producing uropathogenic E. coli in pregnant women with UTIs in Ondo and Ekiti StatesTable1shows the prevalence of antibiotic resistance among ESBL-producing UPEC isolates in Ondo and Ekiti States.Sixty-nine (26.1%) of the UPEC were ESBL-producing strains with concomitant multiple antibiotic resistance profiles.Isolates that produced ESBL-enzymes showed high rate of resistance to the extended spectrum β-lactam and other classes of antibiotics as well.There was a significant statistical difference in the incidence of resistance between ESBL-and non-ESBL-producing E. coli isolates in Ondo and Ekiti States (P ˂ 0.05) (Table

Table 3 .
The molecular weight of plasmid DNA in the representative MAR uropathogenic E. coli in pregnant women with UTIs in Ondo and Ekiti States Yushau et al. (2007)L enzymes detected in E. coli from UTI patients in the East, South and Northern Nigeria, respectively.Prevalence of ESBL-producing UPEC in this study is higher than reports from other countries and other parts of Nigeria.For instance, in Cameroun, Gangoue-Pieboji (2005) reported 12.0% prevalence of E. coli with ESBL, El-Khizzi and Bakheshwain (2006) obtained 15.8% prevalence in Riyadh, Saudi Arabia andYushau et al. (2007)reported 9.3% prevalence in Kano, Nigeria.In general, prevalence of ESBLs in E. coli in UTIs cases varies from country to country and from health institution to another