Extensive use of antibacterials in clinical practice has been associated with increasing frequency of multi-resistant E. coli strains. Genetic elements such as Class 1 integrons have an important role in resistance development. In the current study, a total of 84 E. coli clinical isolates from Mansoura hospitals patients in Egypt were screened for antibacterial susceptibility against 12 different antibacterials. High resistance rates were identified for Ampicillin (92.9%) and sulfamethoxazole/ trimethoprim (84.5%). Class 1 integron was investigated in E. coli isolates by PCR. As a result, Class 1 integron was identified in 51.2% of these isolates. The contents of amplified integron varriable regions, were digested by Alu I restriction endonuclease. Cluster analysis of Class 1 integron digested varriable regions revealed that RFLP digested fragments generated could be classified into 9 different patterns, namely A, B, C, D, E, F, G, H and I. The most prevalent genotype was identifed in Group D. PCR and sequencing were used for detection of antimicrobial resistance genes harbored in integrons of Group D. As a result, main phylogenetic group identified harbored integron cassette carrying resistance gene for two antimicrobial groups namely aminoglycoside and trimethoprim. Multiresistance profiles in Group D exhibited association between antimicrobial resistance and integron presence. These findings suggest that the strategy for treatment of patients with E. coli infections needs to be revised. Furthermore, the high prevalence of Class 1 integron carrying gene confirms the importance of integron-mediated antimicrobial gene cassettes.
The level of antibiotic resistance among hospital and community-acquired isolates has steadily increased and has become a major global health problem. E. coli is responsible for numerous diseases in humans and animals [
In Enterobacteriaceae, conjugative plasmids and transposons are considered as the major agents of spread and maintenance of multidrug resistance genes within species and between different species. In addition, resistance genes can be integrated into DNA elements designated as integrons [
Multi-drug resistance in Enterobacteriaceae is strongly associated with integrons which confers resistance to different antimicrobial agents [
The rates of drug resistant clones of E. coli have been continously increasing over the last few years [
Eighty four clinical E. coli isolates were collected from Mansoura University hospitals namely: Emergency Hos- pital, Urology and Nephrology Center and Mansoura Specialized Medical Hospital in Dakahlia governorate, Egypt during March 2013 to February 2014. The isolates were collected from urine (53), wound (20) and sputum (11) samples. These isolates were identified using standard biotyping methods [
Susceptibility to fifteen antimicrobials was determined under the recommendations of the Clinical laboratory Standards Institute (CLSI, formerly NCCLS) guidelines [
Screening for integrons was carried out by polymerase chain reaction (PCR) amplification of IntM1 using the primer sets listed in
Primer | Sequence | Size of Amplicon (bp) | Targets | Gen Bank Number | References |
---|---|---|---|---|---|
IntM1-U | 5'-ACGAGCGCAAGGTTTCGGT-3' | 565 | Inti1 | AF550415 | [ |
IntM1-D | 5'-GAAAGGTCTGGTCATACATG-3' | ||||
5'-CS | 5'-GGCATACAAGCAGCAAGC-3' | Variable | Gene Cassette(s) of Class 1 Integron | U12338 | [ |
3'-CS | 5'-AAGCAGACTTGACCTGAT-3' |
nealing at 62˚C for 30 s, and extension at 72˚C for 1 min. Final extension was carried out at 72˚C for 5 min. PCR products were analyzed by agarose gel electrophoresis on 1% agarose gel (Sigma-Aldrich, USA) and visu- alized by ethidium bromide staining (Sigma-Aldrich, USA). All tests were twicely done for verification of re- sults.
Amplification of the variable region of Class 1 integrons was performed using two primers CS'3 and CS'5. The reaction mixture was prepared as described previously. PCR reactions began with 10 min of primary denaturation at 94˚C followed by 40 cycles of 94˚C for 30 s, 50˚C for 30 s and finally 72˚C for 30 s. Final extension was performed at 72˚C for 5 min.
To detect variation in size of the amplified integron variable regions, RFLP analysis was performed using Alu I (Fermentas, Lithuania) as digesting restriction endonuclease. Briefly, each 30 μl of the restriction mixture contained 1 μl (20 U) of enzyme, 7 μl of PCR-amplified product, 2 μl of enzyme buffer and 20 μl of double-distilled water. According to manufacturer’s guidelines, restriction mixtures were incubated at 37˚C for one hour and the digested PCR products were electrophoresed on 2% agarose gel at 90 V. Cluster analysis for analyzing the digested fragment was performed using the un-weighted pair-group method with average linkages (UPGMA).
Amplified gene fragments were purified from one strain in the most common RFLP pattern D using the PCR Purification Kit (MEGA quick-spin fragment DNA purification INTRON biotechnology, Sangdaewon-dong, Korea) for subsequent Sequencing. Purified PCR products were used as a template in sequencing reactions carried out with the ABI PRISM® BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Bio-Sys- tems, Foster City, USA). The reaction mixtures were analysed on an ABI 3730 DNA analyzer (Applied Bio- systems, Foster City, USA). Amplicons were sequenced on both strands and the predicted peptide sequences were analyzed by the online BLAST of the NCBI website software (http://www.ncbi.nlm.nih.gov/BLAST/).
Among the drugs under the study, ampicillin has the least antimicrobial effect as 92.9% of the total isolates were resistant. On the other hand, all E. coli isolates were sensitive to imipenem. In addition, the highest resistance rate was recorded to sulfamethoxazole/trimethoprim (84.5%), ceftriaxone (81%), ceftazidime (78.5%), norfloxacin and ciprofloxacin (71.4%), levofloxacin (60.7%), tobramycin (57%), Gentamicin (53.6%) and cefotaxime (46.4%). In contrast, resistance to amikacin was less common but was seen in only 20.2% of the isolates.
Class 1 integron was detected in 43 (51.2%) isolates.
A 2000 bp PCR product was obtained from 10 isolates, 1500 bp amplicon was obtained from 10 isolates, 6
isolates gave a 700 bp PCR product, an amplicon of 400 bp was obtained from 9 isolates, 800 bp amplicon was obtained from 4 isolates. 3 isolates gave 1800 bp and amplicon of 500 bp was obtained from one isolate.
For further classification of Class 1 integron fragments, the amplicons of the integron varriable region IVRs, were digested by Alu I restriction endonuclease (
The most prevalent restriction pattern in IVRs is identified in Group D which included 9 urine and 3 wound isolates. DNA of Class 1 integron fragment of E. coli isolates in group D was subjected to further analysis for identification of the gene sequence of such cassette.
DNA sequencing analysis indicated that these isolates harbored genes were (dfrA17)-(aadA5) which encode dihydrofolate reductase and aminoglycosides adenyltransferase, conferring resistance to trimethoprim and aminoglycosides respectively (
Antimicrobial resistance in human pathogens has become a major public health issue [
High incidence of multi-drug resistant (MDR) strains was also detected among the E. coli isolates in this study, where 84% of the isolates were resistant to at least three of the tested antimicrobials. The level of multidrug resistance among E. coli varies from country to country. In previuos reports, multidrug resistance was identified as 7.1% in 2000 in USA [
selection of antimicrobial resistance mediated by multidrug resistance mediated by multidrug resistance plasmids. Integrons are usually located within transposons or conjugative plasmids. These genetic elements are capable of capturing genes encoding antimicrobial resistance by site-specific recombination system. In addition, they contribute to the acquisition of new genes in bacteria. Class 1 integron is prevalent on plasmids in both Gram-positive and especially in Gram-negative bacteria [
[
Antibiotic | Total resistant n (%) | Positive Class I integron n (%) |
---|---|---|
Ampicillin | 78 (92.9%) | 42 (53.8%) |
Ceftazidime | 66 (78.5%) | 35 (53%) |
Ceftriaxone | 68 (81%) | 35 (51.5%) |
Cefotaxime | 39 (46.4%) | 30 (77 %) |
Norfloxacin | 60 (71.4%) | 34 (56.7%) |
Ciprofloxacin | 60 (71.4%) | 34 (56.7%) |
Levofloxacin | 51 (60.7%) | 34 (66.7 %) |
Tobramycin | 48 (57%) | 29 (60.4%) |
Gentamicin | 45 (53.6%) | 27 (60%) |
Amikacin | 17 (20.2%) | 15 (88.2%) |
Sulfamethoxazole/Trimethoprim | 71 (84.5%) | 32 (45.1%) |
Imipenem | 0 | 0 |
showing the existence of amplicons of 2000 bp, 1800 bp, 1500 bp, 800 bp, 700 bp, 500 bp and 400 bp in 10, 3, 10, 4, 6, 1 and 9 isolates respectively. RFLP digested fragments which are generated could be classified into 9 different patterns, namely A, B, C, D, E, F, G, H and I. Most identified patterns were Group B and Group D. Group B included 6 urine isolates: 27, 79, 3, 22, 57 and 12 in addition to two wound isolates 74 and 78. However, 9 urine isolates : 6, 8, 39, 76, 31, 54, 20, 50, 15 and 3 wound isolates 24, 53 and 55 were classified under Group D. The dfrA17-aadA5 cassette was detected in Group D E. coli strains conferring resistance to trimethoprim and aminoglycosides. Previous studies demonstrated that this cassette was identified in isolates from different sources [
High resistance rates were identified in E. coli isolates in this study which occurs mainly through antibiotic resistance genes. These genes are present on mobile elements that could be transmitted to different pathogens. Si- milar integron components could be proved among strains of different clinical sources. It seems that imipenem can serve as drug of choice for treatment of multi resistant E. coli infections
All thanks and appreciation Department of Microbiology, Faculty of Medicine, Mansoura University, Egypt for providing clinical isolates of E. coli. This work was performed at Microbiology Department, Faculty of Pharmacy, Mansoura University, Egypt.
The authors declare that they have no conflict of interest.