Sabrina Suhani, Auditi Purkaystha, Musammat Kulsuma Begum, Md. Jahidul Islam and Abul Kalam Azad*
Department of Genetic Engineering & Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh
Received date: June 07, 2017; Accepted date: June 30, 2017; Published date: July 05, 2017
Citation: Suhani S, Purkaystha A, Begum MK, Islam MJ, Azad AK (2017) Plasmids for Amoxicillin and Ciprofloxacin Resistance in Escherichia coli Isolate Causing Urinary Tract Infection. Clin Microbiol 6:284. doi: 10.4172/2327-5073.1000284
Copyright: © 2017 Suhani S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Background: Escherichia coli causes urinary tract infection (UTI), one of the most frequent bacterial infections in humans, shows resistance to various commonly prescribed antibiotics. This is termed as multi drug resistance, which is increasing among E. coli. The multi-drug resistant E. coli isolates harbor multiple plasmids that might be responsible for antibiotic resistance. The resistance can be transferred among bacteria through horizontal transfer of the genetic materials.
Objectives: We undertook this study 1) to find a link between antibiotic resistance and plasmid of E. coli from UTI patients of Sylhet region of Bangladesh 2) to analyze the spread of the plasmid mediated antibiotic resistant gene between E. coli isolates.
Methods: Conjugation experiment was carried out in Luria Broth with E. coli DH5α as recipient. For transformation, competent cells were prepared using E. coli DH5α. Plasmid isolation was done by mini alkaline lysis method and plasmid was extracted from agarose gel and transferred to the competent cell.
Results: After conjugation, the donor E. coli isolate that showed resistance to amoxicillin (AMX), ciprofloxacin (CIP) and ceftriaxone (CTR) tranferred its AMX and CIP resistance to the recipient E. coli DH5α, that was previously sensitive to all antibiotics. After transformation, the recipient E. coli DH5α became resistant to CIP and AMX, while the donor showed resistance to gentamycin (CN), ceftriaxone (CTR), amoxicillin (AMX), ciprofloxacin (CIP), cefixime CFM, and cotrimoxazole (COT). Plasmid extracted from the transformant revealed that, three genes (3 kb, 5 kb and 20 kb) have been transferred from the donor to recipient.
Conclusion: Plasmids responsible for amoxicillin and ciprofloxacin were transferred
Antimicrobial resistance; Horizontal plasmid transfer; MDR; UTI
Urinary tract infection (UTI), usually caused by Escherichia coli, is the most common bacterial infection in humans. The risk factors for UTIs include sexual activity, gender, genetics and presence of urinary catheters [1,2]. Females are more prone to UTIs than male because their urethra is much shorter and closer to the anus. Females further lack the bacteriostatic properties of prostatic secretions .
E. coli that causes UTI belongs to several subgroups that are selected by some factors, which enhance extra intestinal survival. These factors include structural features such as fimbriae or pili for adherence, flagella for motility and chemical adhesion . The type-1 pili of uropathogenic E. coli are known to be associated with increased severity of UTI that binds to mannose containing glycoprotein receptors on facet cells lining the bladder or vaginal epithelial cells . Other virulence factors which confer the ability to fecal E. coli to colonize the vaginal mucosa and cause symptomatic UTI has also been identified . However, the ability of E. coli to cause UTIs is on the increase, while the ease of treating these infections due to resistance to first generation antibiotics such as cotrimoxazole, ampicillin and nitrofuranton is becoming increasingly elusive. The greater concern is the recent increase in resistance to second or even third generation antibiotics such as ciprofloxacin, levofloxacin and ceftriaxone [2,5,6].
From studies of the following few decades, it has been clear that multidrug resistance phenomena is due to chromosomal genetic elements as well as existing plasmids and their association with other genetic mobile elements. In Japan, the role of plasmids in antibiotic resistance was first recognized by Watanabe and Fukasawa in 1961 . They reported that transfer of a plasmid, which is known as resistance transfer factor or R-factor, that carried the resistance genes, is a single step process. Further reports showed that plasmids were carriers of not only multi-drug resistance genes, but also genes associated to contribute to the virulence of the host bacteria [8,9].
Antibiotic resistant bacterial infection has been identified as one of the greatest epidemic threats to human health by the World Health Organization. Within this epidemic, a group of pathogens has been individualized and collectively named ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter). These are opportunistic pathogens; the majority of them cause hospital-acquired infection that is resistant to antibiotic treatment. Plasmids play a central role in the dissemination and acquisition of the resistant determinants in these bacteria .
The increasing ability of E. coli to cause UTIs and the difficulty encountered in treating these infections due to multidrug resistance necessitates updating the knowledge of their drug resistance in a given environment. Horizontal gene transfer between bacterial cells contributes to bacterial adaptation to various environments and, in the long term, to bacterial evolution . In human environments, it results in the undesirable spread of pathogenic, antibiotic resistance, or artificially engineered genes. Three mechanisms of horizontal gene transfer in bacteria are generally known: conjugation, transduction and transformation. Conjugation and transduction involve specific apparatus for DNA transfer from donor cells to recipient cells; they are conjugative pili and phage capsids, respectively. However, transformation is mainly performed by the recipient cells that express genetic competence to take up extracellular free DNA . Competence for transformation can be induced naturally and artificially but not all bacterial species develop natural competence.
E. coli is not assumed to be naturally transformable; it develops high genetic competence only under artificial conditions, e.g. exposure to high Ca2+ concentrations . However, several reports have shown that E. coli can express modest genetic competence in certain conditions that can arise in its environment and make it subjected to horizontal transformation [11,12,14]. We herein report that inheritance of antibiotic resistance occurs due to transconjugation or transformation of plasmids. It has been further discussed that a particular plasmid is responsible for resistance against a specific antibiotic.
E. coli isolates and their maintenance
The multi-drug resistant E. coli isolates were obtained from urine samples of UTI patients in North East region of Bangladesh . E. coli isolates were identified based on morphological, cultural and biochemical characteristics as described by Cappuccino et al.  and Bergey’s manual of systematic bacteriology . The reference strain, E. coli DH5α, was collected from department of microbiology, University of Dhaka, Bangladesh. The pure culture of E. coli isolates and E. coli DH5α was routinely sub-cultured on nutrient agar. The pure culture was preserved at -30°C with 20% glycerol.
The plasmid transfer in E. coli DH5α through conjugation
A single colony of multidrug resistant E. coli isolate (donor) and that of antibiotic sensitive E. coli DH5α (recipient) were inoculated into luria broth (LB) separately and incubated in a shaker at 37°C for overnight. Five hundred micro litre of donor and recipient cultures was added to 4 ml fresh LB and incubated overnight at 37°C without shaking . A 10-fold serial dilution was then done with LB and 100 µl from each dilution was plated into sorbitol-MacConkey agar (SMAC medium)  containing selected antibiotics. SMAC medium was used as a differential color forming medium for detection of sorbitol fermenting (donor) isolate with characteristic red to pink colonies and non-sorbitol-fermenting (recipient) isolate with characteristic colorless colonies. In SMAC, the lactose of MacConkey agar was replaced by sorbitol. After conjugation, the recipient E. coli DH5α transformed with plasmid produced pink colonies on the antibiotic selection plate.
Extraction and purification of plasmids
Plasmids were extracted by mini alkaline lysis method  from 16 h cultured cells of E. coli. Plasmids were then subjected to electrophoresis onto 0.7% agarose gel using standard protocol. The gels stained with ethidium bromide were visualized on an ultraviolet transilluminator (UVP, High Performance transilluminator; USA) and recorded with a canon camera (PowerShort A3200 IS). A particular plasmid band was excised from the gel and purified with the FavorPrep™ plasmid DNA extraction mini kit (Favorgen Biotech Corp., Taiwan).
Competent cell preparation, plasmid transformation and antibiotic susceptibility
Competent cells of E. coli DH5α were prepared as described by Sambrook et al. . For transformation, 50 µl of plasmid DNA was added to a tube containing 0.2 ml of competent cells. The mixture was placed on ice for 20 min, and then exposed to heat shock at 42 °C for 1 min and kept immediately in ice for 10 min. A 800 µl LB broth was then added to transformation mixture and incubated at 37ºC for 60 min. About 100 µl from transformation mixture was spread on nutrient agar plates containing the appropriate antibiotic. An aliquot (100 µl of 5-10 fold diluted) of competent cells spread on nutrient agar containing the same antibiotics was used as a control. All plates were incubated at 37°C for 48 h. The transformant colonies were scored and fresh cultured several times on plates containing the desired antibiotics. The transformant was investigated for the presence of target plasmid. The susceptibility of transformant and non-transformant E. coli DH5α to different antibiotics was investigated by the modified Kirby-Bauer disc diffusion method .
Inherence of antibiotic resistance in E. coli DH5α through
E. coli isolate resistant to amoxicillin (AMX), ceftriaxone (CTR) and ciprofloxacin (CIP) and E. coli DH5α sensitive to these antibiotics were used as donor and recipient, respectively (Figure 1).
SMAC media was used for selecting the transconjugants. The donor E. coli isolate produced red to pink colonies (Figure 2a) due to acid production by fermentation of sorbitol in the media. The recipient E. coli DH5α cannot ferment sorbitol, and use peptone instead . This forms ammonia, which raises the pH of the agar and lead to the formation of white/colorless colonies (Figure 2b).
Following conjugation, less-whitish colonies of E. coli DH5α were observed on SMAC media in the presence of AMX and CIP (Figure 3a and b) but not in the presence of CTR (Figure 3c). Growth of E. coli DH5α in the presence of AMX and CIP indicated that resistance to these antibiotics was inherited to them due to transfer of plasmid form the donor through conjugation.
Plasmid DNA transformation makes E. coli DH5α resistant to AMX and CIP
After plasmid DNA transformation, the transformant E. coli DH5α was screened in the presence of AMX and CIP separately. It was observed that some transformants could grow in the presence of these two antibiotics (Figure 4).
After selection in the media containing antibiotic, the transformants were subjected to antibiogram of some antibiotics to determine which antibiotic resistant gene had been transferred to transformant E. coli DH5α. For this, eight antibiotic disks were used to compare the susceptibility of the transformants (Figure 5b) to that of E. coli DH5α (Figure 5c) and multidrug resistant E. coli isolate from which plasmid was extracted (Figure 5a).
It was observed that the MDR E. coli isolates were resistant to six antibiotics and E. coli DH5α was sensitive to all antibiotics. The transformant E. coli DH5α inherited resistance to two antibiotics, CIP and AMX. This result indicated that CIP and AMX resistance genes might have been transferred to E. coli DH5α.
Plasmid profiling of transconjugates and transformants
Plasmids were isolated from the donor or MDR E. coli isolate, transconjugates, transformants and recipient E. coli DH5α and their plasmid profiling were compared. It was observed that the donor E. coli isolate harbored multiple plasmids of different sizes, whereas, the recipient E. coli DH5α had no plasmid (Figure 6).
However, AMX and CIP resistant transconjugates and transformants harbored plasmids of about 3 kb, 5 kb and 20 kb, which were present in the donor or MDR E. coli isolate. This result indicated that the recipient or the competent cells obtained these plasmids during conjugation and transformation.
The emergence, proliferation, aggregation, and management of antimicrobial-resistant pathogenic bacteria have become a worldwide health concern. When the resistance associated with the presence of transposable DNA elements, such as plasmids, it may be difficult to eliminate resistance, which tends to increase over time . Our previous study depicted that E. coli isolates showing MDR were found to harbor multiple plasmids of different sizes and left an assumption that the MDR genes might be in the plasmid or chromosomal DNA . It has been reported that Enterobacteriaceae isolates might be potential carrier of antibiotic resistant genes that could be transferred between bacterial strains of the same or different species of UTI pathogens [22-24]. In the present study, we investigated the plasmid associated antibiotic resistance in E. coli isolates causing UTI.
The present study showed that plasmids responsible for AMX and CIP resistance were transferred to the recipient E. coli DH5α. Many studies have shown that the dissemination of resistant genes mediated by plasmids may occur by conjugation [25-28].
Genetic transformation is an important mechanism of the horizontal exchange of genes. Natural genetic transformation is a gene transfer process where the bacteria can pick up the naked DNA from their environment and the DNA may come from a variety of sources The most frequent source is remnants from dead bacterial cells. The exogenous DNA bind to specific cell surface receptors, then the DNA is transported across the membrane and one strand of the DNA is digested away. As a consequence, the single stranded DNA enters the cell. Recombination occurs between the incorporated DNA and homologous host DNA and the new DNA replaces a strand of the host DNA. This new DNA may contain noble and different genes compared to the host DNA, including antibiotic resistance gene . An investigation carried out in the UK revealed that Enterobacteriaceae isolates from 43 hospitals during 1990–1991 showed antibiotic resistance due to plasmid DNA by transformation . Another study reported transformation process responsible for chloramphenicol, tetracycline and sulfonamide resistance transferred with the CTX-M-1 enzyme as co-transformant .
The present study showed that three plasmids, eg. 3 kb, 5 kb and 20 kb were transferred to the recipient cell, and thereby, the recipient E. coli DH5α became resistant to CIP and AMX. However, the non transformant recipient cells remained sensitive to these antibiotics. These three or any of these plasmids might carry CIP and AMX resistance gene. This report reveals the spread of the plasmid mediated antibiotic resistant genes between E. coli isolates of UTI patient and E. coli DH5α. Nevertheless, further research is needed to determine the specific gene in these plasmids associated with CIP and AMX resistance.
Our study revealed that, following conjugation and transformation, amoxicillin and ciprofloxacin resistance was inherited by E. coli DH5α that was previously sensitive to all antibiotics, from the MRD E. coli isolates obtained from UTI patients. This finding indicates that, plasmids responsible for antibiotic resistance can be transferred among bacteria through horizontal gene transfer. Careful use of antimicrobial agents, developing standard treatment guidelines and increasing awareness among people can ensure proper health care and reduce the spread of antimicrobial resistance among bacteria.