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ORIGINAL ARTICLE
Year : 2016  |  Volume : 4  |  Issue : 1  |  Page : 5-9

Presence of Qnr genes related to resistance to quinolones, first-, second- and third-generation in diarrhoeagenic Escherichia coli


1 Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
2 Biotechnology Research Center, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran

Date of Web Publication31-Mar-2017

Correspondence Address:
Abbas Doosti
Biotechnology Research Center, Islamic Azad University, Shahrekord Branch, P.O. Box 166, Shahrekord
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2214-207X.203541

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  Abstract 


Background: Resistance genes transferred by plasmids are important factors that can contribute to the occurrence of quinolone resistance, specifically in Escherichia coli strains.
Methods: A total number of 117 diarrhoeagenic E. coli strains were tested for the resistance to nalidixic acid, ofloxacin, ciprofloxacin and levofloxacin and for the presence of qnrA, qnrB and qnrS genes determinants by polymerase chain reaction. Antibiotic susceptibility test was performed using the Clinical and Laboratory Standard Institute standard method.
Results: The highest resistance belonged to the nalidixic acid (52.14%) and the least resistance to levofloxacin (37.61%). In this study, among a total of 117 samples, 23 (19.66%) strains were susceptible to all the studied antibiotics. Among the remainder, 94 (80.34%) samples resistant to at least one quinolone, three genes including qnrA, qnrB and qnrS were present in 19.15%, 88.30% and 78.72% isolates, respectively. Furthermore, 51.06% of strains had A, B+ and S+ pattern that seems to have a significant association (P < 0.001) with resistance to quinolones.
Conclusions: The results of present study show the presence of a high frequency of qnr genes in E. coli strains resistant to quinolones in clinical samples from southwest of Iran. In addition, this study approved that plasmid-mediated quinolone resistance is a possible mechanism among the quinolones-resistant E. coli isolated from patients with diarrhoea in the study, and also qnrB and qnrS genes seem to be more important in resistance to quinolones.

Keywords: Antibiotic resistance, diarrhoea, Escherichia coli, plasmid-mediated quinolone-resistance, qnr genes


How to cite this article:
Mokhtari-Farsani A, Doosti A, Mohammadalipour Z. Presence of Qnr genes related to resistance to quinolones, first-, second- and third-generation in diarrhoeagenic Escherichia coli. J Patient Saf Infect Control 2016;4:5-9

How to cite this URL:
Mokhtari-Farsani A, Doosti A, Mohammadalipour Z. Presence of Qnr genes related to resistance to quinolones, first-, second- and third-generation in diarrhoeagenic Escherichia coli. J Patient Saf Infect Control [serial online] 2016 [cited 2017 Jun 28];4:5-9. Available from: http://www.jpsiconline.com/text.asp?2016/4/1/5/203541




  Introduction Top


Diarrhoeal diseases remain a public health problem worldwide with more than two million deaths occurring each year, especially in developing countries.[1] The bacterial pathogens responsible for diarrhoeal disease include diarrhoeagenic escherichia coli (dec), yersinia, shigella, salmonella, campylobacter, aeromonas, etc.[2]

Six major classes of DEC including enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enteroaggregative E. coli, enterohaemorrhagic E. coli and diffuse adhering E. coli are the most common microorganisms that cause diarrhoeal illness.[3],[4] These bacteria are the major target of antimicrobial therapy.[1] It is common to use quinolones for the treatment of gastrointestinal tract infections.[3] Quinolones' family consists of four generations including: first- (nalidixic acid, oxolinic acid and cinoxacin), second- (norfloxacin, lomefloxacin and ofloxacin), third- (sparfloxacin, gatifloxacin, grepafloxacin, levofloxacin) and fourth-generation (trovafloxacin, moxifloxacin and gemifloxacin). Fluoroquinolones (quinolones such as ciprofloxacin which have a fluorine atom attached to the central ring system) initiated in the 1980s could be suitable for the treatment of Gram-negative infections.[5] However, in recent years, resistance to quinolones has increased worldwide.

Fluoroquinolone resistance in the Enterobacteriaceae family has until recently been ascribed to mutations in the topoisomerase (the parC and parE genes) and gyrase (the gyrA and gyrB genes) genes, the quinolone resistance-determining regions.[6] In recent times, one cause for the increasing quinolone resistance which is a plasmid-mediated quinolone resistance (PMQR) has been delineated together with a quinolone-protective mechanism encoded by the qnr genes and its prevalence is raising worldwide.[7],[8] Several PMQR genes (qnr) have been discovered. Five different groups of qnr genes (qnrA, qnrB, qnrC, qnrD and qnrS) have been shown to aid resistance to fluoroquinolones.[9] In 1998, Martínez-Martínez et al. found the qnrA gene in Klebsiella pneumoniae, in the United States. Later on, it was also found in Asia and Europe.[3],[10]

Since little is known about the presence of qnr-mediated quinolone resistance in DEC strains, this study aims to evaluate the prevalence of qnrA, qnrB and qnrS genes among DEC strains isolated from patients in Iran hospitals and its association with resistance to quinolones, first-, second- and third-generation.


  Methods Top


Ethics

Clinical research was conducted according to the Declaration of Helsinki principles. Protocols for sample collection, storage and detection of DEC in samples obtained from patients were approved by the Institutional Review Boards (IRB approval number: 18 on July 10, 2014 by Ethical Committee, Shahrekord University of Medical Sciences) at Kashani and Hajar Hospitals, located in Shahrekord, Iran. All patients gave written informed consent. Verbal consent was not accepted. The Ethics Committee of Shahrekord University of Medical Sciences agreed on the procedure.

Bacterial strains

This study was performed on a total of 117 fecal samples, recovered from patients referred to Iran hospitals, in a period of 3 months (March to May 2015). Patients' infection to DEC was confirmed by the doctor and hospital laboratory.

Samples were seeded on Eosin Methylene Blue agar plates and were incubated at 37°C for 24 h. The strains were biochemically confirmed as E. coli-positive. Then, DECs were characterised by a single multiplex polymerase chain reaction (PCR) technique as previously described.[1] Furthermore, Shiga toxin-producing E. coli bacteria isolated from patients were further characterised by the expression of the O157 lipopolysaccharide antigen and the enterohemolysin gene (hlyA), using a latex particle agglutination kit (Oxoid Limited, Basingstoke, United Kingdom) and PCR, respectively.

Antimicrobial susceptibility tests

Antimicrobial susceptibility of all isolates was determined using the standard Kirby-Bauer disk diffusion method according to the guidelines of Clinical and Laboratory Standard Institute (CLSI). Antimicrobial agents tested were nalidixic acid (30 μg), ofloxacin (5 μg), ciprofloxacin (5 μg) and levofloxacin (5 μg) (Padtanteb, Iran). The susceptibility of the E. coli isolates to each antimicrobial agent was measured and the results were interpreted in accordance with interpretive criteria provided by CLSI.[11]

DNA extraction

Bacterial strains were subcultured overnight in Luria-Bertani broth agar (Sigma-Aldrich, Germany); then, genomic DNA was extracted from typical colonies of DEC using DNA extraction kit (DNP™, CinnaGen, Iran), according to manufacturer's instruction.

Detection and sequencing of qnr genes

The presence of qnrA, qnrB, and qnrS genes in all DEC isolates was subjected to PCR. Primer sequences, sizes of PCR products and PCR conditions are shown in [Table 1]. A tube containing PCR reaction without any DNA template was used as negative control. All primers were obtained from Cinnagene Co., (Tehran, Iran). PCR products were resolved by electrophoresis on 1% w/v agarose gel in TBE buffer and visualised with ethidium bromide under ultraviolet transilluminator. Then, the five PCR-amplified products from each of the qnr gene (qnrA, qnrB and qnrS) were extracted from agarose gel using a DNA extraction gel kit (Bioneer Co., Korea) according to the manufacturer's protocol and finally were subjected to DNA sequencing and sequence similarity was checked using nucleotide BLAST analysis at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi.
Table 1: Primer sequences and polymerase chain reaction conditions

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Statistical analysis

All data for the presence of qnr genes and its association with resistance to quinolones were analysed by MS Excel 2007 and the Chi-square test using the SPSS 17 (SPSS Inc., Chicago, IL, USA) software. P< 0.05 was considered statistically significant.


  Results Top


Ninety-four isolated strains (80.34%) were designated as resistant to at least one antibiotic. The percentage of the resistant isolates to the tested antimicrobials is presented in [Figure 1].
Figure 1: The percentages of antimicrobial resistance detected among Escherichia coli isolates

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Analysis of PCR products for the presence of qnr genes DNA on 1% agarose gel revealed the product length of 542 bp for qnrA, 266 bp for qnrB and 431 bp for qnrS [Figure 2]. The nucleotide sequence obtained from amplified fragments of the qnr genes was found to be qnr genes-specific based on BLAST searches with 99% identity to qnr genes published in the GenBank database (accession numbers: qnrA: KC788562.1, qnrB: KJ415247.1 and qnrS: KM094211.1). From the 94 E. coli isolates resistant to at least one quinoline, 18 (19.15%) isolates were identified positive for qnrA. Furthermore, qnrB and qnrS were detected in 83 (88.30%) and 74 (78.72%) isolates, respectively.
Figure 2: Agarose gel electrophoresis of polymerase chain reaction amplification products for detection of qnr genes (Lane 1 shows fermentas 100 bp DNA molecular marker, Lane 2 is positive samples for qnrA gene, Lane 3 is positive samples for qnrB gene, Lane 4 is positive samples for qnrS gene, Lane 5 is negative sample, Lane 6 is positive control for qnrB gene and Lane 7 is negative control)

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PCR-directed screening of all 23 susceptible E. coli isolates showed that 3 (13.04%), 4 (17.39%) and 2 (8.69%) isolates were positive for qnrA, qnrB and qnrS, respectively.


  Discussion Top


DEC affect an important proportion of the population.[12] It is common to use quinolones or fluoroquinolones such as nalidixic acid, ciprofloxacin and norfloxacin for the treatment of gastrointestinal tract infections. During the 1960s, quinolones were preferred as extremely important treatments in health care. However, in the 1990s, quinolone-resistant isolates spread throughout the world.[13] Increasing frequency of drug resistance among bacteria in recent years is a major public health concern.

The spread of quinolones resistance in bacteria is a complex process involving a diversity of different mechanisms such as chromosomal mutations and resistance genes transferred by plasmids.[3],[13] Previous reports suggest that qnr genes were commonly found in Enterobacteriaceae.[14],[15] The qnr genes have been poorly studied in Iran; therefore, this study aimed to investigate the prevalence of nalidixic acid, ciprofloxacin, ofloxacin and levofloxacin resistance and the presence of qnrA, qnrB and qnrS genes in DEC strains.

In the present study, among 117 E. coli isolates, 61 (52.14%), 55 (47.01%), 49 (41.88%) and 44 (37.61%) isolates were found resistant to nalidixic acid ( first), ofloxacin (second), ciprofloxacin (second) and levofloxacin (third-generation quinolones), respectively. Similarly, Wang et al.[16] reported a 31.43% resistance to ciprofloxacin and Mansouri Jamshidi et al.[17] showed 69.3, 40 and 46% resistance to nalidixic acid, ofloxacin and ciprofloxacin, respectively. In another articles by Jones et al.[18] and Murshed et al.,[19] resistance rates to nalidixic acid were noted as 68% and 77%. Again Mahamat et al.[20] reported that resistance rates to nalidixic acid and ofloxacin were 17.3% and 10.9%, respectively. The results of our study and other studies in recent years show that highest resistance to quinolones in the first-generation slightly decreases for the next generation (second- and third-generation, respectively). In addition, it is noteworthy that the divergent resistance rate at different parts of the world is associated with the differences in clinical features, differences in the advancement of antibiotic consumption rates in each country and geographic location.

In this study, from total 117 isolates, the frequency of qnrA, qnrB and qnrS genes was estimated as 17.94%, 74.35% and 64.95%, respectively. However, from the 94 E. coli isolates resistant to at least one quinoline, 19.15%, 88.30% and 78.72% isolates and from the 23 susceptible E. coli isolates 13.04%, 17.39% and 8.69% were identified as being positive for qnrA, qnrB and qnrS, respectively. In addition, results showed a significant association (P < 0.001) in the frequency of qnr genes in susceptible and resistant samples.

In the present study, the prevalence of qnr genes was relatively higher than in other investigations, except the prevalence of qnrA gene. For example, Mansouri Jamshidi et al.[17] reported that 31.8%, 56.5% and 28.9% of resistance isolates carried qnrA, qnrB and qnrS, respectively. Furthermore, research by Wang et al.[16] found frequencies of 72.73% for qnrA, 45.45% for qnrB and 45.45% for qnrS. In other research by Winissorn et al.,[3]qnrA prevalence was reported to be 8%.

Our results as well as other studies indicated that there are some strains containing more than one qnr genes; sixteen isolated (17.02%) had all of the three genes (qnrA, qnrB and qnrS), two (2.13%) isolates had two of the genes (qnrA and qnrB), forty-eight (51.06%) isolates had another two of the genes (qnrB and qnrS), and there was no sample with qnrA and qnrS pattern. These results are similar to the findings of other studies, for example, a research from Mansouri Jamshidi et al.[17] wherein authors reported that 10.1% isolates had all of the three genes and 28.9%, 15.9% and 11.5% isolates had two of the genes (qnrA, B), (qnrB, S) and (qnrA, S), respectively. Furthermore, our results show that 51.06% of isolated strains resistant to at least one antibiotic have qnrA, qnrB + and qnrS + (A , B + and S +) pattern. This pattern seems to have a significant association (P < 0.001) with resistance to quinolones family because there was no quite susceptible isolated with this pattern.


  Conclusion Top


This study showed the presence of a high frequency of the resistant qnrA, qnrB and qnrS gense in DEC strains isolated from clinical samples from southwest of Iran and suggests that qnr genes are common among quinoline resistance isolates. This could lead to a serious threat of an outbreak of antimicrobial resistance development, which complicates the treatment of infections in the future. Eventually, we suggest further research on resistance genes transferred by plasmids (qnr) and any change in resistance pattern and also limiting the use of antimicrobial drugs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Kargar M, Moein Jahromi F, Doosti A, Mohammadalipour Z, Lorzadeh S. Resistance to different generations of quinolones in Streptococcus pneumoniae strains isolated from hospitals in Shiraz. Comp Clin Pathol 2015; 24:533-6.  Back to cited text no. 13
    
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Mahamat A, Lavigne JP, Fabbro-Peray P, Kinowski JM, Daurès JP, Sotto A. Evolution of fluoroquinolone resistance among Escherichia coli urinary tract isolates from a French university hospital: Application of the dynamic regression model. Clin Microbiol Infect 2005;11:301-6.  Back to cited text no. 20
    


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