|Year : 2014 | Volume
| Issue : 3 | Page : 285-289
Investigation of plasmid-mediated quinolone resistance in Pseudomonas aeruginosa clinical isolates
Y Tanriverdi Cayci1, AY Coban2, M Gunaydin2
1 Medical Microbiology Specialist, Ankara Occupational Diseases Hospital, Ankara, Turkey
2 Ondokuz May?s University, Faculty of Medicine, Department of Medical Microbiology, Samsun, Turkey
|Date of Submission||11-Mar-2013|
|Date of Acceptance||29-Aug-2013|
|Date of Web Publication||10-Jul-2014|
A Y Coban
Ondokuz May?s University, Faculty of Medicine, Department of Medical Microbiology, Samsun
Source of Support: This study supported with project no.
PYO.1904.04.10.018 by Scientific Research Project Office of Ondokuz
Mayıs University, Samsun, Turkey., Conflict of Interest: None
Aims: To investigate plasmid-mediated quinolone resistance in clinical isolates of Pseudomonas aeruginosa with the polymerase chain reaction (PCR). The plasmid-mediated quinolone resistance genes have been identified in many bacteria within the Enterobactericeae family, they have not been detected in P. aeruginosa isolates. Subjects and Methods : Identification of the isolates and testing of antibiotic susceptibility was performed in Vitek2 Compact (Biomeriux, France) and Phoinex (BD, USA) automated systems. Screening for the qnrA, qnrB, qnrS, qnrC, aac (6′)-Ib-cr and qepA genes was carried out by PCR amplification and aac (6′)-Ib-cr DNA sequencing. Results: The qnr and the qepA genes were not detected in any of P. aeruginosa isolates. The aac (6')-Ib gene was detected in six of the isolates and positive isolates for aac (6')-Ib were sequenced for detection of the aac (6')-Ib-cr variant but aac (6')-Ib-cr was not detected in any isolates. Conclusions: Plasmid-mediated quinolone resistance genes have so far not been identified in P. aeruginosa isolates. However, qnrB have detected in P. florescens and P. putida isolates. This is the first study conducted on the qnrA, qnrB, qnrS and qnrC genes as well as the qepA and aac (6')-Ib-cr genes in P. aeruginosa clinical isolates.
Keywords: aac (6′)- Ib-cr, Pseudomonas aeruginosa, qepA, quinolones, Qnr
|How to cite this article:|
Cayci Y T, Coban A Y, Gunaydin M. Investigation of plasmid-mediated quinolone resistance in Pseudomonas aeruginosa clinical isolates. Indian J Med Microbiol 2014;32:285-9
|How to cite this URL:|
Cayci Y T, Coban A Y, Gunaydin M. Investigation of plasmid-mediated quinolone resistance in Pseudomonas aeruginosa clinical isolates. Indian J Med Microbiol [serial online] 2014 [cited 2019 Aug 18];32:285-9. Available from: http://www.ijmm.org/text.asp?2014/32/3/285/136567
| ~ Introduction|| |
Quinolones are synthetic agents that have been developed to rival beta-lactams in clinical usage.  Ciprofloxacin is the most effective quinolone against Pseudomonas aeruginosa. Quinolones achieve their bactericidal effects through the inhibition of DNA gyrase and topoisomerase IV.  The widespread use of quinolones inevitably results in increasing cases of resistance. The resistance mechanisms against quinolones are chromosomal mutations in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE), decreased drug accumulation within the cell through the hyperactivation of efflux pumps and reduced cell wall permeability, and the activity of plasmid-mediated qnr, qepA and aac (6')-Ib-cr genes.  Plasmid-mediated quinolone resistance resulting from the resistance determinant known as 'qnr' was first reported in Klebsiella pneumoniae in 1998.  In time, other qnr genes have been identified in the Enterobactericeae family, and the first determinant was named as qnrA, while subsequent determinants were named qnrB, qnrC, qnrS and qnrD.  Yamane et al., have also identified the plasmid-mediated qepA gene in 2007, which triggers and increases efflux pump activity.  Aac (6')-Ib is an enzyme that causes aminoglycoside resistance through acetylation, and a variant of this enzyme, aac (6')-Ib-cr, was found to be responsible for plasmid-mediated quinolone resistance. The aac (6')-Ib-cr enzyme was first described in a qnrA positive Escherichia More Details coli isolate.  Aac (6')-Ib is an enzyme that causes aminoglycoside resistance through acetylation, and a variant of this enzyme, aac (6')-Ib-cr, was found to be responsible for plasmid-mediated quinolone resistance. Although plasmid-mediated quinolone resistance genes have been identified in many bacteria within the Enterobactericeae family, they have not been detected in P. aeruginosa isolates until now. , Based on our review of the literature, we have determined that that no studies have yet been conducted on the qnrA, qnrB, qnrS and qnrC genes as well as the qepA and aac (6')-Ib-cr genes in P. aeruginosa clinical isolates. In this study, our aim was to investigate plasmid-mediated quinolone resistance in clinical isolates of P. aeruginosa.
| ~ Meterials and Methods|| |
P. aeruginosa clinical isolates (n = 300) were collected from a university hospital microbiology laboratory between November 2009 and April 2010. Identification of the isolates and testing of antibiotic susceptibility was performed in Vitek2 Compact (Biomeriux, France) and Phoinex (BD, USA) automated systems. In accordance with the recommendations of the Clinical and Laboratory Standarts Institute (CLSI), the gentamicin, amikacin and tobramycin susceptibility of aac (6')-Ib positive isolates were studied by using the disc diffusion method. 
Polymerase chain reaction
DNA preparation was performed through a boiling technique. Colonies freshly obtained from Mueller-Hinton agar were dissolved in 500 μl sterile distilled water and incubated at 100°C for 20 minutes. Afterwards, the bacterial solution was centrifuged at 15000 g for 20 min in order to obtain a supernatant that was then used as template DNA in polymerase chain reaction (PCR). Well-characterised qnr-positive strains were used as positive controls for optimisation of multiplex PCR. These strains were provided by Prof. GA Jacoby (Lahey Clinic, Burlington, Massachusetts, USA), Prof. P Nordmann (Service de Bactιriologie-Virologie, INSERM U914 "Emerging Resistance to Antibiotics", Hτpital de Bicκtre, Assistance Publique/Hτpitaux de Paris, Facultι de Mιdecine et Universitι Paris-Sud, K.-Bicκtre, France), while the qnrC plasmid was provided by Prof. M Wang (Institute of Antibiotics, Huashan Hospital, Fudan University, 12 M. Wulumuqi Rd., Shanghai 200040, People's Republic of China) [Table 1].
The qnrA, qnrB, qnrC and qnrS determinants were studied in multiplex PCR, and primer pairs were used as described by Kim et al. [Table 2]  Amplification was performed with the following thermal cycling profile: 1 min at 95°C, and 35 cycles of amplification consisting of 1 min at 95°C, 1 min at 60°C and 1 min at 72°C and 10 min at 72°C for the final extension.
The qepA and aac (6')-Ib-cr genes were studied using a different PCR method. PCR primer pairs were same as those described in the Yamane et al.'s study for the determination of qepA [Table 2].  Amplification was performed with the following thermal cycling profile: 1 min at 96°C, and 30 cycles consisting 1 min at 96°C, 1 min at 60°C, 1 min at 72°C and 5 min at 72°C for the final extension. The aac (6')-Ib-cr gene region was amplified using the primer aac (6')-Ib identified by Kim et al.  Amplification was performed with the following thermal cycling profile: 1 min at 96°C, and 30 cycles consisting 1 min at 96°C, 1 min at 59°C, 1 min at 72°C and 5 min at 72°C for the final extension. Aac (6')-Ib-cr differs from aac (6')-Ib by two amino acids, Trp102Arg and Asp179Tyr, because of this aac (6')-Ib positive isolates were sequenced with Macrogen (Korea) using the primer identified by Park et al. for detection of the aac (6')-Ib-cr variant. 
| ~ Results|| |
P. aeruginosa isolates were obtained from various clinical specimens [Table 3]. P. aeruginosa was isolated mostly from endotracheal aspirate (31.4%), urine (22.4%) and sputum (13.3%) samples. Clinical specimens were the most frequently obtained from internal medicine clinic (14%) [Table 4].
With regards to the ciprofloxacin susceptibility of the isolates, resistance was observed in 64 (21.4%) of the isolates, intermediate resistance in 8 (2.6%) of the isolates and susceptibility in 228 (76%) of the isolates. The 6 aac (6')-Ib positive isolates were resistant to gentamicin and tobramycin. While five of these isolates had intermediate resistance to amikacin, one isolate was susceptible to amikacin.
|Table 4: Distribution of clinics providing specimens from which P. aeruginosa was isolated |
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Polymerase chain reaction
Positive control strains yielded the expected bands [Figure 1]. The qnrA, qnrB, qnrC, qnrS genes and the qepA gene were not detected in any of P. aeruginosa isolates. The aac (6')-Ib gene was detected in six of the isolates [Figure 2]. Positive isolates for aac (6')-Ib were sequenced for detection of the aac (6')-Ib-cr variant (Macrogen, Korea). However, aac (6')-Ib-cr was not detected in any isolates.
|Figure 1: Agarose gel electrophoresis of the qnr A, qnrB, qnrC and qnrS positive control strains PCR products. M: marker, Lane 1: qnrA positive E. coli ref: 20, Lane 2: qnrB positive K. pneumoniae ref:15, Lane 3: qnrC positive pHS1, Lane 4: qnrS positive E. cloacae ref: 287, Lane 5: negative control|
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|Figure 2: Agarose gel electrophoresis of Aac(6')-Ib positive strains. M: marker, Lane 1; Aac(6')-Ib positive strain K. pneumoniae ref:15, Lane 2: P213, Lane 3: P2, Lane 4: P229, Lane 5: P65, Lane 6: P64, Lane 7: P71, Lane 8: negative control|
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| ~ Discussion|| |
P. aeruginosa is an important infectious agent that is usually associated with healthcare settings. It causes a variety of clinical manifestations in hospitalised patients, especially among immunocompromised individuals.  P. aeruginosa is responsible between 10% and 25% of hospital-acquired infections with high mortality and morbidity rates.  Beta-lactam antibiotics with anti-pseudomonal activity are often used for the treatment of P. aeruginosa infections. Quinolones, especially ciprofloxacin, also have anti-pseudomonal activity, and can effectively inhibit DNA synthesis and demonstrate bactericidal effect.  The main mechanisms for quinolone resistance in P. aeruginosa are chromosomal mutations in DNA gyrase and topoisomerase IV enzymes, and decreased drug accumulation within the cell through hyper-activation of efflux pumps and reduced cell wall permeability.  In addition to these mechanisms, plasmid-mediated quinolone resistance encoded by the qnr genes was first described in 1998.  Following the first identification of the qnr genes, different sub-types (qnrA, qnrB, qnrC, qnrS and qnrD) have also been defined.  In 2007, an efflux pump gene 'qepA' was identified in Japan.  In 2006, an unusual ciprofloxacin Minimum inhibitory concentration (MIC) was identified in an E. coli isolate that carried the qnrA gene. Random transposon mutagenesis was applied to this isolate, and the aac (6')-Ib gene was subsequently detected and identified. By sequencing aac (6')-Ib, it was determined that this allele had two different codons for aac (6')-Ib and aac (6')-Ib-cr, the latter being responsible for ciprofloxacin resistance. , Seven qnrA, 27 qnrB, 1 qnrC, 2 qnrS and 1 qnrD subtypes have been identified to date. ,
However, numerous additional qnr genes have recently been identified, suggesting that they have existed in nature for many years. In a study that analysed the sequence of 48 Gram-negative bacteria, qnrA (qnrA3-qnrA5) variants were detected in Shewanella algae chromosome. It was reported that the MIC value of this isolate was 4-8 fold greater than that of S. putrefaciens, which did not harbour this gene. Shewanella spp. can be found in marine and fresh water sources. The qnrB5 and qnrB19 genes were also identified in microbial population samples obtained from sea water.  The qnrS2 gene was also detected in Aeromonas punctata subsp. punctata and A. media isolates obtained from the Seine River.  These data suggest that qnr genes originate from the chromosomes of water-borne micro-organisms.
The qnr determinants have been investigated among members of the Enterobactericeae family at different centres in Turkey. In 2005, Nazik et al., investigated the qnrA gene in 49 isolates and detected a qnrA determinant in one E. cloacae and one C. freundii isolates.  The qnrA, qnrB and qnrS genes were also investigated in 356 Enterobactericeae spp. isolated from blood cultures, and qnrA was detected in 61 isolates, while qnrS was detected in 3 isolates.  Similarly, Oktem et al., investigated qnrA, qnrB and qnrS in 460 Gram-negative bacteria isolated from intensive care unit patients, and detected 1 qnrB1 and 2 qnrS1 genes in 3 (0.65%) of the E. cloacae isolates.  The frequency of qnrA, qnrB, qnrS and aac (6')-Ib-cr genes were investigated in 248 E. coli and K. pneumoniae clinical isolates collected from different centres in Turkey, and the qnrB1 and aac (6')-Ib-cr genes were detected on different plasmids in 1 K. pneumoniae isolate. 
Many of the bacteria from which the qnr determinants were isolated from belong to Enterobacteriaceae family, which includes K. pneumoniae, Enterobacter spp., E. coli and Salmonella More Details enterica. The prevalence of qnr determinants among these bacteria varies between 0.2% and 94%. This wide range in prevalence is believed to be caused by differences in the strain selection criteria that are employed (ceftazidime, nalidixic acid resistance, ESBL harbouring bacteria, etc.).  Plasmid-mediated quinolone resistance determinants were also investigated in P. aeruginosa; however, such determinants were not detected. ,,, Nevertheless, Martinez et al. experimentally transferred the qnr gene carried by pMG252 plasmid to the PU21 strain of P. aeruginosa. Following this conjugation, it was observed that ciprofloxacin, levofloxacin and norfloxacin MIC values of the PU21 strained increased 4-fold.  The results of this study have demonstrated that the transfer of qnr determinants to P. aeruginosa clinical isolates was possible. Thus, it is possible for qnr determinants to be spread between P. aeruginosa clinical isolates. If qnr determinants along with other resistance mechanisms were to spread to P. aeruginosa clinical isolates, this situation would severely limit the effectiveness of quinolones in pseudomonas infections. In 2007, qnr determinants were detected in 10 isolates of Gram-negative bacteria isolated from zoo animals. One of these isolates was a qnrB harbouring P. fluorescens obtained from a turtle, which was shown to be resistant to nalidixic acid and trimethoprim-sulfamethoxazole. This study represented the first case in which a qnr determinant was identified in a Pseudomonas species.  Following this study, Tran et al., reported that qnrA and qnrB were detected in two isolates of P. putida isolated from raw shrimps. Upon transferring these qnrA and qnrB-carrying plasmids to E. coli J53, it was observed that the value for nalidixic acid MIC increased from 8 to 32 μg/ml, while the value for ciprofloxacin MIC increased from 0.06 to 0.25 μg/ml. 
Aac (3) and aac (6') are the main enzymes responsible for aminoglycoside acetylation in P. aeruginosa. In our study, the Aac (6')-Ib variant of aac (6) was detected in six P. aeruginosa isolates. As expected, these isolates were determined to be tobramycin-resistant.
Plasmid-mediated quinolone resistance genes have so far not been identified in P. aeruginosa isolates. However, recent studies have detected qnrB in P. florescens and P. putida isolates. It will hence come as no surprise if plasmid-mediated quinolone resistance determinants were to be detected in P. aeruginosa in the near future.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]