|Year : 2014 | Volume
| Issue : 1 | Page : 31-34
Plasmid-mediated quinolone resistance in typhoidal Salmonellae: A preliminary report from South India
VK Geetha, T Yugendran, R Srinivasan, BN Harish
Department of Microbiology, Jawaharlal Institute of Post Graduate Medical Education and Research, Gorimedu, Puducherry, India
|Date of Submission||07-May-2013|
|Date of Acceptance||10-Oct-2013|
|Date of Web Publication||4-Jan-2014|
B N Harish
Department of Microbiology, Jawaharlal Institute of Post Graduate Medical Education and Research, Gorimedu, Puducherry
Source of Support: Jawaharlal Institute of Post Graduate Medical
Education and Research, Gorimedu, Puducherry, India,, Conflict of Interest: None
Background: Fluoroquinolones are the drugs extensively employed for the treatment of Salmonella infections. Over the couple of decades that have elapsed since the introduction of fluoroquinolones, resistance to these agents by Enterobacteriaceae family members has become common and widespread. Although fluoroquinolone resistance is mediated by genomic DNA (deoxyribonucleic acid) as well as plasmid DNA, the plasmid-mediated quinolone resistance (PMQR) facilitates higher level resistance by interacting with genomic mechanism and is capable of horizontal spread. Materials and Methods: During a period of 1-year, 63 typhoidal Salmonellae were isolated from 14,050 blood cultures and one parietal wall abscess. 36 (56.25%) were Salmonella Typhi and 27 (42%) were Salmonella Paratyphi A. They were all screened for resistance by the disc diffusion method and their minimum inhibitory concentrations were determined using agar dilution, broth dilution and E-strip method. Ciprofloxacin resistant isolates were screened for PMQR determinants by polymerase chain reaction assay. Results: All the 63 isolates were resistant to nalidixic acid. Among the 36 S. Typhi isolates 20 were resistant to ciprofloxacin, of which 14 carried the plasmid gene qnrB and one carried the aac(6')-Ib-cr gene. qnrA and qnrS genes were not detected. Ciprofloxacin resistance was not seen in any of the S. Paratyphi A isolates. Conclusion: The antibiotic sensitivity pattern of typhoidal Salmonellae shows an increasing trend of PMQR. The allele B of qnr gene was found to be the predominant cause of PMQR in this study.
Keywords: Ciprofloxacin, fluoroquinolones, plasmid-mediated quinolone resistance, Salmonella Typhi, typhoid
|How to cite this article:|
Geetha V K, Yugendran T, Srinivasan R, Harish B N. Plasmid-mediated quinolone resistance in typhoidal Salmonellae: A preliminary report from South India. Indian J Med Microbiol 2014;32:31-4
|How to cite this URL:|
Geetha V K, Yugendran T, Srinivasan R, Harish B N. Plasmid-mediated quinolone resistance in typhoidal Salmonellae: A preliminary report from South India. Indian J Med Microbiol [serial online] 2014 [cited 2019 Dec 15];32:31-4. Available from: http://www.ijmm.org/text.asp?2014/32/1/31/124292
| ~ Introduction|| |
Salmonella enterica serotype Typhi and S. enterica serotype Paratyphi A are the main causes of enteric fever in India and S. enterica serovar Typhi being predominant.  Fluoroquinolones are widely used drugs for the treatment of Salmonella infections, since the emergence of multidrug-resistant (MDR) Salmonella strains which showed resistance to all first-line antibiotics.  In developing countries like India, ciprofloxacin continues to be the mainstay in the treatment of enteric fever as it is orally effective and economical. They had two advantages over other antimicrobials. As independent mutations typically arise once per 10 7 cell divisions or less, the likelihood for multiple mutations, which is essential for fluoroquinolone resistance in a single clone is negligible.  Secondly, since the quinolones are fully synthetic, it seemed unlikely that resistance genes would be available for recruitment onto mobile elements.  Thus, the fluoroquinolones were anticipated to thwart resistance. However, with the 26 years that have elapsed since the introduction of fluoroquinolones, resistance to these agents by Enterobacteriaceae has become common and widespread. Three different mechanisms have been found to determine resistance to fluoroquinolones. Primarily, the aas documented in [Figure 1]. isolate was reported from Ko Scientist F, Deputy Director,. of mutations in the quinolone resistance-determining region (QRDR) mutations, which include: mutations in gyrase (gyrA/gyrB) and/or topoisomerase IV (parC/parE) genes encoding the bacterial enzymes targeted by fluoroquinolones. Secondly, by decreasing intracellular drug accumulation by up-regulation of native efflux pumps either alone or together with decreased expression of outer membrane porins. Third one is the plasmid-mediated quinolone resistance (PMQR) encoded by qnr genes A, B and S. Reduced susceptibility to ciprofloxacin in clinical bacterial isolates conferred by a variant of the gene encoding aminoglycoside acetyltransferase, aac(6')-Ib, has also been described. , PMQR facilitates higher level resistance by their interaction with defined chromosomal mechanisms of quinolone resistance. PMQR is the most worrisome element of all the three, as the plasmid determinants of resistance are capable of inter and intra species horizontal gene transfer, which may ensue wide spread dissemination. Hence, its emergence in Salmonella Typhi strains is a cause of worry for clinicians and microbiologists as well as for patients. 
|Figure 1: Gel documentation of polymerase chain reaction assay for aac (6')-Ib-cr gene. 2% agarose gel electrophoresis of aac(6')-Ib-cr amplicons. Amplicons were separated using a 2% agarose gel in Tris-acetate Ethylenediaminetetraacetic acid buffer. (Lane 1: Molecular mass marker (100 bp deoxyribonucleic acid ladder), Lane 2: Positive control (amplicon size – 218 bp), Lane 3: Negative control, Lane 4: Sample negative for aac(6')-Ib-cr, Lane 5: Sample positive for aac(6')-Ib-cr, Lane 6-8: Sample negative for aac(6')-Ib-cr)|
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| ~ Materials and Methods|| |
A total of 62 isolates of typhoidal Salmonellae out of 14,050 blood samples from June 2010 to October 2011, along with one Salmonellae Paratyphi A isolated from patient's parietal wall abscess were collected from patients suspected of enteric fever. Klebsiella pneumoniae (NK 835), Morganella morganii (500314) and Shigella flexneri 3a (IDH6663) harbouring qnr gene alleles A, B and S and aac(6')-Ib-cr gene were also included in this study as positive controls for polymerase chain reaction (PCR) assay. Standard methods were used for isolation and identification of typhoidal Salmonellae from blood cultures. 
Antibiotic susceptibility test
Antibiotic susceptibility test was performed by Kirby-Bauer's disk diffusion method for ampicillin (10 μg), chloramphenicol (30 μg), cotrimoxazole (1.25 μg/23.75 μg), nalidixic acid (NAR) (30 μg), ciprofloxacin (5 μg) and ceftriaxone (30 μg). Susceptibility was interpreted according to zone diameter recommended by Clinical and Laboratory Standards Institute 2012. Agar dilution and broth micro dilution methods were employed for the determination of minimum inhibitory concentrations (MICs) of ciprofloxacin alone. Each plate included a growth control well without antibiotic and a negative well. Escherichia coli ATCC 25922 was included as the quality control organism. MIC value was read as the lowest concentration of antibiotic at which the growth of bacteria has been completely inhibited. The MIC of ciprofloxacin was determined by using the E-test strips for randomly selected 15 isolates, according to the manufacturer's instructions (AB Biodisk, Sweden).
The isolates resistant to ciprofloxacin by micro broth dilution (≥1) were selected for the detection of PMQR genes such as qnrA, qnrB, qnrS and aac(6')-Ib-cr genes. The strains were screened for PMQR determinants using previously described primers are listed in [Table 1]. 
Previously known positive control strains and negative control were included in each run. The amplification was performed using Eppendorf Thermo Cycler with the following condition: Initial denaturation step at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 54°C for 90 s and 72°C for 60 s and a final extension step 72°C for 5 min.
The PCR products along with deoxyribonucleic acid (DNA) ladder of 100-1000 bp were loaded in 2% agarose gel prepared in Tris-borate-ethylenediamine tetraacetic acid (TBE) buffer. Amplicons were loaded in the wells after mixing with gel loading dye. 1 × TBE buffer was used for electrophoresis at 94 V for 1 h. The gels were visualised under ultra violet transilluminator.
| ~ Results|| |
Antimicrobial susceptibility pattern
Out of 63 Salmonellae isolates two of the S. Typhi were MDR showing resistance to ampicillin, cotrimoxazole and chloramphenicol. Three were resistant to ampicillin alone; one was resistant to cotrimoxazole and one showed resistance to chloramphenicol.
All the isolates were resistant to nalidixic acid. Out of 63 isolates of Salmonellae five were resistant, two were susceptible and 56 showed intermediate susceptibility to ciprofloxacin by disk diffusion method.
All the 63 Salmonellae isolates were intermediate sensitive to ceftriaxone, which was determined by agar dilution method showing MIC value ≤ 2 μg/ml. The five isolates showing resistance to ciprofloxacin by disk diffusion showed high MIC values ranging from 2 to 16 μg/ml. The MIC values of ciprofloxacin for the strains, which were resistant by MIC determination method were 1 μg/ml (15 isolates), 2 μg/ml (4 isolates) and 16 μg/ml (1 isolate).
MIC of ciprofloxacin by E-strip test
Randomly, 15 isolates were tested by ciprofloxacin E-strips (AB Biodisk, Sweden). The results are shown in the [Table 2].
PCR for PMQR genes
PCR was done for detection of qnr and aac(6')-Ib-cr genes. 20 isolates showing MIC value of ≥1 μg/ml for ciprofloxacin were included. Out of these 6 were S. Typhi and 14 were S. Paratyphi A. All the S. Typhi and eight of the S. Paratyphi A carried the qnrB gene. All the 20 were negative for qnrA and qnrS plasmids. Only one S. Typhi was positive for aac(6')-Ib-cr genes as documented in ure 1.
| ~ Discussion|| |
The widespread use of fluoroquinolones led to the emergence of S. enterica serovar Typhi and Paratyphi strains with reduced susceptibility to fluoroquinolones. In the past decade the use of fluoroquinolones has been very high. In JIPMER Hospital, Puducherry, India the average monthly consumption of ciprofloxacin tablets (500 mg) and ciprofloxacin intravenous (200 mg) for year 2012 was 25,650 and 2125 in numbers respectively.
PMQR is a supplementary mechanism, which is able to mediate low levels of fluoroquinolone resistance, but can present a higher level resistance by interacting with genomic determinants. First PMQR determinant qnrA was described in Klebsiella pneumoniae from United States in 1992. In Enterobacteriaceae three major groups of qnr determinants, qnrA, qnrB and qnrS, have been identified.  aac(6')-Ib gene encodes a common aminoglycoside acetyltransferase responsible for resistance to the aminoglycosides. A variant of that gene aac(6')-Ib-cr is responsible for low levels of fluoroquinolone resistance. Together with qnr it may cause high levels of fluoroquinolone resistance. 
PMQR has been reported in many bacteria and very few reports are available regarding the same in India but, several of them are from other countries. The work done at NICED Kolkata, India revealed the presence of aac(6')-1b-cr gene in gram negative bacilli but not the qnr gene.  There have been few studies in India for the detection of PMQR in typhoidal Salmonellae, but no report of plasmid encoding qnr genes either in ciprofloxacin-resistant or drug susceptibility to ciprofloxacin strains. The study included strains of typhoidal Salmonellae showing MIC value ≥ 1 μg/ml for the detection of PMQR determinants and has reported the prevalence of PMQR determinants in typhoidal Salmonellae isolates for the first time in India. Out of all the test isolates, fourteen were positive for qnrB determinants and only one showed positive for the cr variant of aac(6')-Ib. All the isolates with a high level of ciprofloxacin resistance showed qnrB determinant. None of the isolates which were sensitive by disk diffusion carried any of the plasmid genes. One qnrS1 poitive S. Paratyphi A isolate was reported from Korea. 
In the present study, only qnrB was detected in 14 strains of typhoidal Salmonellae. The correlation between PMQR and MIC indicates that plasmid mediated resistance is not the only mechanism, because certain strains with MIC value 1 μg/ml were negative for plasmid gene; mutation in QRDR of DNA gyrase and topoisomerase or efflux pump mechanism may be responsible.
The fact that high MIC is always due to target mutations in the QRDR and low level MIC is due to PMQR is contested by our findings; strains with MIC of 2 μg/ml were constantly positive for qnrB. However we must admit that the study did not include search for mutations and/or efflux pumps.
| ~ Conclusion|| |
During the study period, 36 (57.14%) of S. Typhi and 26 (41.27%) of S. Paratyphi A were isolated from blood culture. Affected groups were younger adults and children with an average age of 23.33 years. Males were predominantly affected than females. Isolations were more during summer which is evident from [Figure 2].
|Figure 2: Month wise distribution of the enteric fever occurrence in Puducherry|
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Of the 63 isolates two isolates of S. Typhi were MDR. None of the S. Paratyphi A ware MDR. All the isolates were NAR. 20 isolates were resistant to ciprofloxacin by MIC determination method. Most of the isolates showed intermediate sensitivity towards ciprofloxacin by disc diffusion method. The MIC values of ciprofloxacin were 1 μg/ml (15 isolates), 2 μg/ml (4 isolates) and 16 μg/ml (1 isolate).
Of the 20 isolates 14 carried the plasmid gene qnrB and only one S. Typhi carried aac(6')-Ib-cr gene, which was negative for plasmid gene qnrB. qnrA, qnrS plasmids were not detected in this study. The antibiotic sensitivity pattern of typhoidal Salmonellae shows a trend similar to that observed in the past. However, the significant finding of this study is that the qnrB gene, which is responsible for PMQR was detected in a good number of strains. This is worrisome because ciprofloxacin is a useful orally administrated antibiotic for the treatment of enteric fever. Future research should focus more on this and other genes responsible for PMQR.
| ~ Acknowledgments|| |
The authors would like to acknowledge Dr. T. Ramamurthy, Scientist F, Deputy Director, NICED, Kolkata, West Bengal, India who generously, provided us with the control strains for PCR assay.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]
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