|Year : 2013 | Volume
| Issue : 4 | Page : 374-378
Study of the role of efflux pump in ciprofloxacin resistance in Salmonella enterica serotype Typhi
V Sharma, S Dahiya, P Jangra, BK Das, R Kumar, S Sood, A Kapil
Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
|Date of Submission||07-Feb-2013|
|Date of Acceptance||13-Jul-2013|
|Date of Web Publication||25-Sep-2013|
Department of Microbiology, All India Institute of Medical Sciences, New Delhi
Source of Support: This study was supported by Indian Council of
Medical Research, Conflict of Interest: None
Purpose: There are increasing reports on failure of clinical response to ciprofloxacin in typhoid fever despite the strain being sensitive to drug in in-vitro using standard guidelines and showing mutations in DNA gyrase. But this increased MIC and clinical failures with ciprofloxacin are not always co-related with mutations presently identified in gyrA and parC genes. This shows that there may be other mechanisms such as an active drug efflux pump responsible as has been shown in other Enterobacteriaceae. This study was carried out to determine the role of efflux pump in Salmonella Typhi isolates. Materials and Methods : Total 25 already characterized nalidixic acid sensitive and nalidixic acid resistant S. Typhi strains with different range of ciprofloxacin MIC were included to study the role of efflux pump in the presence of CCCP (efflux pump inhibitor). For genotypic characterization, the entire acrR gene was sequenced to confirm the presence of any mutation in the gene. Results: The MIC of ciprofloxacin remained same in the presence and absence of CCCP in the studied strains and no significant mutations were found in the acrR gene in any of the isolates studied. Conclusions: No role of efflux pump in ciprofloxacin resistance was found in strains studied. There is a need to explore further mechanism of ciprofloxacin resistance in Salmonella Typhi.
Keywords: AcrR, carbonyl cyanide m-chlorophenylhydrazone, ciprofloxacin, efflux pump, minimum inhibitory concentration, polymerase chain reaction, resistance, S. typhi
|How to cite this article:|
Sharma V, Dahiya S, Jangra P, Das B K, Kumar R, Sood S, Kapil A. Study of the role of efflux pump in ciprofloxacin resistance in Salmonella enterica serotype Typhi. Indian J Med Microbiol 2013;31:374-8
|How to cite this URL:|
Sharma V, Dahiya S, Jangra P, Das B K, Kumar R, Sood S, Kapil A. Study of the role of efflux pump in ciprofloxacin resistance in Salmonella enterica serotype Typhi. Indian J Med Microbiol [serial online] 2013 [cited 2020 Sep 30];31:374-8. Available from: http://www.ijmm.org/text.asp?2013/31/4/374/118898
| ~ Introduction|| |
Typhoid fever continuous to be a major health concern in developing countries despite the availability of good antibiotics. After the emergence of multiple drug resistance (MDR) strains, ciprofloxacin was widely used but was soon followed by reports on clinical failure to the treatment of patients with ciprofloxacin. ,, This happened even though the strains of Salmonella More Details enterica serovar Typhi (hereafter called Salmonella Typhi) causing these infections were susceptible to ciprofloxacin using Clinical and Laboratory Standards Institute breakpoints in the laboratory tests.
In light of this, Nalidixic Acid (NA)-another quinolone-was used as surrogate marker to depict decreased susceptibility to ciprofloxacin thus dividing Salmonella Typhi in to Nalidixic acid resistant Salmonella Typhi (NARST) and Nalidixic acid sensitive Salmonella Typhi (NASST) with infections due to NARST being indicative of possible clinical failure. ,,, We have previously reported NARST strains having decreased susceptibility to ciprofloxacin and had mutations in gyrA gene commonly at Ser83 to Phe/Tyr followed by Asp87 to Asn/Gly/Tyr with few strains showing double mutations. Although, ciprofloxacin minimum inhibitory concentration (MIC) ranged from 0.023 μg/ml to 1.0 μg/ml in NARST strains, there were no additional mutations in gyrA gene correlating with increasing MICs to ciprofloxacin. Additionally, some infections due to NASST strains without gyrA mutation showed no response to ciprofloxacin therapy.
This led us to look for some additional mechanisms for resistance to ciprofloxacin in Salmonella Typhi in our isolates. Efflux pump has been shown to be responsible for ciprofloxacin resistance in enterobacteriaceae. ,, There are few studies showing the presence of efflux pump activity in Salmonella Typhi.,
The aim of the present study was to look for the role of efflux pump in the Salmonella Typhi strains which were also characterised for gyrA mutation and isolated from patients with documented clinical failure to treatment with ciprofloxacin.
| ~ Materials and Methods|| |
Selection of strains
A total of 25 previously characterised Salmonella Typhi strains were selected for the study. These strains were selected based on varying levels to MIC to ciprofloxacin and representing NARST as well as NASST  [Table 1]. Nalidixic acid sensitive strains were used as negative control.
|Table 1: The characterisation of strains included in the present study *(Renuka et al., 2004)|
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Phenotypic expression of efflux pump
Ciprofloxacin standard curve
Ciprofloxacin solution of different concentrations (μg/ml) was prepared in 0.1 M glycine hydrochloride (pH 3.0) (Sigma, USA). The fluorescence of the solution was measured at λexcitation and λemission of 279 nm and 447 nm, respectively. Fluorescence intensity was plotted against ciprofloxacin concentration to obtain a standard curve [Figure 1] and [Table 2].
|Table 2: The different concentration of ciprofloxacin for standard curve|
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|Figure 1: Ciprofloxacin standard curve: Ciprofloxacin concentration v/s fluorescence intensity|
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Accumulation of ciprofloxacin
Ciprofloxacin uptake was assayed by the method described by Mortimer and Piddock.  Briefly, bacteria grown till late logarithmic phase were harvested by centrifugation and resuspended in sodium phosphate buffer. To this suspension 10 μg/ml ciprofloxacin was added. Subsequently, samples were removed at different time intervals. After 5 min, efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma, USA) was added to the reaction mixture. Samples were diluted with ice-cold sodium phosphate buffer, harvested by centrifugation and resuspended in glycine hydrochloride buffer (pH 3.0). This causes the lysis of the cells. The suspension was centrifuged and fluorescence of the supernatant (containing the ciprofloxacin released by the cells after lysis) was measured at λexcitation and λemission of 279 nm and 447 nm, respectively.
Results were compared with the standard curve for ciprofloxacin to obtain the amount of ciprofloxacin accumulated.
Minimum inhibitory concentration of ciprofloxacin in presence of carbonyl cyanide m-chlorophenylhydrazone
To investigate the presence of active efflux mechanism, the MIC of ciprofloxacin was determined in combination with 0.5 μg of CCCP per ml. An efflux mechanism was inferred to be present when the quinolone MIC in the presence of CCCP was at least fourfold less (two doubling dilutions) than the corresponding MIC in the absence of these compounds, which also served as a internal control.
Genotypic study of efflux pump
The entire acrR gene was amplified to ensure the detection of any mutation in the gene including at the hot spot. The forward primer: 5′-GGTCCTTAAACCC ATTGCTG-3′ was designed in house by using Primer 3 software, to bind upstream flanking region of acrR gene sequence of S. Typhi strain CT18 available through GenBank (Accession no. NC_003198.1). The primer for its suitability was analysed by using Integrated DNA Technologies OligoAnalyzer 3.1 software. The designed forward primer was used with the reverse primer: 5′-ACAGAATAGCGACACAGAAA-3′  to obtain a 816 bp amplicon.
Polymerase chain reaction for acrR
The polymerase chain reaction (PCR) was carried at the following reaction conditions: Initial PCR activation for 5 min at 94°C, enrichment cycling for 30 cycles of (15 sec at 94°C, 60 sec at 57°C, 60 sec at 72°C), and final extension for 10 min at 72°C. The product was examined in 2% agarose gel seen under ultraviolet (UV) rays with the help of ethidium bromide to confirm the product at 816 bp.
The PCR product was sequenced by dideoxy- chain termination method with ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit, Version 2.0 (Perkin-Elmer Applied Biosystems, Foster city, California) using the same primers as were used for PCR. Sequencing was performed with both forward and reverse primers on GeneAmp 2400 DNA Thermal Cycler and analyzed in an automatic deoxyribonucleic acid (DNA) sequencer ABI PRISM 310 Genetic Analyzer (Perkin-Elmer Applied Biosystems).
| ~ Results|| |
Phenotypic expression of efflux pump
The experiment was performed in two sets: Initially ciprofloxacin (10 μg/ml) was added in both the set. CCCP was added only in Set II after 5 min. At regular time intervals, 0.5 ml aliquots were taken from the two sets and the fluorescence was obtained as a measure of ciprofloxacin accumulated inside the cell, as shown in [Table 3].
|Table 3: The fluorescence obtained from a representative strains (3238/96) with and without adding CCCP, measured at regular time intervals|
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MIC of ciprofloxacin in presence of carbonyl cyanide m-chlorophenylhydrazone
The MIC was determined in the presence and absence of CCCP (final conc. 0.5 μg/ml) by agar dilution method [Table 4]. However, the MIC of most of the strains remained same in the presence and absence of CCCP and for some of the strains varied by less than or equal to twofold.
Genotypic study of efflux pump
The PCR product obtained was examined by agarose gel electrophoresis, sequenced and analysed with the GeneDoc software. On alignment with the acrR gene sequence of S. Typhi strain CT18 available in GenBank, no significant mutations were found in the gene in any of the clinical strains [Figure 2].
| ~ Discussion|| |
Ciprofloxacin resistance in typhoid fever is being increasingly reported from developing countries.  Ciprofloxacin act by inhibiting DNA synthesis due to its action mainly on gyrA and parC. The reports of mutations in gyrA and parC contributing to the drug resistance have already been reported in clinical isolates of Salmonella Typhi by using Nalidixic acid as surrogate marker for resistance called as NARST. However, these mutations do not always explain different level of ciprofloxacin MIC and clinical failure to the treatment even with infections due to NASST strains. 
So to understand the possibility of role of efflux pump to impart resistance or reduced susceptibility to ciprofloxacin, we carried out the present study in strains isolated from patients with documented clinical failure to the treatment with ciprofloxacin from our hospital which were already characterised for gyrA mutations.
In Enterobacteriaceae, efflux pump has been reported to be responsible for ciprofloxacin resistance. However, few reports are available which shows the contribution of efflux activity to ciprofloxacin resistance in S. Typhi., In a recent study, active efflux pump was found to contribute to ciprofloxacin resistance S. Typhi, which also carried gyrA gene mutation. However, the mechanism was not studied at molecular level.  Phenotypically, we did not found any efflux pump activity.
With the molecular techniques available, it is possible to study mutations in the local repressor acrR as it controls the expression of AcrAB-TolC efflux pump present in Salmonella spp. and Escherichia More Details coli., The 45 th amino acid (arginine) in the structure of acrR lies within the middle of a putative helix-turn-helix DNA binding motif and is highly conserved. Mutation at this position is known to lead to the loss of repressive function and thus over-expression of the efflux pump, which is responsible for fluoroquinolone resistance in many organisms. So, therefore, any mutation in this regulator gene would reflects the role with the following aims. In our isolates, we did not found any mutation.
In our study, we made an effort to detect active efflux pump in ciprofloxacin resistant S. Typhi strains. We did not find significant efflux pump activity in Salmonella Typhi strains with increased MIC of ciprofloxacin and no spontaneous mutations were encountered in the repressor gene of efflux pump in these strains.
This study was carried out in selected strains. In the absence of this mechanism, there is a need to study the other mechanisms responsible to ciprofloxacin resistance, e.g., the role of qnr plasmids and also Pharmacokinetics/Pharmacodynamics (PK/PD) parameters. The spread of resistant Salmonella Typhi necessitates the need of continuous surveillance of antimicrobial resistance and study of mechanisms for ciprofloxacin resistance.
Efflux pump was not responsible for increasing concentration of ciprofloxacin in S. Typhi in this study.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]