Abstract: The aim of the study was to determine antimicrobial susceptibility pattern and to examine the mechanism of increasing quinolone resistance of S. typhi isolates in Kerala, South India. Ciprofloxacin resistant isolates were characterized by mutation analysis of quinolone resistance determining region (QRDR) of gyrA, gyrB, parC and parE genes and the results showed that majority of the isolates were resistant to nalidixic acid. There was an increase in the number of isolates showing resistance to ciprofloxacin, ceftriaxone and ofloxacin. Multi drug resistant (MDR) S. enterica serovar Typhi were not isolated. Majority of ciprofloxacin resistant isolates carried mutation in quinolone resistance determining region (QRDR) of gyrA and strains with high level ciprofloxacin resistance carried double mutation in quinolone resistance determining region (QRDR) of gyrA and single mutation in quinolone resistance determining region (QRDR) of parC. In conclusion we reported that there was an increase in the number of S. typhi isolates showing resistance to flouroquinolones and ceftriaxone. We have found out that flouroquinolone resistance in S. typhi isolates was not plasmid encoded. We had also found out that mutations in quinolone resistance determining region (QRDR) region of gyrA play an important role in flouroquinolone resistance of S. typhi isolates and parC mutation facilitate increasing level of resistance.
INTRODUCTION
Salmonella enterica serovar Typhi is the causative agent of typhoid fever which cause 16 million cases with 6,00,000 deaths worldwide annually (Crump et al., 2004). Majority of these cases were reported in developing countries (Zulfiqar, 2006). Until the development of multi drug resistant (MDR) S. enterica serovar Typhi strains in 1989, chloramphenicol was the drug of choice for treating typhoid fever (Rowe et al., 1997). Since then, flouroquinolones particularly ciprofloxacin was the drug of choice in the treatment of typhoid fever (Rowe et al., 1991). But reports have indicated the emergence of Salmonella enterica serovar Typhi strains that exhibited decreased susceptibility to flouroquinolones (Kenji et al., 2001; Parry et al., 1998). The emergence of S. enterica serovar Typhi strains with decreased flouroquinolone susceptibility and nalidixic acid resistance have been reported in India (Tamilarasu et al., 2005). High level ciprofloxacin resistance in S. enterica serovar Typhi has also been recently reported by Hasan et al. (2005). Since, physicians still administer ciprofloxacin for the treatment of typhoid fever ciprofloxacin resistance and reduced susceptibility to ciprofloxacin is a matter of concern.
Resistance to quinolones arise mainly due to a number of mechanisms, including point mutations that result in amino acid substitutions in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) which maintain the level of super coiling of bacterial DNA, reduced outer membrane permeability, increased efflux of drugs and plasmid encoded gene (Katie et al., 2005; George, 2005; Piddock, 2002; John et al., 2005; George et al., 2006). Since, there is an increase in the occurrence of antibiotic resistant S. typhi isolates routine surveillance should be performed to determine the extent of antibiotic resistance and to provide suitable guidelines for treatment. The aim of the study was to examine the mechanism of increase in ciprofloxacin resistance in S. typhi isolates in Kerala by testing antimicrobial susceptibility, MIC determination, plasmid analysis and mutation analysis of quinolone resistance determining region (QRDRs) of gyrA, gyrB, parC and parE gene of flouroquinolone non-susceptible isolates.
MATERIALS AND METHODS
Bacterial Strains
A total of 48 strains of S. enterica serovar Typhi were isolated
from blood cultures of febrile patients from various hospitals in Kerala,
India from the period 2006-2007. Strains were tested biochemically and
confirmation was done using agglutinating O antisera.
Antimicrobial Susceptibility Testing
S. enterica serovar Typhi isolates were tested for susceptibility
to antimicrobials by disc diffusion technique according to guidelines
provided by the Clinical and Laboratory Standards Institute, CLSI (Formerly
National Committee for Clinical Laboratory Standards) (NCCLS, 2006) using
commercially available antimicrobial discs (Hi Media Laboratories, India).
Following antimicrobials were tested: chloramphenicol (30 μg), ampicillin
(10 μg), amoxycillin (30 μg), gentamycin (10 μg), co-trimoxazole
(25 μg), tetracycline (30 μg), nalidixic acid (10 μg),
ciprofloxacin (5 μg), ofloxacin (5 μg) and ceftriaxone (25 μg).
Escherichia coli ATCC 25922 was used as control strain for antimicrobial
susceptibility testing.
Determination of Minimum Inhibitory Concentration (MIC)
Minimum Inhibitory Concentration (MIC)s of ampicillin, amoxycillin,
tetracycline, gentamycin, chloramphenicol, ciprofloxacin, ofloxacin, nalidixic
acid and ceftriaxone were determined for each strain by broth macrodilution
method according to guidelines provided by CLSI (NCCLS, 2000).
Plasmid DNA Analysis
All isolates of S. enterica serovar Typhi isolates were subjected
to plasmid profiling. Plasmid DNA was extracted by rapid procedure using
alkaline lysis method (Kado and Len, 1981). Plasmid DNA was analyzed on
its electrophoretic movement on 0.8% agarose gel at 100 V for 2 h. Molecular
mass of plasmid DNA was estimated by Hind III digested Lambda DNA marker.
Transformation Experiments
Transformation experiments with plasmid DNA from ciprofloxacin resistant
S. enterica serovar Typhi isolates were done to study whether genes
encoding flouroquinolone resistance is plasmid borne. Plasmid DNA isolated
from ciprofloxacin resistant isolates was transformed in to E. coli
DH5α using standard protocol (Sambrook et al., 1989). Transformants
were grown on LB plates containing 5 mg L-1 of ciprofloxacin.
Amplification of Quinolone Resistance Determining Region of gyrA,
gyrB, parC and parE Genes
Polymerase chain reaction (PCR) primers were designed using Primer
3 programme. (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_results.cgi).
Primers were synthesized using DNA synthesizer (AB1 3900 DNA synthesizer).
Chromosomal DNA from ciprofloxacin resistant isolates was prepared by
boiling the strains for 10 min, followed by centrifugation at 13,000 rpm
for 2 min to obtain the supernatant. PCR was performed on MJ Research
thermal cyclerTM with the four primer pairs shown in Table
1.
| Table 1: | Primers used for amplification and sequencing of gyrA, gyrB, parC and parE genes |
PCR was performed in a total volume of 25 μL which contained 5 μL of supernatant, 25 pmol of each primer, 200 μM dNTPs, 1.5 mM MgCl2 and 0.5 U of Taq polymerase (Bangalore Genei, India). After an initial denaturation step of 3 min at 94°C, amplification was performed over 30 cycles, each consisting of 1 min at 94°C, 1 min at hybridization temperature (56°C for gyrA, 58°C for gyrB and 53°C for parC and parE) and 1 min at 72°C, with a final extension step of 10 min at 72°C. PCR products were purified according to standard procedures (Sambrook et al., 1989).
Sequencing and Analysis of Quinolone Resistance Determining Rregion
of gyrA, gyrB, parC, parE Genes
Sequencing of amplified quinolone resistance determining region of
gyrA, gyrB, parC and parE genes was carried
out by the method of Sanghavi et al. (1999) in an automated DNA
sequencer (ABI 3100 Genetic Analyzer) with primers given in Table
1. DNA sequences of amplified regions were analyzed using commercial
software (Chromas software). Sequences were compared using BLAST analysis
with nucleotide sequence database of gyrA, gyrB, parC
and parE under the following accession numbers: S. enterica
serovar Typhi gyrA accession No: AB071870, S. enterica
serovar Typhi gyrB accession No: AB072396, S. enterica
serovar Typhi parC accession No: AB071987 and S. enterica
serovar Typhi parE accession No: AB072701.
RESULTS
The study revealed that 68.7% S. enterica serovar Typhi isolates were resistant to nalidixic acid (Fig. 1). S. typhi isolates were categorized in to ciprofloxacin sensitive, intermediately susceptible and resistant isolates by disk diffusion technique. Fifty percent isolates showed susceptibility to ciprofloxacin, 50% were intermediately susceptible and none were resistant. 16.7% of isolates showed resistance to ofloxacin. None of the isolates exhibited multiple drug resistance. All strains were sensitive to chloramphenicol. Fifty percent strains were resistant to tetracycline. 8.3% isolates showed resistance to ceftriaxone and 23% showed intermediate resistance to ceftriaxone. Antibiotyping of S. typhi strains yielded 18 biotypes (Table 2).
A summary of MIC values and number of strains is shown in Table 3. MIC values of ciprofloxacin ranged between 0.25-16 mg L-1. According to NCCLS break point the criteria for ciprofloxacin resistance was an MIC value of ≥4 mg L-1, for decreased susceptibility MIC value was in range between 1 and <4 mg L-1 and for susceptible strains MIC value was <1 mg L-1. So, in this study out of 48 isolates, 23 (47.9%) isolates showed ciprofloxacin resistance and 10 (20.8%) isolates showed decreased susceptibility to ciprofloxacin. 36 (75%) isolates showed MIC value of >4 mg L-1 for ofloxacin. One strain had MIC value of 16 mg L-1 for ciprofloxacin and 64 mg L-1 for ofloxacin.
| Fig. 1: | Antimicrobial susceptibility pattern of Salmonella enterica serotype Typhi isolates from Kerala during 2006-2007 (n = 48) |
| Table 2: | Antibiotyping of S. typhi isolates from Kerala (n = 48) |
| n = Total number of strains analyzed. Values in parentheses indicates percentage; Amp: Ampicillin, Chl: Chloramphenicol, Tet: Tetracycline, Amx: Amoxycillin, Gen: Gentamycin, Sxt: Co-trimoxazole, Cro: Ceftriaxone, Nal: Nalidixic acid, Ofl: Ofloxacin, Cip: Ciprofloxacin | |
| Table 3: | Minimum inhibitory concentration of S. typhi isolates (n = 48) |
| |
|
| n = Total number of strains analyzed | |
Plasmid profile analysis revealed that none of the isolates carried high molecular weight plasmids. Eighty five percent of isolates carried plasmid with 23 Kb molecular weight and all the isolates had smaller bands of plasmids.
To find out whether flouroquinolone resistance associated with each strain was located on a transformable plasmid, plasmid DNA from ciprofloxacin resistant S. typhi isolates was transformed to E. coli DH5α. The transformants was not able to grow on media containing 5 mg L-1 of ciprofloxacin.
Quinolone resistance determining region (QRDR) of gyrA, gyrB, parC and parE genes of 20 ciprofloxacin resistant isolates (13 strains that showed resistance to ciprofloxacin and 7 strains that showed decreased susceptibility to ciprofloxacin) and two ciprofloxacin susceptible strains were amplified and sequenced. DNA sequences were compared using BLAST analysis with that of nucleotide sequence database of 4 genes. Out of 20 ciprofloxacin resistant isolates 18(90%) isolates contained single amino acid substitution at 83 position in quinolone resistance determining region of gyrA and 60% isolates with high level ciprofloxacin resistance (MIC≥4 mgL-1) contained a double amino acid alteration at 83 and 87 position in quinolone resistance determining region of gyrA and single alteration at 84 position in quinolone resistance determining region of parC. Substitutions were Ser 83→Phe and Asp87→Asn, Tyr in gyrA, or Glu84→Lys in parC. Amino acid substitutions were not detected in quinolone resistance determining region of gyrB and parE. No alteration in quinolone resistance determining region of gyrA, gyrB, parC and parE were detected in two ciprofloxacin susceptible strains.
DISCUSSION
Recently, there is an increase in the occurrence of antibiotic resistant bacteria of all genera. So, scientific community should perform routine surveillance of microbial population to determine the extent of antibiotic resistance to provide suitable guidelines for treatment. In the present study, 48 clinical isolates of S. typhi from Kerala were tested for their susceptibility to 10 antibiotics used in the treatment of typhoid fever. MDR S. enterica serovar Typhi were not isolated in this study. Studies conducted at various regions in India also shows there is a decrease in the occurrence of MDR S. typhi (Madhulika et al., 2004; Sanghavi et al., 1999; Saha et al., 2002). Reoccurrence of resistant strains has also been reported by Kumar et al. (2002). Studies in U.S reported an increase in the number of MDR nalidixic acid resistant S. typhi (Marta-Louise et al., 2000; Jennifer et al., 2007) whereas studies in Bangladesh reported an decrease in MDR isolates with no corresponding increase in sensitive strains (Rahman et al., 2002).
There was an increase in the number of strains showing resistance to nalidixic acid, ciprofloxacin and ceftriaxone. Fifty percent of isolates were identified as intermediately susceptible to ciprofloxacin by disk diffusion technique. There was an increased value of MICs of ciprofloxacin. MIC values of ciprofloxacin of isolates ranged between 0.25-16 mg L-1. This finding was similar to earlier studies in UK and India (Threlfall and Ward, 2001; Jesudason et al., 1996). MIC values showed that 48% isolates were ciprofloxacin resistant and 21% showed decreased susceptibility to ciprofloxacin. 8.3% strains showed resistance and 23% strains showed intermediate resistance to ceftriaxone which was in contrast to earlier study in which all strains were reported as ceftriaxone sensitive (Pallab et al., 2006).
The present study also focused to examine the genetic locus of flouroquinolone resistance. Flouroquinolone resistance usually arises spontaneously due to mutations within the Type II topoisomerase genes often in combination with decreased expression of multidrug efflux pump. Some studies reports that quinolone resistant gene is located on plasmids. Plasmid mediated quinolone resistance was first reported in a clinical isolate of Salmonella enterica strain in U.K in 2007 (Katie et al., 2007). To find out whether flouroquinolone resistance associated with S. typhi isolates was located on a transformable plasmid we conducted transformation experiment. The result showed that none of the isolates carry ciprofloxacin resistance genes on plasmid.
Earlier studies reported that flouroquinolone resistance arises due to mutations in quinolone resistance determining region of genes encoding DNA gyrase (gyrA, gyrB) or DNA topoisomerase IV (parC, parE) (Katie et al., 2005; George, 2005; Piddock, 2002). We tried to characterize gyrA, gyrB, parC and parE mutations in ciprofloxacin resistant isolates in Kerala. Ninty percent of ciprofloxacin resistant isolates had single mutation in the quinolone resistance determining region of gyrA. Sixty percent isolates with high level of ciprofloxacin resistance carried double mutation in gyrA and single mutation in parC. The result is in consistent with earlier findings that gyrA mutations are necessary for quinolone resistance. parC mutations facilitate increasing levels of resistance (Piddock, 2002).
CONCLUSION
There was an increase in the number of isolates showing resistance to flouroquinolones and ceftriaxone. We have found out that flouroquinolone resistance in S. typhi isolates was not plasmid encoded. We had also found out that mutations in quinolone resistance determining region of gyrA play an important role in flouroquinolne resistance of S. typhi isolates and parC mutation facilitate increasing level of resistance.
ACKNOWLEDGMENTS
Junior fellowship grant from Indian Council of Medical Research (ICMR) to N. Ayana is gratefully acknowledged. Also grateful to the Department of Biotechnology and Microbiology, Kannur University for the facilities and scientific support.