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Antimicrobial resistance trends in blood culture positive Salmonella Typhi isolates from Pondicherry, India, 2005–2009
ORIGINAL ARTICLE
BACTERIOLOGY
Antimicrobial resistance trends in blood culture positive Salmonella Typhi isolates from Pondicherry, India, 2005–2009 G. A. Menezes1,2, B. N. Harish1, M. A. Khan3, W. H. F. Goessens3 and J. P. Hays3 1) Department of Microbiology, Institute of National Importance, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India, 2) Department of Microbiology, SSR Medical College, Belle Rive, Phoenix, Mauritius and 3) Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Centre, ¢s Gravendijkwal, Rotterdam, the Netherlands
Abstract Typhoid fever is caused by Salmonella enterica serovar Typhi, a major public health concern in developing countries. Recently, there has been an upsurge in the occurrence of bacterial isolates that are resistant to ciprofloxacin, and the emergence of broad spectrum b-lactamases in typhoidal salmonellae constitutes a new challenge for the clinician. A total of 337 blood culture isolates of S. Typhi, isolated from Pondicherry, India, between January 2005 and December 2009, were investigated using phenotypic, molecular and serological methods. Of the 337 isolates, 74 (22%) were found to be multidrug resistant (MDR) and 264 (78%) nalidixic acid resistant (NAR). Isolates with reduced susceptibility to ciprofloxacin possessed single mutations in the gyrA gene. A high rate of resistance (8%) was found to ciprofloxacin. All isolates with a ciprofloxacin MIC ‡ 4 mg/L possessed both double mutations in the QRDR of the gyrA gene and a single mutation in the parC gene. Active efflux pump mechanisms were also found to be involved in ciprofloxacin resistance. Finally, a large number of PFGE patterns (non-clonal genotypes) were observed among the S. Typhi isolates. In conclusion, a high rate of ciprofloxacin resistance was observed in comparison to other endemic areas in blood culture isolates of S. Typhi from Pondicherry, India, with steadily increasing NAR but decreasing MDR isolations over the study period. This is most likely to be due to an increased use of ciprofloxacin as a first-line drug of choice over more traditional antimicrobial agents for the treatment of typhoid fever. Keywords: Fluoroquinolones, gyrA, multidrug resistant, nalidixic acid resistant, pulsed field gel electrophoresis, quinolone resistancedetermining region, Salmonella Typhi Original Submission: 15 December 2010; Revised Submission: 10 April 2011; Accepted: 11 April 2011 Editor: G. Pappas Article published online: 25 April 2011 Clin Microbiol Infect 2012; 18: 239–245 10.1111/j.1469-0691.2011.03546.x Corresponding author: B. N. Harish, Department of Microbiology, Jipmer, Pondicherry 605006, India E-mail: [email protected]
Introduction Typhoid fever, caused by Salmonella enterica serovar Typhi, is a major health problem in developing countries, particularly in the Indian subcontinent and in southeast Asia. Typhoid fever is the most serious form of enteric fever and in the year 2000 it was estimated that the global number of typhoid cases exceeded 21 000 000, with more than 200 000 deaths [1]. Further, if not treated properly, enteric fever carries a mortality rate of 30%, whilst appropriate antimicrobial
treatment reduces the mortality rate to as low as 0.5% [2]. In cases of enteric fever, it is often necessary to commence treatment before the results of laboratory sensitivity testing become available, and the fluoroquinolone ciprofloxacin has become the first-line drug for treatment, especially since the widespread emergence of S. Typhi isolates that are multidrug resistant (MDR) to the more traditional antimicrobial agents comprising chloramphenicol, ampicillin and trimethoprimsulphamethoxazole (cotrimoxazole) [3]. However, this switch to ciprofloxacin has led to a subsequent increase in the occurrence of S. Typhi isolates resistant to this antimicrobial agent, including in India [4,5]. In salmonellae, quinolone resistance is usually associated with mutations in the quinolone resistance-determining region (QRDR) of the A subunit target site of DNA gyrase, though the presence of plasmid-mediated quinolone
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resistance qnr genes and aac(6¢)-Ib-cr has also been described in quinolone-resistant non-Typhi Salmonella [6]. The exact mechanism of this DNA gyrase-mediated resistance in S. Typhi is not fully understood, though various studies have found that single point mutations in this region confer resistance to nalidixic acid and hence reduced susceptibility to fluoroquinolones [7]. In contrast, high-level ciprofloxacin resistance may be due to either (i) the cumulative impact of mutations in many genes, (ii) decreased membrane permeability, (iii) active efflux pump, and/or (iv) the presence of plasmid-encoded qnr genes [8]. Worldwide, there are sporadic reports of high-level resistance to beta-lactam antibiotics, including ceftriaxone in typhoidal salmonellae, due to the presence of CTX-M-15 and SHV-12 extended spectrum b-lactamases (ESBLs) [9,10]. Further, we recently reported an ACC-1 AmpC b-lactamase producing S. Typhi that had been isolated from the blood of a girl aged 14 years [11]. Therefore, a study was undertaken to characterize trends in antimicrobial resistance in clinically relevant S. Typhi isolates originating from Pondicherry, India, to help guide clinicians in successful treatment therapies.
Materials and Methods Bacterial culture and identification
Blood cultures from a total of 3744 patients presenting with fever at the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, and Government General Hospital, Pondicherry, during the period January 2005 to December 2009 were taken for microbial diagnosis. The patients ranged in age from 2 to 74 years (median, 43 years). Standard blood culture protocols were followed, including culture on a biphasic medium consisting of Brain Heart Infusion (BHI) agar and BHI broth (HiMedia Laboratories Ltd, Mumbai, India). Colonies were identified as S. Typhi using standard biochemical methods [12], and confirmed using Salmonella polyvalent O, O9 and H:d antisera (Murex Biotech, Dartford, England).
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tration (MIC) of 0.125–1.0 mg/L. The breakpoints for ofloxacin and gatifloxacin were £2 mg/L (susceptible) and ‡8 mg/L (resistant), and for ciprofloxacin, £1 mg/L (susceptible) and ‡4 mg/mL (resistant) (CLSI, 2007). Escherichia coli ATCC 25922 was used as a control isolate. Nalidixic acid susceptibility was used as a screening test for reduced susceptibility to ciprofloxacin [13]. MICs were determined by both agar dilution and E-test (AB Biodisk, Solna, Sweden) for the antibiotics ciprofloxacin, ampicillin, chloramphenicol and ceftriaxone. MICs against gatifloxacin and ofloxacin were determined by E-test only. Efflux pumps
Testing for antimicrobial resistance via efflux pump activity was performed using the cyclohexane tolerance test [14], and via the detection of the MIC in the absence and presence of the efflux pump inhibitor (EPI) PabN (Phe-Arg-bnaphthylamide) at 20 mg/L [15]. Molecular analysis of quinolone resistance
Molecular analysis of quinolone resistance was performed on 57 (27 ciprofloxacin resistant, five ciprofloxacin intermediate and 25 ciprofloxacin susceptible) isolates. The molecular mechanism of quinolone resistance was determined by investigating mutations in the QRDRs of DNA gyrase (gyrA and gyrB) and DNA topoisomerase IV (parC and parE) genes, according to previously described protocols [16–18]. The possible presence of the plasmid-mediated quinolone resistance qnr gene (qnrA, qnrB, and qnrS), and aac(6¢)-Ib-cr gene was determined using PCR [19,20]. Detection of beta-lactamases
PCR screening and sequencing was performed to identify the b-lactamase resistance genes blaTEM, blaSHV, blaOXA-1 group and blaCTX-M [21–23]. Beta-lactamase screening was performed for 61 isolates that were highly resistant to ampicillin (MIC ‡ 256 mg/L). Isoelectric focusing of b-lactamase enzymes was performed using standard methods [24]. Isolate genotyping
Antimicrobial susceptibility testing
Isolates were tested for susceptibility to antimicrobials using the Kirby Bauer disk diffusion method. Antibiotic disks contained ampicillin (10 lg), cotrimoxazole (25 lg), chloramphenicol (30 lg), ciprofloxacin (5 lg), ceftazidime (30 lg), ceftriaxone (30 lg) and nalidixic acid (10 lg), and results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (2007). Reduced susceptibility to ciprofloxacin was determined as a minimum inhibitory concen-
Genotyping was performed on a representative sample of 20 S. Typhi isolates using pulsed field gel electrophoresis (PFGE). Briefly, isolates were incubated overnight at 37C in 7 mL Mueller Hinton broth. After incubation, 1 mL of bacterial cells was harvested, pelleted and washed three times using 1 mL EET (Na2EDTA 100 mM, EGTA 10 mM, Tris HCl 1 M) buffer, before being adjusted to a cell density of 0.5 at 560 nm. One hundred microlitres of cell suspension and 100 lL of 1.4% PFGE grade agarose in EET buffer were mixed and
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poured into PFGE plug moulds. The plugs were incubated at 4C for 30 min to harden and 1 mL of lysozyme (5 mg/mL) was added before incubation at 37C for 3–4 h. Lysozyme was removed from the plugs and 1 mL of deproteinising solution (per plug 3 mg/mL proteinase K and 1% SDS) was added prior to overnight incubation at 37C. Next, the plugs were washed six times every 30 min with T10E1 (10 mM Tris, 1 mM EDTA) buffer, and then soaked in T10E0.1 (10 mM Tris, 0.1 mM EDTA) buffer for 30 min. Restriction digestion was performed using 40 U of XbaI (Fermentas, St. Leon-Rot, Germany) at 37C. PFGE was performed in a 1% agarose gel in a CHEF DRII system (BioRad, Hercules, CA, USA) with the following conditions: 0.5· Tris–Borate EDTA buffer, 14C, 6 V/cm for 22 h (with switch times ranging from 5 to 40 s). Lambda ladder PFGE marker (BioRad) was used as a molecular weight standard.
Antimicrobial resistance in Salmonella Typhi
phi isolates during the study period Period of isolation
No. (%) NAR
No. (%) CIPR
2005 2006 2007 2008 2009 Total
51 57 48 58 50 264
0 5 8 9 5 27
(6.6) (12.7) (13) (8.5) (8)
100 90 80 70 MDR NAR
60 50 40 30
Trends in antimicrobial susceptibility of S. Typhi isolates
20
A total of 337 S. Typhi isolates from two hospitals recovered during the period 2005–2009 were included in this study, representing 100% of the S. Typhi isolates recovered during this period. Of these isolates, 222 (65.9%) were fully susceptible to ampicillin, chloramphenicol and cotrimoxazole; 74 (22.0%) were MDR; 264 (78.3%) were nalidixic acid resistant (NAR); and 27 (8.0%) were both MDR and NAR (MDR being defined as resistance to all first-line antimicrobials, i.e. ampicillin, cotrimoxazole and chloramphenicol). The results are summarized in Table 1. Twenty-seven isolates (8.0%) were resistant to ciprofloxacin. There was a steady decline in the number of MDR isolates over the 5-year study period, as well as a parallel increase in NAR (non-MDR) isolates (Tables 1 and 2 and Fig. 1). The susceptibility results for different antibiotics as well as the MIC50 and MIC90 (MICs at which 50% or 90% of the isolates were inhibited) values obtained for 337 S. Typhi isolates are shown in Table 3. Of the 27 ciprofloxacin-resistant
10 0
2005
2006
[70]
[76]
2007 Year [63]
2008
2009
[69]
[59]
FIG. 1. Proportions that were nalidixic acid resistant (NAR) and multidrug resistant (MDR) among the isolates of S. Typhi. Numbers in brackets indicate the total number of blood culture positive S. Typhi isolates cultured in each year at Jawaharlal Institute of Postgraduate Medical Education and Research and the Government General Hospital, Pondicherry, India.
isolates recovered in this study, a single isolate was found to possess an MIC of >32 mg/L (E-test), whilst the corresponding agar dilution method and HIMEDIA HiComb MIC test (HiMedia) revealed an MIC of 64 mg/L. In the same isolate, the MIC of ofloxacin and gatifloxacin (E-test), was >32 and 8 mg/L, respectively. For gatifloxacin, the MIC50 and MIC90 were one to two dilution steps lower than those of cipro-
antimi-
crobial resistance to ampicillin, cotrimoxazole and chloramphenicol among S. Typhi isolates
(72.9) (75) (76.2) (84) (83.3) (78.3)
CIPR, ciprofloxacin resistant; NAR, nalidixic acid resistant.
Results
TABLE 1. Distribution of
241
TABLE 2. Trends in resistance to quinolones among S. Ty-
Percentage of isolates tested
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Period of isolation
No. (%) susceptiblea
No. (%) with single resistanceb
No. (%) paired resistancec
No. (%) MDRd
Total no. (%)
2005 2006 2007 2008 2009 Total
24 40 37 65 56 222
9 7 8 2 3 29
6 3 1 2 0 12
31 26 17 0 0 74
70 76 63 69 59 337
(34.3) (52.6) (58.7) (94.2) (94.9) (65.9)
(12.4) (9.2) (12.7) (2.9) (5.1) (8.6)
(9) (4) (1.6) (2.9) (3.5)
(44.2) (34.2) (27)
(22)
(20.8) (22.6) (18.7) (20.4) (17.5) (100)
a
Susceptible to ampicillin, chloramphenicol and cotrimoxazole. b Single resistance – resistance to only one antimicrobial (ampicillin or cotrimoxazole). c Paired resistance – resistance to two antimicrobials (ampicillin and chloramphenicol; ampicillin and cotrimoxazole; and chloramphenicol and cotrimoxazole). d MDR – defined as resistance to ampicillin, chloramphenicol and cotrimoxazole.
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TABLE 3. Antimicrobial sensitivity patterns obtained for S. Typhi isolates recovered from blood cultures of patients in Pondicherry, India,
between
(n = 337)
2005
and
2009
MIC (mg/L) Antimicrobial agents
Sensitive No. (%)
Intermediate No. (%)
Resistant No. (%)
Range
MIC50
MIC90
Ampicillin Chloramphenicol Cotrimoxazole Nalidixic acid Ciprofloxacin Gatifloxacin Ofloxacin Ceftazidime Ceftriaxone
243 (72.1) 255 (75.7) 234 (69.4) 73 (21.7) 305 (90.5) 324 (96.1) 310 (92.0) 337 (100) 337 (100)
12 (3.6) 6 (1.8) 0 0 5 (1.5) 6 (1.8) 4 (1.2) 0 0
82 (24.3) 76 (22.6) 103 (30.6) 264 (78.3) 27 (8.0) 7 (2.1) 23 (6.8) 0 0
1–‡256 1–‡256 – – 0.016–64 0.016–8 0.016–32 0.064–2 0.064–2
1 2 ND ND 0.5 0.125 0.5 0.25 0.25
‡256 ‡256 ND ND 1 1 4 0.25 0.25
ND, not determined. Seventy-four (22.0%) isolates were multidrug resistant (MDR), defined as being resistant to ampicillin, chloramphenicol and cotrimoxazole. MIC50 and MIC90 = MIC at which 50% and 90% of the isolates were inhibited, respectively.
floxacin and ofloxacin with respect to all isolates tested. Ciprofloxacin MICs for susceptible isolates ranged between 0.016 and 1 mg/L. A correlation was observed between reduced ciprofloxacin susceptibility and nalidixic acid resistance (231/234 isolates with ciprofloxacin MICs ranging from 0.125 to 2 mg/L were NAR). The exceptions were three isolates with 1 mg/L MIC that were nalidixic acid sensitive (NAS) and MDR. Seventy-one of the isolates possessing ciprofloxacin MICs of £0.25 mg/L were all NAS and susceptible to all other antimicrobial agents tested. All 337 isolates were found to be susceptible to ceftazidime and ceftriaxone, though interestingly there was a gradual increase in MIC90 values for both ceftazidime and ceftriaxone during the study period from 0.064 to 0.25 mg/L. Efflux pumps
Using the cyclohexane tolerance test, only a single S. Typhi isolate (with a ciprofloxacin MIC of 64 mg/L) showed evidence of an efflux pump-mediated contribution to resistance. In the presence of EPI, the MIC to ciprofloxacin and nalidixic acid decreased four-fold in all of the 27 isolates possessing an MIC of ‡4 mg/L. Molecular analysis of quinolone resistance
The relationship between ciprofloxacin MIC and nucleotide changes within DNA gyrase and topoisomerase IV subunit genes of 11 isolates, representing different resistance profiles, is shown in Table 4. Isolates with ciprofloxacin MICs of ‡1 to <4 mg/L, exhibited a single point mutation in gyrA and parC genes. NAR isolates with a comparatively reduced susceptibility to ciprofloxacin (MIC 0.125–0.5 mg/L), possessed a single gyrA mutation (either at Ser83 or Asp87) only. All ciprofloxacin-resistant isolates (MICs ‡ 4 mg/L) showed three mutations, two mutations within the QRDR of gyrA, at positions 83 and 87, and a single mutation in parC, at position 80. No mutations were detected in QRDRs of gyrA and
TABLE 4. Relationship between ciprofloxacin MIC, point mutations within the QRDRs of DNA gyrase and topoisomerase IV subunit genes and testing for the effect of efflux pumps in 11 representative S. Typhi isolates Isolate no.
CIP MIC (mg/L)
T20
64
T85 T86 T9
1 2 4
T1 T2 T7
2 2 6
T13 T14 T18 T21
0.015 0.5 1 0.25
Amino acid change in GyrAa
Amino acid change in ParCb
Efc
(Ser 83 fi Phe), (Asp 87 fi Asn) (Ser 83 fi Phe) (Ser 83 fi Phe) (Ser 83 fi Leu), (Asp 87 fi Asn) (83 Ser fi Tyr) (Ser 83 fi Phe) (Ser 83 fi Phe), (Asp 87 fi Asn) None (Ser 83 fi Phe) (Ser 83 fi Phe) (Ser 83 fi Tyr)
(Ser 80 fi Ile)
+
(Thr 57 fi Ser) (Ser 80 fi Ile) (Ser 80 fi Ile)
) ) +
(Thr 57 fi Ser) (Ser 80 fi Ile) (Ser 80 fi Ile)
) ) +
None None (Thr 57 fi Ser) None
) ) ) )
CIP, ciprofloxacin; MIC, minimum inhibitory concentration. a Nucleotide changes in gyrA, 83-TCC (Ser) fi TTC (Phe); 83-TCC (Ser) fi TAC (Tyr); 83-TCC (Ser) fi TTG (Leu); 87-GAC (Asp) fi AAC (Asn). b Nucleotide changes in parC, 80- AGC (Ser) fi ATC (Ile); 57- ACC (Thr) fi AGC (Ser). Alterations in the QRDRs of gyrB and parE genes were not found in any of the strains tested. c Ef – phenotypic test for elucidation of efflux mechanism.
parC genes of nalidixic-acid-susceptible isolates, for which ciprofloxacin MICs were <0.125 mg/L. Sequence mutations in the QRDRs of gyrB and parE genes were not found in any of the isolates tested. In some NAS and NAR isolates, irrespective of their MICs against ciprofloxacin (0.015–£4 mg/L; data not shown), a mutation was found outside the QRDR of gyrA. This substitution, found outside the QRDR of GyrA, was Gly133 fi Glu. All isolates were negative for qnr (qnrA, qnrB, and qnrS) and aac(6¢)-Ib-cr genes. Detection of beta-lactamase genes
Twelve (20%) of 61 isolates highly resistant to ampicillin isolates were found to be positive for blaTEM-1, with isoelectric
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FIG. 2. Pulsed field gel electrophoresis fingerprinting of 20 S. Typhi isolates that exhibited a range of antimicrobial susceptibility profiles identified in this study. Cluster analysis was performed using the method of dice coefficient with unweighted pair group method with arithmetic mean with band tolerance set to 2.0%. All isolates were cultured from blood. Antimicrobial drugs: ciprofloxacin (CIP), ampicillin (AMP), chloramphenicol (CHL), trimethoprim/ sulphamethoxazole (SXT), nalidixic acid (NAL), ceftriaxone (CRO).
focusing experiments showing a b-lactamase with a pI of 5.4 (TEM-1). Four (7%) of the 61 isolates were positive for blaOXA-1-like genes, but none of the isolates were positive for both blaTEM-1 and blaOXA-1. Of the 12 blaTEM-1-positive isolates, five were also found to be ciprofloxacin resistant. Genotyping
Twenty isolates were selected for PFGE genotyping, being representative of the different antibiotic susceptibility profiles obtained from the culture isolates. Overall, 13 different (non-clonal) PFGE patterns were observed at 95% (Fig. 2).
Discussion Typhoid fever remains a public health problem in developing countries such as India. In this study, we investigated 337 blood culture-positive S. Typhi isolates, and similar to previous research [25] a higher number of culture-positive cases occurred in persons aged 6–20 years. A gradual decline in the number of S. Typhi MDR isolates was also observed, corresponding to the decrease in occurrence of MDR S. Typhi observed in south India, in a previous publication [4]. This was accompanied by an increase in non-MDR isolates, though the majority of these (78%) were resistant to NAR and showed reduced susceptibility to ciprofloxacin. Further, antimicrobial resistance was not associated with a particular clonal genotype of S. Typhi. This finding is most likely to be due to decreased prescribing of traditional antimicrobial
agents, and an increasing reliance on ciprofloxacin as the first-line treatment for S. Typhi in Pondicherry [5]. Indeed, 8% of S. Typhi isolates from blood cultures in Pondicherry were found to be highly resistant to ciprofloxacin (MIC ‡ 4 mg/L), and the emergence of such resistant bacteria is a cause of concern. In recent years, there have been some sporadic reports of high-level ciprofloxacin resistance in Salmonella enterica [5]. Raveendran et al. [26] in 2008 reported a ciprofloxacin resistance rate of 5.6% in S. Typhi isolates of New Delhi. However, isolates with reduced susceptibility to ciprofloxacin (‡0.125; NAR isolates) have a less favourable response to ciprofloxacin [3]. In our study, in gyrA, double point mutations were found in isolates with ciprofloxacin MICs ‡ 4 mg/L, with single mutations in isolates with lower MICs (0.25–2.0 mg/L), with evidence for the contribution of efflux pumps in all isolates with an MIC ‡ 4 mg/L, similar to an earlier report [27]. All the isolates with MIC ‡ 1 mg/L, irrespective of their higher MICs against ciprofloxacin, had a single mutation in the parC gene. Resistance mutations of gyrA have been clustered in a region of the gene product between amino acids 67 and 106, termed the QRDR. The substitution Gly133 fi Glu, found in our study, has been seen in very few sequences in GyrA from S. Typhi that cover position 133 (GenBank accession no. AY302588, AY302585, EF176634 and GU190905). Although this mutation was found in the gene outside of the QRDR, it was not seen in isolates with MIC > 4 mg/L against ciprofloxacin and hence it is probably not associated with quinolone resistance in these isolates.
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Several studies have also reported that the first and essential step towards the development of a (quinolone) resistance phenotype in Salmonella spp. is the acquisition of mutations that give rise to an increase in efflux, with mutations in the QRDR regions only representing a second step in enhancement of resistance [28]. Bacterial efflux pumps are important mechanisms involved in resistance to many classes of antimicrobial agents, including fluoroquinolones [29]. With respect to ampicillin resistance, this was attributable to the production of TEM-1 or OXA-1 b-lactamases, similar to the findings of Ling et al. [30]. There was a gradual increase in MICs against ceftazidime and ceftriaxone (a third-generation cephalosporin) during the study period, from a MIC90 value of 0.064 mg/L in 2005 to 0.25 mg/L in 2009, though still well within the susceptible range. This type of resistance to quinolones, and more recently, increases in MIC levels to third- and fourthgeneration cephalosporins, re-emphasize the importance of continued surveillance in the treatment of enteric fever [11]. Indeed, there are sporadic reports of high MICs to ceftriaxone in typhoidal salmonellae, [9,10] via the presence of CTX-M-15 and SHV-12 ESBLs. In our opinion, the spread of ESBLs in S. Typhi isolates is deeply worrying, greatly limiting available therapeutic options, and leaving only the carbapenems and tigecycline as possible treatment options. Interestingly, according to our study, there was genotypic diversity, as determined by PFGE studies among the typhoidal salmonellae circulating in our region. They seem to indicate that multiple, independent clones of S. Typhi exist in different parts of the world. In order to better manage and prevent the spread of antimicrobial resistance, both clinicians and governments require accurate epidemiological information. At the present moment, this information tends to be lacking, especially in countries with large populations and unrestricted ‘over the counter’ prescription policies, such as India. The spread of fluoroquinolone-resistant S. Typhi necessitates a change towards ‘evidence-based’ treatment practices for the treatment of typhoid fever, not least in southern India.
Transparency Declaration This work was financially supported by the Indian Council of Medical Research (ICMR), New Delhi, India, and a European Union FP6 grant (DRESP2). The authors declare that they have no conflicting interests in relation to this work.
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19. Wu JJ, Ko W, Tsai SH, Yan JJ. Prevalence of plasmid-mediated quinolone resistance determinants QnrA, QnrB, and QnrS among clinical isolates of Enterobacter cloacae in a Taiwanese hospital. Antimicrob Agents Chemother 2007; 51: 1223–1227. 20. Robicsek A, Strahilevitz J, Jacoby GA et al. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 2006; 12: 83–88. 21. Tasli H, Bahar IH. Molecular charatcterisation of TEM and SHV derived extended spectrum beta lactamases in hospital based Enterobacteriaceae in Turkey. Jpn J Infect Dis 2005; 58: 162–167. 22. Karisik E, Ellington MJ, Pike R et al. Molecular characterisation of plasmids encoding CTX-M-15 b-lactamase from Escherichia coli strains in the United Kingdom. J Antimicrob Chemother 2006; 58: 665–668. 23. Woodford N, Fagan EJ, Ellington MJ. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum b-lactamases. J Antimicrob Chemother 2006; 57: 154–155. 24. Paterson DL, Rice LB, Bonomo RA. Rapid method of extraction and analysis of extended spectrum b-lactamases from clinical strains of Klebsiella pneumoniae. Clin Microbiol Infect 2001; 7: 709– 711.
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25. Mohanty S, Renuka K, Sood S, Das BK, Kapil A. Antibiogram pattern and seasonality of Salmonella serotypes in a North Indian tertiary care hospital. Epidemiol Infect 2006; 134: 961–966. 26. Raveendran R, Wattal C, Sharma A et al. High level ciprofloxacin resistance in Salmonella enterica isolated from blood. Indian J Med Microbiol 2008; 26: 50–53. 27. Keddy KH, Smith AM, Sooka A, Ismail H, Oliver S. Fluoroquinoloneresistant typhoid, South Africa. Emerg Infect Dis 2010; 16: 879–880. 28. Chen S, Cui S, McDermott PF et al. Contribution of target gene mutations and efflux to decreased susceptibility of Salmonella enterica serovar Typhimurium to fluoroquinolones and other antimicrobials. Antimicrob Agents Chemother 2007; 51: 535–542. 29. Lomovskaya O, Warren MS, Lee A et al. Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrob Agents Chemother 2001; 45: 105–116. 30. Ling JM, Zhou GM, Woo TH, French GL. Antimicrobial susceptibilities and b-lactamase production of Hong Kong isolates of gastroenteric salmonellae and Salmonella typhi. J Antimicrob Chemother 1991; 28: 877–885.
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BACTERIOLOGY
Antimicrobial resistance trends in blood culture positive Salmonella Typhi isolates from Pondicherry, India, 2005–2009 G. A. Menezes1,2, B. N. Harish1, M. A. Khan3, W. H. F. Goessens3 and J. P. Hays3 1) Department of Microbiology, Institute of National Importance, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India, 2) Department of Microbiology, SSR Medical College, Belle Rive, Phoenix, Mauritius and 3) Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Centre, ¢s Gravendijkwal, Rotterdam, the Netherlands
Abstract Typhoid fever is caused by Salmonella enterica serovar Typhi, a major public health concern in developing countries. Recently, there has been an upsurge in the occurrence of bacterial isolates that are resistant to ciprofloxacin, and the emergence of broad spectrum b-lactamases in typhoidal salmonellae constitutes a new challenge for the clinician. A total of 337 blood culture isolates of S. Typhi, isolated from Pondicherry, India, between January 2005 and December 2009, were investigated using phenotypic, molecular and serological methods. Of the 337 isolates, 74 (22%) were found to be multidrug resistant (MDR) and 264 (78%) nalidixic acid resistant (NAR). Isolates with reduced susceptibility to ciprofloxacin possessed single mutations in the gyrA gene. A high rate of resistance (8%) was found to ciprofloxacin. All isolates with a ciprofloxacin MIC ‡ 4 mg/L possessed both double mutations in the QRDR of the gyrA gene and a single mutation in the parC gene. Active efflux pump mechanisms were also found to be involved in ciprofloxacin resistance. Finally, a large number of PFGE patterns (non-clonal genotypes) were observed among the S. Typhi isolates. In conclusion, a high rate of ciprofloxacin resistance was observed in comparison to other endemic areas in blood culture isolates of S. Typhi from Pondicherry, India, with steadily increasing NAR but decreasing MDR isolations over the study period. This is most likely to be due to an increased use of ciprofloxacin as a first-line drug of choice over more traditional antimicrobial agents for the treatment of typhoid fever. Keywords: Fluoroquinolones, gyrA, multidrug resistant, nalidixic acid resistant, pulsed field gel electrophoresis, quinolone resistancedetermining region, Salmonella Typhi Original Submission: 15 December 2010; Revised Submission: 10 April 2011; Accepted: 11 April 2011 Editor: G. Pappas Article published online: 25 April 2011 Clin Microbiol Infect 2012; 18: 239–245 10.1111/j.1469-0691.2011.03546.x Corresponding author: B. N. Harish, Department of Microbiology, Jipmer, Pondicherry 605006, India E-mail: [email protected]
Introduction Typhoid fever, caused by Salmonella enterica serovar Typhi, is a major health problem in developing countries, particularly in the Indian subcontinent and in southeast Asia. Typhoid fever is the most serious form of enteric fever and in the year 2000 it was estimated that the global number of typhoid cases exceeded 21 000 000, with more than 200 000 deaths [1]. Further, if not treated properly, enteric fever carries a mortality rate of 30%, whilst appropriate antimicrobial
treatment reduces the mortality rate to as low as 0.5% [2]. In cases of enteric fever, it is often necessary to commence treatment before the results of laboratory sensitivity testing become available, and the fluoroquinolone ciprofloxacin has become the first-line drug for treatment, especially since the widespread emergence of S. Typhi isolates that are multidrug resistant (MDR) to the more traditional antimicrobial agents comprising chloramphenicol, ampicillin and trimethoprimsulphamethoxazole (cotrimoxazole) [3]. However, this switch to ciprofloxacin has led to a subsequent increase in the occurrence of S. Typhi isolates resistant to this antimicrobial agent, including in India [4,5]. In salmonellae, quinolone resistance is usually associated with mutations in the quinolone resistance-determining region (QRDR) of the A subunit target site of DNA gyrase, though the presence of plasmid-mediated quinolone
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resistance qnr genes and aac(6¢)-Ib-cr has also been described in quinolone-resistant non-Typhi Salmonella [6]. The exact mechanism of this DNA gyrase-mediated resistance in S. Typhi is not fully understood, though various studies have found that single point mutations in this region confer resistance to nalidixic acid and hence reduced susceptibility to fluoroquinolones [7]. In contrast, high-level ciprofloxacin resistance may be due to either (i) the cumulative impact of mutations in many genes, (ii) decreased membrane permeability, (iii) active efflux pump, and/or (iv) the presence of plasmid-encoded qnr genes [8]. Worldwide, there are sporadic reports of high-level resistance to beta-lactam antibiotics, including ceftriaxone in typhoidal salmonellae, due to the presence of CTX-M-15 and SHV-12 extended spectrum b-lactamases (ESBLs) [9,10]. Further, we recently reported an ACC-1 AmpC b-lactamase producing S. Typhi that had been isolated from the blood of a girl aged 14 years [11]. Therefore, a study was undertaken to characterize trends in antimicrobial resistance in clinically relevant S. Typhi isolates originating from Pondicherry, India, to help guide clinicians in successful treatment therapies.
Materials and Methods Bacterial culture and identification
Blood cultures from a total of 3744 patients presenting with fever at the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, and Government General Hospital, Pondicherry, during the period January 2005 to December 2009 were taken for microbial diagnosis. The patients ranged in age from 2 to 74 years (median, 43 years). Standard blood culture protocols were followed, including culture on a biphasic medium consisting of Brain Heart Infusion (BHI) agar and BHI broth (HiMedia Laboratories Ltd, Mumbai, India). Colonies were identified as S. Typhi using standard biochemical methods [12], and confirmed using Salmonella polyvalent O, O9 and H:d antisera (Murex Biotech, Dartford, England).
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tration (MIC) of 0.125–1.0 mg/L. The breakpoints for ofloxacin and gatifloxacin were £2 mg/L (susceptible) and ‡8 mg/L (resistant), and for ciprofloxacin, £1 mg/L (susceptible) and ‡4 mg/mL (resistant) (CLSI, 2007). Escherichia coli ATCC 25922 was used as a control isolate. Nalidixic acid susceptibility was used as a screening test for reduced susceptibility to ciprofloxacin [13]. MICs were determined by both agar dilution and E-test (AB Biodisk, Solna, Sweden) for the antibiotics ciprofloxacin, ampicillin, chloramphenicol and ceftriaxone. MICs against gatifloxacin and ofloxacin were determined by E-test only. Efflux pumps
Testing for antimicrobial resistance via efflux pump activity was performed using the cyclohexane tolerance test [14], and via the detection of the MIC in the absence and presence of the efflux pump inhibitor (EPI) PabN (Phe-Arg-bnaphthylamide) at 20 mg/L [15]. Molecular analysis of quinolone resistance
Molecular analysis of quinolone resistance was performed on 57 (27 ciprofloxacin resistant, five ciprofloxacin intermediate and 25 ciprofloxacin susceptible) isolates. The molecular mechanism of quinolone resistance was determined by investigating mutations in the QRDRs of DNA gyrase (gyrA and gyrB) and DNA topoisomerase IV (parC and parE) genes, according to previously described protocols [16–18]. The possible presence of the plasmid-mediated quinolone resistance qnr gene (qnrA, qnrB, and qnrS), and aac(6¢)-Ib-cr gene was determined using PCR [19,20]. Detection of beta-lactamases
PCR screening and sequencing was performed to identify the b-lactamase resistance genes blaTEM, blaSHV, blaOXA-1 group and blaCTX-M [21–23]. Beta-lactamase screening was performed for 61 isolates that were highly resistant to ampicillin (MIC ‡ 256 mg/L). Isoelectric focusing of b-lactamase enzymes was performed using standard methods [24]. Isolate genotyping
Antimicrobial susceptibility testing
Isolates were tested for susceptibility to antimicrobials using the Kirby Bauer disk diffusion method. Antibiotic disks contained ampicillin (10 lg), cotrimoxazole (25 lg), chloramphenicol (30 lg), ciprofloxacin (5 lg), ceftazidime (30 lg), ceftriaxone (30 lg) and nalidixic acid (10 lg), and results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (2007). Reduced susceptibility to ciprofloxacin was determined as a minimum inhibitory concen-
Genotyping was performed on a representative sample of 20 S. Typhi isolates using pulsed field gel electrophoresis (PFGE). Briefly, isolates were incubated overnight at 37C in 7 mL Mueller Hinton broth. After incubation, 1 mL of bacterial cells was harvested, pelleted and washed three times using 1 mL EET (Na2EDTA 100 mM, EGTA 10 mM, Tris HCl 1 M) buffer, before being adjusted to a cell density of 0.5 at 560 nm. One hundred microlitres of cell suspension and 100 lL of 1.4% PFGE grade agarose in EET buffer were mixed and
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poured into PFGE plug moulds. The plugs were incubated at 4C for 30 min to harden and 1 mL of lysozyme (5 mg/mL) was added before incubation at 37C for 3–4 h. Lysozyme was removed from the plugs and 1 mL of deproteinising solution (per plug 3 mg/mL proteinase K and 1% SDS) was added prior to overnight incubation at 37C. Next, the plugs were washed six times every 30 min with T10E1 (10 mM Tris, 1 mM EDTA) buffer, and then soaked in T10E0.1 (10 mM Tris, 0.1 mM EDTA) buffer for 30 min. Restriction digestion was performed using 40 U of XbaI (Fermentas, St. Leon-Rot, Germany) at 37C. PFGE was performed in a 1% agarose gel in a CHEF DRII system (BioRad, Hercules, CA, USA) with the following conditions: 0.5· Tris–Borate EDTA buffer, 14C, 6 V/cm for 22 h (with switch times ranging from 5 to 40 s). Lambda ladder PFGE marker (BioRad) was used as a molecular weight standard.
Antimicrobial resistance in Salmonella Typhi
phi isolates during the study period Period of isolation
No. (%) NAR
No. (%) CIPR
2005 2006 2007 2008 2009 Total
51 57 48 58 50 264
0 5 8 9 5 27
(6.6) (12.7) (13) (8.5) (8)
100 90 80 70 MDR NAR
60 50 40 30
Trends in antimicrobial susceptibility of S. Typhi isolates
20
A total of 337 S. Typhi isolates from two hospitals recovered during the period 2005–2009 were included in this study, representing 100% of the S. Typhi isolates recovered during this period. Of these isolates, 222 (65.9%) were fully susceptible to ampicillin, chloramphenicol and cotrimoxazole; 74 (22.0%) were MDR; 264 (78.3%) were nalidixic acid resistant (NAR); and 27 (8.0%) were both MDR and NAR (MDR being defined as resistance to all first-line antimicrobials, i.e. ampicillin, cotrimoxazole and chloramphenicol). The results are summarized in Table 1. Twenty-seven isolates (8.0%) were resistant to ciprofloxacin. There was a steady decline in the number of MDR isolates over the 5-year study period, as well as a parallel increase in NAR (non-MDR) isolates (Tables 1 and 2 and Fig. 1). The susceptibility results for different antibiotics as well as the MIC50 and MIC90 (MICs at which 50% or 90% of the isolates were inhibited) values obtained for 337 S. Typhi isolates are shown in Table 3. Of the 27 ciprofloxacin-resistant
10 0
2005
2006
[70]
[76]
2007 Year [63]
2008
2009
[69]
[59]
FIG. 1. Proportions that were nalidixic acid resistant (NAR) and multidrug resistant (MDR) among the isolates of S. Typhi. Numbers in brackets indicate the total number of blood culture positive S. Typhi isolates cultured in each year at Jawaharlal Institute of Postgraduate Medical Education and Research and the Government General Hospital, Pondicherry, India.
isolates recovered in this study, a single isolate was found to possess an MIC of >32 mg/L (E-test), whilst the corresponding agar dilution method and HIMEDIA HiComb MIC test (HiMedia) revealed an MIC of 64 mg/L. In the same isolate, the MIC of ofloxacin and gatifloxacin (E-test), was >32 and 8 mg/L, respectively. For gatifloxacin, the MIC50 and MIC90 were one to two dilution steps lower than those of cipro-
antimi-
crobial resistance to ampicillin, cotrimoxazole and chloramphenicol among S. Typhi isolates
(72.9) (75) (76.2) (84) (83.3) (78.3)
CIPR, ciprofloxacin resistant; NAR, nalidixic acid resistant.
Results
TABLE 1. Distribution of
241
TABLE 2. Trends in resistance to quinolones among S. Ty-
Percentage of isolates tested
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Period of isolation
No. (%) susceptiblea
No. (%) with single resistanceb
No. (%) paired resistancec
No. (%) MDRd
Total no. (%)
2005 2006 2007 2008 2009 Total
24 40 37 65 56 222
9 7 8 2 3 29
6 3 1 2 0 12
31 26 17 0 0 74
70 76 63 69 59 337
(34.3) (52.6) (58.7) (94.2) (94.9) (65.9)
(12.4) (9.2) (12.7) (2.9) (5.1) (8.6)
(9) (4) (1.6) (2.9) (3.5)
(44.2) (34.2) (27)
(22)
(20.8) (22.6) (18.7) (20.4) (17.5) (100)
a
Susceptible to ampicillin, chloramphenicol and cotrimoxazole. b Single resistance – resistance to only one antimicrobial (ampicillin or cotrimoxazole). c Paired resistance – resistance to two antimicrobials (ampicillin and chloramphenicol; ampicillin and cotrimoxazole; and chloramphenicol and cotrimoxazole). d MDR – defined as resistance to ampicillin, chloramphenicol and cotrimoxazole.
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TABLE 3. Antimicrobial sensitivity patterns obtained for S. Typhi isolates recovered from blood cultures of patients in Pondicherry, India,
between
(n = 337)
2005
and
2009
MIC (mg/L) Antimicrobial agents
Sensitive No. (%)
Intermediate No. (%)
Resistant No. (%)
Range
MIC50
MIC90
Ampicillin Chloramphenicol Cotrimoxazole Nalidixic acid Ciprofloxacin Gatifloxacin Ofloxacin Ceftazidime Ceftriaxone
243 (72.1) 255 (75.7) 234 (69.4) 73 (21.7) 305 (90.5) 324 (96.1) 310 (92.0) 337 (100) 337 (100)
12 (3.6) 6 (1.8) 0 0 5 (1.5) 6 (1.8) 4 (1.2) 0 0
82 (24.3) 76 (22.6) 103 (30.6) 264 (78.3) 27 (8.0) 7 (2.1) 23 (6.8) 0 0
1–‡256 1–‡256 – – 0.016–64 0.016–8 0.016–32 0.064–2 0.064–2
1 2 ND ND 0.5 0.125 0.5 0.25 0.25
‡256 ‡256 ND ND 1 1 4 0.25 0.25
ND, not determined. Seventy-four (22.0%) isolates were multidrug resistant (MDR), defined as being resistant to ampicillin, chloramphenicol and cotrimoxazole. MIC50 and MIC90 = MIC at which 50% and 90% of the isolates were inhibited, respectively.
floxacin and ofloxacin with respect to all isolates tested. Ciprofloxacin MICs for susceptible isolates ranged between 0.016 and 1 mg/L. A correlation was observed between reduced ciprofloxacin susceptibility and nalidixic acid resistance (231/234 isolates with ciprofloxacin MICs ranging from 0.125 to 2 mg/L were NAR). The exceptions were three isolates with 1 mg/L MIC that were nalidixic acid sensitive (NAS) and MDR. Seventy-one of the isolates possessing ciprofloxacin MICs of £0.25 mg/L were all NAS and susceptible to all other antimicrobial agents tested. All 337 isolates were found to be susceptible to ceftazidime and ceftriaxone, though interestingly there was a gradual increase in MIC90 values for both ceftazidime and ceftriaxone during the study period from 0.064 to 0.25 mg/L. Efflux pumps
Using the cyclohexane tolerance test, only a single S. Typhi isolate (with a ciprofloxacin MIC of 64 mg/L) showed evidence of an efflux pump-mediated contribution to resistance. In the presence of EPI, the MIC to ciprofloxacin and nalidixic acid decreased four-fold in all of the 27 isolates possessing an MIC of ‡4 mg/L. Molecular analysis of quinolone resistance
The relationship between ciprofloxacin MIC and nucleotide changes within DNA gyrase and topoisomerase IV subunit genes of 11 isolates, representing different resistance profiles, is shown in Table 4. Isolates with ciprofloxacin MICs of ‡1 to <4 mg/L, exhibited a single point mutation in gyrA and parC genes. NAR isolates with a comparatively reduced susceptibility to ciprofloxacin (MIC 0.125–0.5 mg/L), possessed a single gyrA mutation (either at Ser83 or Asp87) only. All ciprofloxacin-resistant isolates (MICs ‡ 4 mg/L) showed three mutations, two mutations within the QRDR of gyrA, at positions 83 and 87, and a single mutation in parC, at position 80. No mutations were detected in QRDRs of gyrA and
TABLE 4. Relationship between ciprofloxacin MIC, point mutations within the QRDRs of DNA gyrase and topoisomerase IV subunit genes and testing for the effect of efflux pumps in 11 representative S. Typhi isolates Isolate no.
CIP MIC (mg/L)
T20
64
T85 T86 T9
1 2 4
T1 T2 T7
2 2 6
T13 T14 T18 T21
0.015 0.5 1 0.25
Amino acid change in GyrAa
Amino acid change in ParCb
Efc
(Ser 83 fi Phe), (Asp 87 fi Asn) (Ser 83 fi Phe) (Ser 83 fi Phe) (Ser 83 fi Leu), (Asp 87 fi Asn) (83 Ser fi Tyr) (Ser 83 fi Phe) (Ser 83 fi Phe), (Asp 87 fi Asn) None (Ser 83 fi Phe) (Ser 83 fi Phe) (Ser 83 fi Tyr)
(Ser 80 fi Ile)
+
(Thr 57 fi Ser) (Ser 80 fi Ile) (Ser 80 fi Ile)
) ) +
(Thr 57 fi Ser) (Ser 80 fi Ile) (Ser 80 fi Ile)
) ) +
None None (Thr 57 fi Ser) None
) ) ) )
CIP, ciprofloxacin; MIC, minimum inhibitory concentration. a Nucleotide changes in gyrA, 83-TCC (Ser) fi TTC (Phe); 83-TCC (Ser) fi TAC (Tyr); 83-TCC (Ser) fi TTG (Leu); 87-GAC (Asp) fi AAC (Asn). b Nucleotide changes in parC, 80- AGC (Ser) fi ATC (Ile); 57- ACC (Thr) fi AGC (Ser). Alterations in the QRDRs of gyrB and parE genes were not found in any of the strains tested. c Ef – phenotypic test for elucidation of efflux mechanism.
parC genes of nalidixic-acid-susceptible isolates, for which ciprofloxacin MICs were <0.125 mg/L. Sequence mutations in the QRDRs of gyrB and parE genes were not found in any of the isolates tested. In some NAS and NAR isolates, irrespective of their MICs against ciprofloxacin (0.015–£4 mg/L; data not shown), a mutation was found outside the QRDR of gyrA. This substitution, found outside the QRDR of GyrA, was Gly133 fi Glu. All isolates were negative for qnr (qnrA, qnrB, and qnrS) and aac(6¢)-Ib-cr genes. Detection of beta-lactamase genes
Twelve (20%) of 61 isolates highly resistant to ampicillin isolates were found to be positive for blaTEM-1, with isoelectric
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FIG. 2. Pulsed field gel electrophoresis fingerprinting of 20 S. Typhi isolates that exhibited a range of antimicrobial susceptibility profiles identified in this study. Cluster analysis was performed using the method of dice coefficient with unweighted pair group method with arithmetic mean with band tolerance set to 2.0%. All isolates were cultured from blood. Antimicrobial drugs: ciprofloxacin (CIP), ampicillin (AMP), chloramphenicol (CHL), trimethoprim/ sulphamethoxazole (SXT), nalidixic acid (NAL), ceftriaxone (CRO).
focusing experiments showing a b-lactamase with a pI of 5.4 (TEM-1). Four (7%) of the 61 isolates were positive for blaOXA-1-like genes, but none of the isolates were positive for both blaTEM-1 and blaOXA-1. Of the 12 blaTEM-1-positive isolates, five were also found to be ciprofloxacin resistant. Genotyping
Twenty isolates were selected for PFGE genotyping, being representative of the different antibiotic susceptibility profiles obtained from the culture isolates. Overall, 13 different (non-clonal) PFGE patterns were observed at 95% (Fig. 2).
Discussion Typhoid fever remains a public health problem in developing countries such as India. In this study, we investigated 337 blood culture-positive S. Typhi isolates, and similar to previous research [25] a higher number of culture-positive cases occurred in persons aged 6–20 years. A gradual decline in the number of S. Typhi MDR isolates was also observed, corresponding to the decrease in occurrence of MDR S. Typhi observed in south India, in a previous publication [4]. This was accompanied by an increase in non-MDR isolates, though the majority of these (78%) were resistant to NAR and showed reduced susceptibility to ciprofloxacin. Further, antimicrobial resistance was not associated with a particular clonal genotype of S. Typhi. This finding is most likely to be due to decreased prescribing of traditional antimicrobial
agents, and an increasing reliance on ciprofloxacin as the first-line treatment for S. Typhi in Pondicherry [5]. Indeed, 8% of S. Typhi isolates from blood cultures in Pondicherry were found to be highly resistant to ciprofloxacin (MIC ‡ 4 mg/L), and the emergence of such resistant bacteria is a cause of concern. In recent years, there have been some sporadic reports of high-level ciprofloxacin resistance in Salmonella enterica [5]. Raveendran et al. [26] in 2008 reported a ciprofloxacin resistance rate of 5.6% in S. Typhi isolates of New Delhi. However, isolates with reduced susceptibility to ciprofloxacin (‡0.125; NAR isolates) have a less favourable response to ciprofloxacin [3]. In our study, in gyrA, double point mutations were found in isolates with ciprofloxacin MICs ‡ 4 mg/L, with single mutations in isolates with lower MICs (0.25–2.0 mg/L), with evidence for the contribution of efflux pumps in all isolates with an MIC ‡ 4 mg/L, similar to an earlier report [27]. All the isolates with MIC ‡ 1 mg/L, irrespective of their higher MICs against ciprofloxacin, had a single mutation in the parC gene. Resistance mutations of gyrA have been clustered in a region of the gene product between amino acids 67 and 106, termed the QRDR. The substitution Gly133 fi Glu, found in our study, has been seen in very few sequences in GyrA from S. Typhi that cover position 133 (GenBank accession no. AY302588, AY302585, EF176634 and GU190905). Although this mutation was found in the gene outside of the QRDR, it was not seen in isolates with MIC > 4 mg/L against ciprofloxacin and hence it is probably not associated with quinolone resistance in these isolates.
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Several studies have also reported that the first and essential step towards the development of a (quinolone) resistance phenotype in Salmonella spp. is the acquisition of mutations that give rise to an increase in efflux, with mutations in the QRDR regions only representing a second step in enhancement of resistance [28]. Bacterial efflux pumps are important mechanisms involved in resistance to many classes of antimicrobial agents, including fluoroquinolones [29]. With respect to ampicillin resistance, this was attributable to the production of TEM-1 or OXA-1 b-lactamases, similar to the findings of Ling et al. [30]. There was a gradual increase in MICs against ceftazidime and ceftriaxone (a third-generation cephalosporin) during the study period, from a MIC90 value of 0.064 mg/L in 2005 to 0.25 mg/L in 2009, though still well within the susceptible range. This type of resistance to quinolones, and more recently, increases in MIC levels to third- and fourthgeneration cephalosporins, re-emphasize the importance of continued surveillance in the treatment of enteric fever [11]. Indeed, there are sporadic reports of high MICs to ceftriaxone in typhoidal salmonellae, [9,10] via the presence of CTX-M-15 and SHV-12 ESBLs. In our opinion, the spread of ESBLs in S. Typhi isolates is deeply worrying, greatly limiting available therapeutic options, and leaving only the carbapenems and tigecycline as possible treatment options. Interestingly, according to our study, there was genotypic diversity, as determined by PFGE studies among the typhoidal salmonellae circulating in our region. They seem to indicate that multiple, independent clones of S. Typhi exist in different parts of the world. In order to better manage and prevent the spread of antimicrobial resistance, both clinicians and governments require accurate epidemiological information. At the present moment, this information tends to be lacking, especially in countries with large populations and unrestricted ‘over the counter’ prescription policies, such as India. The spread of fluoroquinolone-resistant S. Typhi necessitates a change towards ‘evidence-based’ treatment practices for the treatment of typhoid fever, not least in southern India.
Transparency Declaration This work was financially supported by the Indian Council of Medical Research (ICMR), New Delhi, India, and a European Union FP6 grant (DRESP2). The authors declare that they have no conflicting interests in relation to this work.
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