JAC Advance Access originally published online on August 8, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):733-740; doi:10.1093/jac/dkl333
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The acquisition of full fluoroquinolone resistance in Salmonella Typhi by accumulation of point mutations in the topoisomerase targets
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus Hinxton, Cambridge CB10 1SA, UK
*Corresponding author. Tel: +44-1223-834244; Fax: +44-1223-494919; E-mail: jw5{at}sanger.ac.uk
Received 27 April 2006; returned 2 July 2006; revised 18 July 2006; accepted 19 July 2006
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Abstract |
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Objectives: To determine the contribution to fluoroquinolone resistance of point mutations in the gyrA and parC genes of Salmonella Typhi.
Methods: Point mutations that result in Ser-83
Phe, Ser-83Tyr and Asp-87Asn amino acid substitutions in GyrA and Glu-84Lys in ParC were introduced into a quinolone-susceptible, attenuated strain of Salmonella Typhi using suicide vector technology. This is the first time that this approach has been used in Salmonella and abrogates the need for selection with quinolone antibacterials in the investigation of resistance mutations.
Results: A panel of mutants was created using this methodology and tested for quinolone resistance. The ParC substitution alone made no difference to quinolone susceptibility. Any single GyrA substitution resulted in resistance to nalidixic acid (MIC 
512 mg/L) and increased by up to 23-fold the MIC of the fluoroquinolones ofloxacin (MIC 
2 mg/L) ciprofloxacin (MIC 1 mg/L) and gatifloxacin (MIC 0.38 mg/L). Among the double substitution mutants, those with a substitution in ParC were less prone to killing with ciprofloxacin. The triple substitution mutants (Ser-83Phe or Tyr and Asp-87Asn in GyrA with Glu-84Lys in ParC) showed high levels of resistance to all the fluoroquinolones tested (MICs: gatifloxacin, 3–4 mg/L; ofloxacin, 32 mg/L; ciprofloxacin, 32–64 mg/L).
Conclusions: In Salmonella Typhi the fluoroquinolones tested act on GyrA and, at higher concentrations, on ParC. The point mutations conferred reduced susceptibility to ofloxacin and ciprofloxacin, and also reduced susceptibility to gatifloxacin. Three mutations conferred resistance to ofloxacin (32 mg/L), ciprofloxacin (32 mg/L) and to the more active fluoroquinolone gatifloxacin (MIC 3 mg/L). These results predict that the use of ofloxacin or ciprofloxacin will select for resistance to gatifloxacin in nature.
Keywords: typhoid fever , antibiotic resistance , fluoroquinolones , nalidixic acid , ofloxacin , ciprofloxacin , gatifloxacin
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Introduction |
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Salmonella enterica subspecies enterica serovar Typhi and Salmonella Paratyphi A are invasive human pathogens, causing the systemic diseases typhoid and paratyphoid fever.1 Due to the increasing resistance to antibacterials used traditionally for therapy (ampicillin, amoxicillin, co-trimoxazole and chloramphenicol), the use of fluoroquinolones, such as ciprofloxacin and ofloxacin, for the treatment of these infections, has become commonplace. This in turn has led to an increase in fluoroquinolone resistance in Salmonella Typhi and Salmonella Paratyphi A.2
Quinolone antibiotics, which include the fluoroquinolones, act by inhibiting the topoisomerase enzymes, DNA gyrase and topoisomerase IV, which maintain the level of supercoiling in the bacterial DNA. Both of these enzymes are tetrameric, being composed of two A subunits and two B subunits encoded by the gyrA and gyrB genes for DNA gyrase and the parC and parE genes for topoisomerase IV.
A number of resistance mechanisms to quinolones have been identified, including point mutations that result in amino acid substitutions in the topoisomerases, reduced outer membrane permeability, increased efflux of antibiotics and other harmful compounds, and the plasmid-encoded Qnr genes.3–7
Point mutations in the topoisomerase genes are generally restricted to certain codons within the ‘quinolone resistance determining region’ (QRDR).8 In Salmonella, some of the more common point mutations found to be associated with resistance to quinolones occur in the gyrA gene resulting in substitutions at the Ser-83 position, often to Tyr, Phe or Ala, and Asp-87 substitutions to Asn, Gly or Tyr. The most common amino acid substitution reported in ParC is Thr-57Ser, with Thr-66Ile or Ser-80Arg being observed as occasional second substitutions.9 Salmonella Typhi and Salmonella Paratyphi A strains showing resistance or reduced susceptibility to fluoroquinolones have been reported, and these almost invariably have point mutations in gyrA.10–16
Investigations of quinolone-resistant salmonellae have been performed almost exclusively on resistant isolates or on strains selected in the laboratory. One limitation of investigating such strains is that the quinolones to which the bacteria are exposed may select for mutations elsewhere in the genome. Thus, using fluoroquinolone-resistant isolates and in vitro selected mutants, it is not possible to determine with confidence the relative contribution to resistance, if any, of the different point mutations, alone or in combination, in the topoisomerase genes. One way to accurately determine the involvement or relative contribution of point mutations to quinolone resistance is to introduce these mutations into the respective genes of a susceptible strain in a directed way that does not involve quinolone selection. There is only one report of such a study, conducted with Escherichia coli, which investigated the effects of point mutations on the structure of the topoisomerase enzymes where some mutants generated by directed mutagenesis were compared with others created by selection.17
The lack of such directed mutagenesis studies is probably a result of the relative difficulty of introducing mutations into essential genes within the bacterial genome; the standard recombinase method18 cannot be used. Here, we describe the use of allelic exchange methodology19,20 to introduce point mutations into the topoisomerase genes of Salmonella Typhi and describe for the first time the resistance conferred by specific point mutations, alone and in combination, in a set of Salmonella Typhi mutants created from the same susceptible parent strain without the use of quinolone selection.
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Materials and methods |
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Bacterial strains and plasmids
These are listed in Table 1. The attenuated Salmonella Typhi Ty2 strain CVD908-htrA, which has deletions in the aroC, aroD and htrA genes, was used.21,22 This strain has been shown to be a safe and effective live oral vaccine in clinical trials, and so overcomes any potential ethical issues associated with the introduction of quinolone resistance into what would otherwise be a dangerous pathogen.
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Growth media and supplements
Bacteria were grown in Luria–Bertani (LB)-broth or on LB-agar. When necessary, growth media were supplemented with chloramphenicol (Sigma) at 15 mg/L, ampicillin at 500 mg/L and ‘aro mix’ (40 mg/L each of L-phe and L-trp, and 10 mg/L each of p-aminobenzoic acid and 2,3-dihydroxybenzoic acid final concentration).23 When medium was supplemented with 5% sucrose, NaCl was excluded from the recipe.24
DNA manipulations
Restriction endonucleases were obtained from New England Biolabs Ltd (Hitchin, UK) or Roche Diagnostics Ltd (Welwyn Garden City, UK). PCR for routine screening and checking of genetic constructs was performed using ‘Platinum® PCR Supermix’ (Invitrogen, Paisley, UK), while ‘PfuUltraTM’ DNA polymerase (Stratagene, La Jolla, USA) was used for making genetic constructs. DNA fragments were extracted from agarose gels using a QIAquick gel extraction kit (QIAGEN, Crawley, UK). Plasmid DNA was prepared using a plasmid midi kit (QIAGEN) and genomic DNA was prepared using a Wizard® genomic DNA purification kit (Promega, Southampton, UK). Electrotransformation was performed as described previously25 using 0.1 cm electrode cuvettes in a GenePulser XcellTM (Bio-Rad Laboratories, Hemel Hempstead, UK) set to 1.6–1.8 kV, 25 µF and 200 
, except that bacteria for transformation were grown in LB-broth supplemented with ‘aro mix’. Nucleotide sequences were determined and analysed as described previously26 and using Vector NTI (Stratagene).
Generation of defined mutants by allelic exchange mutagenesis
Point mutations were introduced into CVD908-htrA by allelic exchange using derivatives of suicide vector pJCB12 as described previously.20 DNA fragments including the 5' region of gyrA or parC and incorporating the point mutation(s) were constructed using overlap extension PCR27 with the appropriate point mutation(s) included in the overlapping oligonucleotides. Details of all oligonucleotides used are given in Table 2. The generated fragments were ligated to pJCB12 using appropriate restriction enzyme sites. Following propagation in E. coli strain CC118
pir, the nucleotide sequence of the inserted DNA of the pJCB12-derivatives was confirmed before the plasmid DNA was introduced into CVD908-htrA by electrotransformation. Chloramphenicol-resistant transformants were selected. In the absence of a functional pir gene, these are generated only if the pJCB12-derivative construct has recombined with a replicon within the cell, usually by homologous recombination. Transformants were then screened by PCR to identify those in which the homologous recombination event had occurred at the correct locus (using oligonucleotides 47 125 and gyrA-11 for gyrA-recombinants, and R6K-01 and parC-23 for parC-recombinants, Figure 1). Transformants that were positive in such PCRs were then grown in the absence of selection to allow recombination between the homologous regions of the introduced and original DNA of gyrA or parC. This occurs in a very small proportion of the growing bacteria and results in excision of the suicide vector construct with either reversion or allelic exchange occurring. Such derivatives are selected by growth in LB-broth supplemented with chloramphenicol and ampicillin, which kills the chloramphenicol-resistant bacteria,28 followed by plating on NaCl-free LB-agar supplemented with 5% sucrose.19 Colonies obtained were then either tested for resistance to a selection of quinolones using antibiotic-containing discs, or, for some parC mutants, by quantitative real-time PCR (see below). Appropriate colonies were then chosen from the NaCl-free 5% sucrose LB-agar and grown up for storage at –80°C. At no time during their construction and growth did the –80°C stored mutant come into contact with quinolone antibiotics; therefore, the resistance observed cannot be due to selection by quinolones. Stored mutants were tested for susceptibility to quinolone antibiotics. In order to reduce the probability of the observed antibiotic resistance being due to a spontaneous mutation elsewhere on the chromosome, and not the point mutation introduced into gyrA or parC, each mutant was constructed twice independently, and antibiotic MICs were determined for each.
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Quantitative real-time PCR for detection of parC15 mutants
This was performed using an Mx3000PTM machine and Brilliant® SYBR® Green QPCR Master Mix in accordance with the supplier's instructions (Stratagene). The parC15 allele was detected in CVD908-htrA derivatives by colony PCR using oligonucleotides parC-22 and parC-23, and the following PCR cycle: 95°C for 10 min; then 30 cycles of 95°C for 30 s, 62°C for 1 min, 72°C for 30 s. Using this method, colonies that carried the parC15 allele gave a Ct of 14–16; for the wild-type allele this value was 22–24.
Antibiotic resistance determination
Antibiotic resistance was determined for the –80°C stored mutants. Antibiotic-containing discs were from Difco (Poole, UK) and included nalidixic acid (30 µg), cinoxacin (100 µg), enrofloxacin (5 µg), moxifloxacin (5 µg), ofloxacin (5 µg) and ciprofloxacin (5 µg). Solid nalidixic acid, ofloxacin and ciprofloxacin powders were obtained from Sigma-Aldrich. MICs of gatifloxacin, ciprofloxacin and ofloxacin were determined using Etest® strips (AB BIODISK, Solna, Sweden) on LB-agar supplemented with aro mix as CVD908-htrA and its derivatives grow poorly on Iso-Sensitest agar, which is normally recommended for antimicrobial testing. The British Society for Antimicrobial Chemotherapy (BSAC) doubling-dilution method (www.bsac.org.uk) was used to confirm MICs 32 mg/L.
Rates of bacterial kill with ciprofloxacin
Iso-Sensitest broth (Oxoid) was inoculated with a portion of five well-separated colonies taken from non-selective solid media. Broths were incubated without shaking at 37°C for 15–18 h and 40 µL diluted in 2.5 mL of warm Iso-Sensitest broth to give 
2 x 106 cfu/mL of the organism in exponential phase growth. This broth was then incubated at 37°C with shaking at 200 rpm for 1 h. A 2.5 mL aliquot of warm Iso-Sensitest broth, containing ciprofloxacin, was then added at time 0 to give a final concentration of 4 mg/L. The cultures were incubated with shaking at 200 rpm at 35–37°C during sampling.
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Results |
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Introduction of point mutations into Salmonella Typhi and confirmation of the nucleotide sequence
Point mutations were constructed that resulted in the substitutions Glu-84Lys (GAA to AAA) for ParC and Asp-87Asn (GAC to AAC), Ser-83Tyr (TCC to TAC), and Ser-83Phe (TCC to TTC) for GyrA. In addition, the Ser-83Phe and Ser-83Tyr substitutions were combined with Asp-87Asn for GyrA. These point mutations were chosen because they have been associated with resistant strains of Salmonella Typhi and Paratyphi A as well as other Salmonella.11–15 In particular, an isolate of Salmonella Paratyphi A with an MIC of ciprofloxacin >128 mg/L isolated from a patient in Japan was found to possess Phe-83 and Asn-87 residues in GyrA, and Lys-84 in ParC.10 All of the parC mutants were generated by introducing the parC mutation into the appropriate gyrA mutant or CVD908-htrA, except WT96, which was constructed by introducing both the gyrA mutations concurrently into strain WT72, which carries the parC point mutation. The presence of the point mutation(s), and the absence of any other alterations across and flanking the manipulated region, was confirmed by nucleotide sequence determinations.
The introduced point mutations are responsible for the observed quinolone resistance
During the construction of any bacterial mutant, there is a small probability that an observed phenotype may be the result of a mutation having occurred elsewhere in the genome. To confirm that the observed quinolone resistance was not due to changes elsewhere in the genome, each mutant was constructed twice, independently. Between these mutant pairs there was no discernable difference in the resistance profiles using (fluoro)quinolone-containing discs, and the MICs of gatifloxacin, ofloxacin and ciprofloxacin obtained using Etest® strips were within the limits of experimental variation (2-fold, Figure 2).
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In addition, for the generation of strain WT96, introduction of the pWT-gyrA45 (coding for Ser-83
Phe, Asp-87Asn) plasmid into WT72 (carrying only the ParC Glu-84Lys mutation) resulted in derivatives with the pWT-gyrA45 plasmid recombined at gyrA that were either susceptible (like the parent strain WT76) or resistant (like WT96) (Figure 1). This is as expected, with susceptibility or resistance being dependent on whether or not the recombination event incorporated the mutations into the functional gene. When treated for removal of the suicide vector construct, as expected each of these derivatives generated both susceptible and resistant recombinants (Figure 1).
In a similar way, a complementation experiment was performed whereby the gene coding for susceptible GyrA was re-introduced into strain WT82. This strain has both GyrA (Ser-83Tyr, Asp-87Asn) substitutions and the ParC (Glu-84Lys) substitution and, as with the other triple point mutants, shows the highest levels of fluoroquinolone resistance. Among the derivatives in which the suicide vector-gyrA plasmid had recombined at the mutant gyrA locus, were both susceptible and resistant recombinants. Again, when treated for removal of the suicide vector each of these generated both susceptible and resistant derivatives, depending on whether or not the cross-over event had incorporated the mutations into the functional gene. In this case the susceptible derivatives had the ParC Glu-80Lys substitution but showed the same susceptibility to quinolones as CVD908-htrA. Thus, such complementation data should be interpreted with caution in undefined resistant strains.
As with the construction of WT96 and the WT82 complementation experiment, during construction of the other strains, both susceptible and resistant derivatives were obtained following selection for removal of the suicide vector constructs from the recombination site in the topoisomerase gene (not shown).
These observations show that conversion between susceptibility and resistance is commensurate with insertion and removal of the suicide vector constructs at gyrA, confirming that the observed resistance is indeed conferred by the introduced point mutations and is not due to changes elsewhere in the genome.
Amino acid substitutions in GyrA confer resistance to nalidixic acid and reduced susceptibility to fluoroquinolones
Using antibiotic-containing discs, there were no discernable differences in resistance profiles between homologous mutants and so photographs of only one mutant of each homologous pair are shown in Figure 2. All the gyrA mutants were resistant to nalidixic acid (30 µg disc) and cinoxacin (100 µg disc), and compared with CVD908-htrA showed smaller zones of growth inhibition around the discs containing 5 µg of the fluoroquinolones enrofloxacin, moxifloxacin, ofloxacin and ciprofloxacin, (Figure 2).
Also shown in Figure 2 are the MICs of gatifloxacin, ofloxacin and ciprofloxacin. Differences between MIC values for homologous mutant pairs were within expected experimental variation (<2-fold). As expected, any one of the single amino acid substitutions conferred resistance to nalidixic acid (MIC 512 mg/L, not shown) and increased the MICs of the fluoroquinolones by between 7- and 24-fold compared with CVD908-htrA (Figure 2). When either of the Ser-83 substitutions was combined with Asp-87Asn in GyrA increases in MIC were at most 2-fold compared with any single substitution (Figure 2).
The Glu-84Lys amino acid substitution in ParC confers high levels of resistance to fluoroquinolones only when present with two GyrA substitutions
Introduction of the Glu-84Lys substitution alone into ParC gave no significant increases in MIC. When the Glu-84Lys substitution in ParC was combined with any one of the other single substitutions in GyrA, all the MICs increased compared with the GyrA substitutions alone, but these increases were mostly of 2-fold or less. For strains with any two mutations, when compared with the GyrA substitution alone, only the combination of GyrA Ser-83Phe and ParC Glu-84Lys substitution caused an MIC increase of more than 2-fold (Figure 2).
When all three amino acid substitutions were incorporated, the MICs of the fluoroquinolones increased by at least 180-fold compared with CVD908-htrA and at least 7-fold (for gatifloxacin) compared with any of the double substitution mutants, giving MICs of 32 mg/L or more for ofloxacin and ciprofloxacin and 3–4 mg/L for gatifloxacin in the triple substitution mutants (Figure 2).
The ParC substitution, in combination with one of the GyrA substitutions, provides greater protection from the bactericidal effects of ciprofloxacin than a second substitution in GyrA
Between mutants with various combinations of single and double substitutions, the MICs of each quinolone differed by relatively small amounts, which may be due to experimental variation (Figure 2). Mutations in essential enzymes as well as conferring resistance may confer a biological cost. As a crude measure of this we found that there were no detectable differences in growth rates in rich media between the CVD908-htrA and single, double or triple topoisomerase mutants (data not shown). We also wanted to know whether any of the mutants had a selective advantage compared with others, in the presence of ciprofloxacin at clinically relevant levels. We therefore performed experiments to determine the killing rates of these mutants in the presence of 4 mg/L ciprofloxacin. Figure 3 shows the rate of kill for the single and double mutants harbouring the Ser-83Phe substitution in GyrA. The presence of a second Asp-87Asn substitution in GyrA had no effect on the rate of kill. However, when the second substitution was Glu-84Lys in ParC the rate of kill was slower such that after 1 h the number of surviving bacteria was at least 30-fold higher than with the other mutants. Similar results were obtained for the single and double mutants harbouring the Ser-83Tyr substitution in GyrA. Thus, when compared in the same experiment, double mutants carrying the ParC substitution were less prone to killing by ciprofloxacin than double mutants carrying two GyrA substitutions.
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In the presence of ciprofloxacin the killing rates of the mutants harbouring the ParC substitution alone were not significantly different to that of CVD908-htrA (not shown).
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Discussion |
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The results presented here are from the first study using a panel of point mutants that have all been created from the same susceptible parent strain without resorting to selection using fluoroquinolones. These mutants therefore confirm the role of these point mutations in the resistance of Salmonella Typhi to fluoroquinolones and allow direct comparison of the contribution to resistance conferred by the point mutations, alone or in combination. Using resistant isolates or mutants that have been selected with quinolones in vitro, such comparisons are not accurate or reliable. Thus, the methodology described here can be used to confirm the role of point mutations suspected of being involved in resistance to fluoroquinolones and will be of use for testing other point mutations in Salmonella Typhi, other Salmonella serotypes and E. coli strains.
One of the single amino acid substitutions in GyrA was sufficient for resistance to the quinolones nalidixic acid and cinoxacin, but resistance to the fluoroquinolones (gatifloxacin, ofloxacin, ciprofloxacin, enrofloxacin and moxifloxacin) required two substitutions in GyrA and one in ParC (Figure 2), confirming that these three point mutations combined are sufficient to confer fluoroquinolone resistance in Salmonella Typhi. The high levels of resistance observed for these triple point mutants indicate that the amino acid substitutions protect GyrA and ParC from relatively high concentrations of fluoroquinolones. The observed susceptibility of the mutants that possess only the ParC substitution (strains WT72 and WT74) must therefore be due to activity against GyrA only. Thus, the fluoroquinolones gatifloxacin, ofloxacin and ciprofloxacin begin to manifest their effects on GyrA when their extracellular concentrations are 0.016, 0.094 and 0.023 mg/L, respectively (Figure 2). Similarly, in the double GyrA substitution mutants (WT28, WT30, WT43 and WT46) the observed susceptibility must be due to activity against ParC only. So, gatifloxacin, ofloxacin and ciprofloxacin begin to manifest their effects on ParC when their extracellular concentrations are 0.25, 0.75 and 0.38 mg/L, respectively (Figure 2). Thus, the results confirm that the quinolones cinoxacin and nalidixic acid act primarily on GyrA and the fluoroquinolones act both on GyrA and, at higher concentrations, ParC. For purified E. coli topoisomerases in vitro, it has been observed that the fluoroquinolones act on the wild-type GyrA at lower concentrations than on the wild-type ParC,6,29 and our data show that this is probably true also for topoisomerases when inside the Salmonella Typhi bacterium. The extracellular MIC for the other GyrA allozymes may be determined similarly from the ParC Glu-84Lys substitution mutants (0.25, 1.5–2 and 0.75–1 for the Ser-83 substitutions, and 0.19, 0.75 and 0.38 for the Asp-87 substitution for gatifloxacin, ofloxacin and ciprofloxacin, respectively).
Salmonella Typhi isolates that show reduced susceptibility to the fluoroquinolones (MIC of ciprofloxacin 1mg/L) have been associated with treatment failure and prolonged fever clearance in typhoid patients, and some of these strains have been found to have point mutations resulting in substitutions at GyrA Ser-83 or Asp-87.11–13,15,30–33 Decreased susceptibility to the fluoroquinolones is difficult to diagnose using antibiotic-containing discs. However, it has been closely correlated to nalidixic acid resistance (MIC 512 mg/L), and so the use of nalidixic acid resistance has been recommended for the identification of reduced susceptibility to fluoroquinolones.34,35 Our data show that the single amino acid substitutions in gyrA that were tested are a mechanism of both nalidixic acid resistance and reduced susceptibility to fluoroquinolones and indicate that single point mutations in gyrA can be the cause of treatment failures in typhoid patients.
It is clear that the triple substitution mutants are resistant to relatively high concentrations of fluoroquinolones, and thus will have a selective advantage in the presence of fluoroquinolones. However, in order to achieve a triple substitution it is likely that selection will be required at each step. The MIC data suggest that the selective advantage between mutants with one and two of the substitutions investigated here may be comparatively small. In addition, there was no significant difference in the growth rates between these mutants (data not shown), indicating no obvious cost to fitness between any of the double substitution mutants. However, comparison of the mutants in experiments to determine the rates of kill by ciprofloxacin demonstrated that double substitution mutants which include the ParC substitution were less susceptible to killing than double substitution mutants with substitutions in GyrA alone. Thus, fluoroquinolone-resistant Salmonella Typhi could evolve by acquisition of these point mutations, for example, with the second being within parC under selection from ciprofloxacin. In this way the stepwise accumulation of single mutations may be driven by selection at each step.
The more active gatifloxacin may be useful for the treatment of enteric fever in regions where treatment failure with ciprofloxacin is common. However our results sound a word of caution. Engineered Salmonella Typhi strains showing high levels of resistance to ciprofloxacin, caused by three mutations in topoisomerase genes, also have raised MICs of gatifloxacin (up to 4 mg/L). Given peak serum concentrations of 3.8–5 mg/L of gatifloxacin following a 400 mg dose36 it seems unlikely that these patients will respond to treatment with that dose of gatifloxacin. Our results suggest that use of this more active fluoroquinolone could delay the emergence of high-level resistance by treating effectively infections caused by strains with one or two mutations but will not prevent the spread of strains which have acquired all three mutations.
Although uncommon at present, our results show that there is no detectable barrier to the evolution of high levels of fluoroquinolone resistance in Salmonella Typhi and we should expect to see the emergence of high-level fluoroquinolone resistance. Only molecular epidemiology of such isolates will tell us whether the strains are transmissible. If they are, then given the overwhelming selective pressure created by the widespread use of fluoroquinolone antibacterials, these isolates will almost certainly spread worldwide unless they can be quickly recognized and appropriately treated.
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No financial or other interest has influenced the conclusions drawn from the work.
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Acknowledgements |
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We are grateful to Nik Matthews and the other members of Team 56 at The Wellcome Trust Sanger Institute for providing us with an excellent nucleotide sequencing service. We would like to thank Professor Laura Piddock (Birmingham University, UK) for comments on the manuscript. This work was funded by The Wellcome Trust of Great Britain.
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References |
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