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Frequency of 1st- and 2nd-step topoisomerase mutations in Streptococcus pneumoniae following levofloxacin and moxifloxacin exposure
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Diagnostic Microbiology and Infectious Disease 60 (2008) 295 – 299 www.elsevier.com/locate/diagmicrobio
Frequency of 1st- and 2nd-step topoisomerase mutations in Streptococcus pneumoniae following levofloxacin and moxifloxacin exposure Laurie B. Hovde, Dana A. Simonson, John C. Rotschafer⁎ Department of Experimental and Clinical Pharmacology, Antibiotic Pharmacodynamic Research Institute, The University of Minnesota College of Pharmacy, Minneapolis, MN 55455, USA Received 27 June 2007; accepted 11 October 2007
Abstract Seven Streptococcus pneumoniae isolates were exposed to inhibitory concentrations of levofloxacin and moxifloxacin in antibioticcontaining agar dilution plates. Colony counts were used to calculate the frequency of mutation. DNA was sequenced to detect mutations in the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes. The wild-type S. pneumoniae isolate developed a parC mutation after exposure to levofloxacin more frequently than it developed a gyrA mutation after exposure to moxifloxacin. The 1st-step gyrA mutant developed a 2nd-step gyrA–parC mutation more frequently after exposure to levofloxacin. Conversely, the transformation from a 1st-step parC mutant to a 2nd-step parC–gyrA mutant occurred more frequently following exposure to moxifloxacin. Our data suggest that the occurrence of a 2nd mutation will be contingent on the location of the 1st mutation and the preferential binding site of the fluoroquinolone that drives the transformation from 1st- to 2nd-step mutant. © 2008 Elsevier Inc. All rights reserved. Keywords: Streptococcus pneumoniae; Levofloxacin; Moxifloxacin; Topoisomerase IV; DNA gyrase
1. Introduction The data presented by Wickman et al. (2002) suggest that moxifloxacin is less likely than ciprofloxacin to select fluoroquinolone-resistant mutants in Streptococcus pneumoniae. The 1st-step mutation frequency with moxifloxacin as the selector was less than with ciprofloxacin as the selector, but, interestingly, the reverse was seen when selecting for 2nd-step mutants. Ciprofloxacin is a poor choice for comparison with the newer fluoroquinolones that have superior activity against S. pneumoniae. Levofloxacin and moxifloxacin are respiratory fluoroquinolones with intrinsic activity against S. pneumoniae and are indicated for treatment of community-acquired pneumonia (Canton et al., 2003). The preferential target for levofloxacin is topoisomerase IV (subunit parC), whereas the preferential target for moxifloxacin is
DNA gyrase (subunit gyrA) (Li et al., 2002). Consequently, in wild-type S. pneumoniae isolates, selective pressure by levofloxacin prompts a single-step parC mutation, and selective pressure by moxifloxacin prompts a single-step gyrA mutation. S. pneumoniae isolates with 1st-step mutations in the quinolone resistance-determining region (QRDR) remain fully susceptible to the respiratory fluoroquinolones according to Clinical and Laboratory Standards Institute (CLSI) breakpoints (CLSI, 2005; Davies et al., 2006; Jorgensen et al., 1999). The purpose of this study was to compare the frequencies of a 2nd mutation when 1st-step mutants of S. pneumoniae were exposed to inhibitory concentrations of levofloxacin and moxifloxacin. 2. Materials and methods 2.1. Bacteria
⁎ Corresponding author. Tel.: +1-612-624-2183; fax: +1-612-626-5082. E-mail address: [email protected] (J.C. Rotschafer). 0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2007.10.007
Seven S. pneumoniae isolates were chosen based on mutations in the QRDR of the gyrA, gyrB, parC, and parE
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genes. Isolate 1 is a wild type (i.e., no QRDR mutations), 2 is a 1st-step parC mutant, 3 is a 2nd-step gyrA–parC mutant, 4 is a 1st-step gyrA mutant created by exposing isolate 1 to moxifloxacin at 4 times the MIC, 5 is a parE mutant, 6 is a parC–parE mutant, and 7 is a gyrA–parC–parE mutant (Table 1, parent). 2.2. Antibiotics Stock solutions (4 mg/mL) of pharmaceutical grade levofloxacin (Ortho-McNeil Pharmaceutical, Raritan, NJ) and moxifloxacin (Bayer, West Haven, CT) were prepared according to the manufacturer's instructions and frozen at −80 °C until needed. 2.3. Agar dilution plates Agar plates containing levofloxacin or moxifloxacin at concentrations equal to 1, 2, 4, and 8 times the MIC (2, 4, 8, and 16 times the MIC for strain 6) of the strain to be exposed were prepared using modified trypticase soy agar (Becton Dickinson, Sparks, MD). The agar was prepared according to the manufacturer's directions and cooled to 50 °C. Defibrinated sheep blood (Hema Resource, Aurora, OR) was
added to a concentration of 5%. An appropriate amount of a stock solution of levofloxacin or moxifloxacin was added to achieve the desired drug concentration. After mixing, 60 mL was dispensed into each 15 × 150-mm Petri dish. The agar was allowed to solidify and stored at 4 °C for less than 7 days before use. Levofloxacin and moxifloxacin stability has not been studied in trypticase soy agar with 5% sheep blood, but levofloxacin remained stable in other types of media for ≥14 days (Sanders et al., 2004). Five agar plates were prepared at each concentration for each strain. 2.4. Inoculum preparation and plating To maximize the number of bacteria in the inoculum, we concentrated an overnight culture of the isolate in 500 mL of Todd–Hewitt broth (Becton Dickinson) supplemented with 0.5% yeast extract (THBY) (Becton Dickinson) by centrifugation at 1890 × g for 20 min at room temperature (Hettich Rotofix 32, Germany). The supernatant was decanted, leaving approximately 5 mL of heavy suspension. An aliquot of the suspension (0.1 mL) was removed, serially diluted in saline, plated onto sheep blood agar plates, and incubated. The colony counts were used to calculate the
Table 1 Susceptibility of S. pneumoniae isolates to levofloxacin and moxifloxacin and detected amino acid substitutions in DNA gyrase and topoisomerase IV Strain
MIC (μg/mL) of
Amino acid substitutions in
LVX
MOX
gyrA
gyrB
parC
parE
1 a parent Post-LVX Post-LVX Post-MOX Post-MOX 2 b parent Post-LVX Post-LVX Post-MOX 3 b parent Post-LVX Post-LVX
1 2 2 2 2 2 8 16 8–16 8 16 16
0.25 0.25 0.5 0.5–1 0.5–1 0.5 2 4 4 4 4 4
0 0 0 0 0 0 0 0 0 0 Leu436→Ile
0 Ser79→Phe 0 0 0 Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe
0 0 0 0 0 0 0 0 0 0 0
Post-MOX Post-MOX 4 c parent Post-LVX Post-MOX 5 b parent Post-LVX Post-MOX 6 b parent Post-LVX Post-LVX Post-MOX Post-MOX 7 b parent Post-LVX Post-MOX
16 16 2 16–32 32 1 2 2 1 8 4 8 4 4 4–8 4
4 4 0.5 4–8 8 0.125 0.25 0.5 0.125 1 2 2 2 1 0.5–1 1
0 0 0 Ser81→Phe Ser81→Tyr 0 Glu85→Gln Ser81→Phe Ser81→Phe Ser81→Phe Ser81→Phe Ser81→Phe Val101→Leu Ser81→Phe Phe81→Leu Ser81→Phe Ser81→Phe Ser81→Phe 0 0 0 0 Glu85→Gly Ser81→Tyr Ser81→Phe Ser81→Tyr Ser81→Ala Ser81→Ala Ala81→Val
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Ser79→Phe Ser79→Phe 0 Ser79→Tyr Ser79→Tyr 0 0 0 Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe Asp83→Asn Asp83→Asn Asp83→Asn
0 0 0 0 0 Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val
LVX = levofloxacin; MOX = moxifloxacin. a Clinical wild-type isolate kindly provided by Ron Jones, MD, JMI Laboratories, North Liberty, IA. b First-step parC mutant from TRUST study, Johnson & Johnson Pharmaceutical Research, Raritan, NJ. c First-step gyrA mutant created by exposing the wild-type isolate to moxifloxacin at 4× MIC.
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number of viable bacteria in the inoculum. The inocula contained between 7.2 × 107 and 6.7 × 109 colony-forming units (CFU)/mL (average = 1.4 × 109 CFU/mL). Two hundred microliters of the inoculum (0.2 mL) were applied to the surface of each of the agar dilution plates, 5 plates per concentration, and spread evenly resulting in a total volume of 1 mL of inoculum exposed to the antibiotic. The plates were incubated at 36 °C with 5% to 10% CO2 and examined for growth at 24, 48, and 72 h. 2.5. Frequency of spontaneous single-step mutation After 72 h of incubation, the frequency of mutation values were calculated from the ratio of the number of CFU(s) on drug-containing plates to the number of CFU(s) on drug-free plates (Pestova et al., 2000). The frequency of mutation was determined using the highest multiple of the MIC that still allowed growth of a countable number of colonies, and all colonies growing at that drug concentration were considered to be mutants. To be consistent, we calculated the frequency of mutation from the same multiple of the MIC for both fluoroquinolones. Thus, if there were no colonies on the plates at the matching multiple of the MIC, the frequency of mutation value for the corresponding drug was expressed as less than the number of CFU(s) exposed. Two to 5 colonies randomly chosen from these plates were individually frozen at −80 °C for later susceptibility testing and DNA sequencing. 2.6. Susceptibility testing MIC testing was performed on parent (or preexposure) isolates and on 2 (4 for isolate 4) of the postexposure isolates that were randomly selected from the agar dilution plates and were viable after freeze. The microdilution broth method was performed using the BioTek Precision 2000 pipetting system (BioTek Instruments, Winooski, VT). CLSI guidelines (CLSI, 2000) were followed for all but 4 of the postexposure isolates. For strain 6, 1 of 2 postmoxifloxacin exposure isolates required incubation with 5% to 10% CO2 to enhance growth. Strain 5 was particularly troublesome; 1 of 2 postlevofloxacin exposure and 1 of 2 postmoxifloxacin exposure isolates would not grow in Mueller–Hinton broth containing 5% lysed horse blood, so MIC testing was performed in THBY in the presence of 5% to 10% CO2. S. pneumoniae ATCC 49619 was used to monitor quality control of CLSI methods and alternative methods. 2.7. Polymerase chain reaction and DNA sequencing A pure culture, arising from 1 colony, was used to inoculate 25 mL of THBY. After overnight incubation at 36 °C with 5% to 10% CO2, 3 mL of culture were separated into 2 microcentrifuge tubes and spun for 5 min at 20 598 × g at room temperature (Hermle Z233-M2, Germany) to pellet the cells. The supernatant was decanted, and the pellets were combined and washed with Tris–EDTA buffer (TE) followed by another centrifugation and decanting step. Cell
297
lysis was achieved by adding 0.150 mL of TE, 0.025 mL of 10 mg/mL lysozyme in TE, and 0.030 mL of 2 mg/mL mutanolysin in TE to the pellet. Lysis took place during incubation for 30 min at 37 °C. DNA was extracted using MoBio UltraClean Microbial DNA Isolation Kit (MO BIO, Carlsbad, CA). The protocol was modified to extract sufficient quantities of DNA. Solution MD1 was added directly to the lysate and centrifuged to remove cell debris. The MoBio protocol was reinstituted at step 9, except that all centrifugation was at 20 598 × g. Gel electrophoresis was performed to determine if a sufficient quantity of DNA was extracted. Polymerase chain reaction (PCR) was performed using 1 μL of the DNA template in a 0.050-mL reaction volume. The QRDRs of gyrA, gyrB, parC, and parE were amplified under the following conditions: 1 denaturization cycle at 95 °C for 5 min; 30 cycles at 94 °C for 60 s, 53 °C for 60 s, 72 °C for 3 min for gyrA and parC; and 30 cycles at 92 °C for 60 s, 48 °C for 60 s, 72 °C for 2 min for gyrB and parE, each followed by 1 extension cycle at 72 °C for 10 min. A 382-bp fragment of gyrA was amplified using primers described by Pan and Fisher (1999), and a 458-bp fragment of gyrB was amplified using primers described by Munoz et al. (1995). The primers used to amplify both the 367-bp fragment of parC and the 291-bp fragment of parE were described by Pan and Fisher (1996). Gel electrophoresis was performed to determine if the desired product was amplified. PCR products were purified using a Qiaquick PCR purification kit (QIAGEN, Valencia, CA) and sequenced at the Advanced Genetic Analysis Center (St. Paul, MN). The nucleotide sequences of the parent strains were compared with published sequences for gyrA (GenBank accession no. AJ005815), gyrB (GenBank accession no. Z67740), parC, and parE (GenBank accession no. Z67739) using T-COFFEE (Notredame et al., 2000). The nucleotide sequences of the postexposure strains were compared with the sequences of the parent strains. 3. Results 3.1. Frequency of mutation Frequency of mutation values are reported for isolate 1 (wild type), isolate 2 (parC mutant), and isolate 4 (gyrA mutant). Frequency of mutation values for the other isolates studied was determined but not reported because the results were not interpretable. At 4 times the MIC, the frequency of mutation from wild-type to 1st-step parC mutant after exposure to levofloxacin was 1.5 × 10−7. The frequency of mutation from wild-type to 1st-step gyrA mutant after moxifloxacin exposure was 2.6 × 10−8. The mutation from a 1st-step parC to 2nd-step parC–gyrA occurred more frequently with moxifloxacin than with levofloxacin, 4.0 × 10−7 and b2.0 × 10−8, respectively. At 8 times the MIC, the frequency of mutation from a 1st-step gyrA mutant to a 2nd-step gyrA–parC mutant was 3.3 × 10−7 for levofloxacin and 3.0 × 10−8 for moxifloxacin (Table 2).
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Table 2 Frequencies of levofloxacin- and moxifloxacin-selected mutations in the wild-type isolate and the parC and gyrA 1st-step mutants Strain
1a 2b 4c
Levofloxacin
Moxifloxacin
Multiple of MIC
Mutation frequency
Multiple of MIC
Mutation frequency
4× 4× 8×
1.5 × 10−7 b2.0 × 10−8 3.3 × 10−7
4× 4× 8×
2.6 × 10−8 4.0 × 10−7 3.0 × 10−8
2nd-step gyrA–parC mutant strains, plus/minus an additional parE mutation, showed no change in the parC or parE amino acid sequence between pre- (parent) and postexposure isolates. However, there were some changes in gyrA, either in the nucleotide sequence, resulting in a change in the amino acid at position 81, or an additional gyrA mutation at position 101. Also, the gyrA–parC mutant developed a gyrB mutation after exposure to levofloxacin.
a
Clinical wild-type isolate kindly provided by Ron Jones, MD, JMI Laboratories. b First-step parC mutant from TRUST study, Johnson & Johnson Pharmaceutical Research. c First-step gyrA mutant created by exposing the wild-type isolate to moxifloxacin at 4× MIC.
3.2. Susceptibility testing The 1st-step parC mutant, which was still sensitive to levofloxacin and moxifloxacin, developed a secondary gyrA mutation when exposed to either fluoroquinolones, and the MIC increased 4- to 8-fold, resulting in postexposure isolates that were no longer sensitive to levofloxacin or moxifloxacin. Similarly, the 1st-step gyrA mutant that was sensitive to levofloxacin and moxifloxacin developed a secondary parC mutation after exposure to either fluoroquinolones. The MICs increased 8- to 16-fold, resulting in resistance to levofloxacin and moxifloxacin. The parC–parE mutant developed a gyrA mutation resulting in a 4- to 8-fold increase of the levofloxacin MIC and subsequent resistance. The 8- to 16-fold increase of the moxifloxacin MIC resulted in 3 of 4 isolates becoming nonsusceptible and 1 isolate remaining sensitive. The pre- (parent) and postexposure MIC results are listed in Table 1. 3.3. PCR and DNA sequencing A sufficient quantity of DNA was extracted, and sequencing of the product obtained via PCR, using the primers and conditions listed above, confirmed that the desired gene fragments were amplified. Sequence comparisons revealed the mutations listed in Table 1. In some cases, postexposure isolates chosen from the same agar dilution plate had different amino acid substitutions, and in those cases, both mutations are listed. After exposing the wild-type isolate to levofloxacin, only 1 of 2 isolates chosen for postexposure testing had developed a 1st-step parC mutation, whereas all 4 isolates exposed to moxifloxacin and chosen for postexposure testing developed a 1st-step gyrA mutation. When this 1st-step gyrA mutant was further exposed to either levofloxacin or moxifloxacin, a parC mutation developed. The isolate with a parE mutation only did not develop additional topoisomerase mutations after exposure to levofloxacin or moxifloxacin. The parent strains with a 1st-step parC mutation, plus/minus an additional parE mutation, all developed a mutation in gyrA when under pressure from either levofloxacin or moxifloxacin. The
4. Discussion The frequency of mutation calculation assumes that all colonies present on plates containing inhibitory concentrations of drug are mutants (Pan and Fisher, 1998; Pestova et al., 2000). Pan et al. states that “mutant frequencies were determined by comparing the number of colonies that grew on plates containing drug with the number colonies obtained in the absence of drug”. In our study, some of the postexposure isolates randomly chosen for MIC testing and DNA sequencing revealed only slight (2- to 4-fold) MIC increases, remaining susceptible, and additional QRDR mutations were not detected. These isolates may have mutations outside the QRDR or may have initiated active efflux allowing them to grow in the presence of inhibitory concentrations of drug. It is also possible that some of the parent isolates survived and subsequently grew as the drug concentration diminished with time (i.e., breakthrough growth). This causes speculation regarding the “all-inclusive” nature of frequency of mutation calculations. Opinions differ regarding the role of the prevalent parE Ile460Val amino acid substitution in regard to fluoroquinolone resistance (Kawamura-Sato et al., 2005; Pestova et al., 2000). This mutation was present in 3 of the isolates we studied. Notably, the isolate that possessed the Ile460Val parE mutation only and, as such, may be considered a wild type by some did not develop any further mutations when exposed to selective concentrations of levofloxacin or moxifloxacin. These results suggest that 1st-step Ile460Val parE mutants may not be able to develop a secondary mutation in gyrA or parC. Additional studies need to be done to elucidate the bona fide role of the Ile460Val parE mutation. We questioned whether the frequency of the 2nd mutation would differ depending on the fluoroquinolone used as the selecting agent. Our results indicated that moxifloxacin selected a 2nd-step mutant from a 1st-step parC mutant more frequently than levofloxacin, similar to observations by Wickman et al. (2002). Conversely, levofloxacin selected a 2nd-step mutant from a 1st-step gyrA mutant more frequently than moxifloxacin. In conclusion, our data suggest that the occurrence of a 2nd mutation will be contingent on the 1st mutation and the preferential binding site of the fluoroquinolone that drives the transformation from 1st- to 2nd-step mutant. If a binding site has been altered, the fluoroquinolone that prefers the
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altered site is less likely than a fluoroquinolone that targets a remote site to cause a 2nd mutation, and the frequency of the mutation is less. Acknowledgments The authors thank Ortho-McNeil Pharmaceuticals for providing the funding for this project. References Canton R, Morosini M, Enright MC, Morrissey I (2003) Worldwide incidence, molecular epidemiology and mutations implicated in fluoroquinolone-resistant Streptococcus pneumoniae: data from the PROTEKT surveillance programme. J Antimicrob Chemother 52:944−952. Clinical and Laboratory Standards Institute (2000) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—fifth edition. NCCLS Document M7-A5. Wayne (PA): CLSI. Clinical and Laboratory Standards Institute (2005) Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement. M100-S15. Wayne (PA): CLSI. Davies TA, Yee YC, Goldschmidt R, Bush K, Sahm DF, Evangelista A (2006) Infrequent occurrence of single mutations in topoisomerase IV and DNA gyrase genes among US levofloxacin-susceptible clinical isolates of Streptococcus pneumoniae from nine institutions (1999– 2003). J Antimicrob Chemother 57:437−442. Jorgensen JH, Weigel LM, Ferraro MJ, Swenson JM, Tenover FC (1999) Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates including those with mutations in the gyrA, parC, and parE loci. Antimicrob Agents Chemother 43:329−334.
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Kawamura-Sato K, Hasegawa T, Torii K, Ito H, Ohta M (2005) Prevalence of Ile-460-Val/ParE substitution in clinical Streptococcus pneumoniae isolates that were less susceptible to fluoroquinolones. Curr Microbiol 51:27−30. Li X, Zhao X, Drlica K (2002) Selection of Streptococcus pneumoniae mutants having reduced susceptibility to moxifloxacin and levofloxacin. Antimicrob Agents Chemother 46:522−524. Munoz R, Bustamante M, de la Campa AG (1995) Ser-127-to-Leu substitution in the DNA gyrase B subunit of Streptococcus pneumoniae is implicated in novobiocin resistance. J Bacteriol 177:4166−4170. Notredame C, Higgins D, Heringa J (2000) T-Coffee: a novel method for multiple sequence alignments. J Mol Biol 302:205−217. Pan XS, Fisher LM (1996) Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance. J Bacteriol 178:4060−4069. Pan XS, Fisher LM (1998) DNA gyrase and topoisomerase IV are dual targets of clinafloxacin action in Streptococcus pneumoniae. Antimicrob Agents Chemother 42:2810−2816. Pan XS, Fisher LM (1999) Streptococcus pneumoniae DNA gyrase and topoisomerase: overexpression, purification, and differential inhibition by fluoroquinolones. Antimicrob Agents Chemother 43:1129−1136. Pestova E, Millichap J, Noskin G, Peterson L (2000) Intracellular targets of moxifloxacin: a comparison with other fluoroquinolones. J Antimicrob Chemother 45:583−590. Sanders CA, Nieda RR, Desmond EP (2004) Validation of the use of Middlebrook 7H10 agar, BACTEC MGIT 960, and BACTEC 460 12B media for testing the susceptibility of Mycobacterium tuberculosis to levofloxacin. J Clin Microbiol 42:5225−5228. Wickman PA, Hossain A, Thomson KS, Hanson ND (2002) Moxifloxacin has reduced potential to select mutational fluoroquinolone resistance in Streptococcus pneumoniae. Abstracts of the 102nd General Meeting of the American Society for Microbiology, Park City, Utah, A-17.
Diagnostic Microbiology and Infectious Disease 60 (2008) 295 – 299 www.elsevier.com/locate/diagmicrobio
Frequency of 1st- and 2nd-step topoisomerase mutations in Streptococcus pneumoniae following levofloxacin and moxifloxacin exposure Laurie B. Hovde, Dana A. Simonson, John C. Rotschafer⁎ Department of Experimental and Clinical Pharmacology, Antibiotic Pharmacodynamic Research Institute, The University of Minnesota College of Pharmacy, Minneapolis, MN 55455, USA Received 27 June 2007; accepted 11 October 2007
Abstract Seven Streptococcus pneumoniae isolates were exposed to inhibitory concentrations of levofloxacin and moxifloxacin in antibioticcontaining agar dilution plates. Colony counts were used to calculate the frequency of mutation. DNA was sequenced to detect mutations in the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes. The wild-type S. pneumoniae isolate developed a parC mutation after exposure to levofloxacin more frequently than it developed a gyrA mutation after exposure to moxifloxacin. The 1st-step gyrA mutant developed a 2nd-step gyrA–parC mutation more frequently after exposure to levofloxacin. Conversely, the transformation from a 1st-step parC mutant to a 2nd-step parC–gyrA mutant occurred more frequently following exposure to moxifloxacin. Our data suggest that the occurrence of a 2nd mutation will be contingent on the location of the 1st mutation and the preferential binding site of the fluoroquinolone that drives the transformation from 1st- to 2nd-step mutant. © 2008 Elsevier Inc. All rights reserved. Keywords: Streptococcus pneumoniae; Levofloxacin; Moxifloxacin; Topoisomerase IV; DNA gyrase
1. Introduction The data presented by Wickman et al. (2002) suggest that moxifloxacin is less likely than ciprofloxacin to select fluoroquinolone-resistant mutants in Streptococcus pneumoniae. The 1st-step mutation frequency with moxifloxacin as the selector was less than with ciprofloxacin as the selector, but, interestingly, the reverse was seen when selecting for 2nd-step mutants. Ciprofloxacin is a poor choice for comparison with the newer fluoroquinolones that have superior activity against S. pneumoniae. Levofloxacin and moxifloxacin are respiratory fluoroquinolones with intrinsic activity against S. pneumoniae and are indicated for treatment of community-acquired pneumonia (Canton et al., 2003). The preferential target for levofloxacin is topoisomerase IV (subunit parC), whereas the preferential target for moxifloxacin is
DNA gyrase (subunit gyrA) (Li et al., 2002). Consequently, in wild-type S. pneumoniae isolates, selective pressure by levofloxacin prompts a single-step parC mutation, and selective pressure by moxifloxacin prompts a single-step gyrA mutation. S. pneumoniae isolates with 1st-step mutations in the quinolone resistance-determining region (QRDR) remain fully susceptible to the respiratory fluoroquinolones according to Clinical and Laboratory Standards Institute (CLSI) breakpoints (CLSI, 2005; Davies et al., 2006; Jorgensen et al., 1999). The purpose of this study was to compare the frequencies of a 2nd mutation when 1st-step mutants of S. pneumoniae were exposed to inhibitory concentrations of levofloxacin and moxifloxacin. 2. Materials and methods 2.1. Bacteria
⁎ Corresponding author. Tel.: +1-612-624-2183; fax: +1-612-626-5082. E-mail address: [email protected] (J.C. Rotschafer). 0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2007.10.007
Seven S. pneumoniae isolates were chosen based on mutations in the QRDR of the gyrA, gyrB, parC, and parE
296
L.B. Hovde et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 295–299
genes. Isolate 1 is a wild type (i.e., no QRDR mutations), 2 is a 1st-step parC mutant, 3 is a 2nd-step gyrA–parC mutant, 4 is a 1st-step gyrA mutant created by exposing isolate 1 to moxifloxacin at 4 times the MIC, 5 is a parE mutant, 6 is a parC–parE mutant, and 7 is a gyrA–parC–parE mutant (Table 1, parent). 2.2. Antibiotics Stock solutions (4 mg/mL) of pharmaceutical grade levofloxacin (Ortho-McNeil Pharmaceutical, Raritan, NJ) and moxifloxacin (Bayer, West Haven, CT) were prepared according to the manufacturer's instructions and frozen at −80 °C until needed. 2.3. Agar dilution plates Agar plates containing levofloxacin or moxifloxacin at concentrations equal to 1, 2, 4, and 8 times the MIC (2, 4, 8, and 16 times the MIC for strain 6) of the strain to be exposed were prepared using modified trypticase soy agar (Becton Dickinson, Sparks, MD). The agar was prepared according to the manufacturer's directions and cooled to 50 °C. Defibrinated sheep blood (Hema Resource, Aurora, OR) was
added to a concentration of 5%. An appropriate amount of a stock solution of levofloxacin or moxifloxacin was added to achieve the desired drug concentration. After mixing, 60 mL was dispensed into each 15 × 150-mm Petri dish. The agar was allowed to solidify and stored at 4 °C for less than 7 days before use. Levofloxacin and moxifloxacin stability has not been studied in trypticase soy agar with 5% sheep blood, but levofloxacin remained stable in other types of media for ≥14 days (Sanders et al., 2004). Five agar plates were prepared at each concentration for each strain. 2.4. Inoculum preparation and plating To maximize the number of bacteria in the inoculum, we concentrated an overnight culture of the isolate in 500 mL of Todd–Hewitt broth (Becton Dickinson) supplemented with 0.5% yeast extract (THBY) (Becton Dickinson) by centrifugation at 1890 × g for 20 min at room temperature (Hettich Rotofix 32, Germany). The supernatant was decanted, leaving approximately 5 mL of heavy suspension. An aliquot of the suspension (0.1 mL) was removed, serially diluted in saline, plated onto sheep blood agar plates, and incubated. The colony counts were used to calculate the
Table 1 Susceptibility of S. pneumoniae isolates to levofloxacin and moxifloxacin and detected amino acid substitutions in DNA gyrase and topoisomerase IV Strain
MIC (μg/mL) of
Amino acid substitutions in
LVX
MOX
gyrA
gyrB
parC
parE
1 a parent Post-LVX Post-LVX Post-MOX Post-MOX 2 b parent Post-LVX Post-LVX Post-MOX 3 b parent Post-LVX Post-LVX
1 2 2 2 2 2 8 16 8–16 8 16 16
0.25 0.25 0.5 0.5–1 0.5–1 0.5 2 4 4 4 4 4
0 0 0 0 0 0 0 0 0 0 Leu436→Ile
0 Ser79→Phe 0 0 0 Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe
0 0 0 0 0 0 0 0 0 0 0
Post-MOX Post-MOX 4 c parent Post-LVX Post-MOX 5 b parent Post-LVX Post-MOX 6 b parent Post-LVX Post-LVX Post-MOX Post-MOX 7 b parent Post-LVX Post-MOX
16 16 2 16–32 32 1 2 2 1 8 4 8 4 4 4–8 4
4 4 0.5 4–8 8 0.125 0.25 0.5 0.125 1 2 2 2 1 0.5–1 1
0 0 0 Ser81→Phe Ser81→Tyr 0 Glu85→Gln Ser81→Phe Ser81→Phe Ser81→Phe Ser81→Phe Ser81→Phe Val101→Leu Ser81→Phe Phe81→Leu Ser81→Phe Ser81→Phe Ser81→Phe 0 0 0 0 Glu85→Gly Ser81→Tyr Ser81→Phe Ser81→Tyr Ser81→Ala Ser81→Ala Ala81→Val
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Ser79→Phe Ser79→Phe 0 Ser79→Tyr Ser79→Tyr 0 0 0 Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe Ser79→Phe Asp83→Asn Asp83→Asn Asp83→Asn
0 0 0 0 0 Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val Ile460→Val
LVX = levofloxacin; MOX = moxifloxacin. a Clinical wild-type isolate kindly provided by Ron Jones, MD, JMI Laboratories, North Liberty, IA. b First-step parC mutant from TRUST study, Johnson & Johnson Pharmaceutical Research, Raritan, NJ. c First-step gyrA mutant created by exposing the wild-type isolate to moxifloxacin at 4× MIC.
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number of viable bacteria in the inoculum. The inocula contained between 7.2 × 107 and 6.7 × 109 colony-forming units (CFU)/mL (average = 1.4 × 109 CFU/mL). Two hundred microliters of the inoculum (0.2 mL) were applied to the surface of each of the agar dilution plates, 5 plates per concentration, and spread evenly resulting in a total volume of 1 mL of inoculum exposed to the antibiotic. The plates were incubated at 36 °C with 5% to 10% CO2 and examined for growth at 24, 48, and 72 h. 2.5. Frequency of spontaneous single-step mutation After 72 h of incubation, the frequency of mutation values were calculated from the ratio of the number of CFU(s) on drug-containing plates to the number of CFU(s) on drug-free plates (Pestova et al., 2000). The frequency of mutation was determined using the highest multiple of the MIC that still allowed growth of a countable number of colonies, and all colonies growing at that drug concentration were considered to be mutants. To be consistent, we calculated the frequency of mutation from the same multiple of the MIC for both fluoroquinolones. Thus, if there were no colonies on the plates at the matching multiple of the MIC, the frequency of mutation value for the corresponding drug was expressed as less than the number of CFU(s) exposed. Two to 5 colonies randomly chosen from these plates were individually frozen at −80 °C for later susceptibility testing and DNA sequencing. 2.6. Susceptibility testing MIC testing was performed on parent (or preexposure) isolates and on 2 (4 for isolate 4) of the postexposure isolates that were randomly selected from the agar dilution plates and were viable after freeze. The microdilution broth method was performed using the BioTek Precision 2000 pipetting system (BioTek Instruments, Winooski, VT). CLSI guidelines (CLSI, 2000) were followed for all but 4 of the postexposure isolates. For strain 6, 1 of 2 postmoxifloxacin exposure isolates required incubation with 5% to 10% CO2 to enhance growth. Strain 5 was particularly troublesome; 1 of 2 postlevofloxacin exposure and 1 of 2 postmoxifloxacin exposure isolates would not grow in Mueller–Hinton broth containing 5% lysed horse blood, so MIC testing was performed in THBY in the presence of 5% to 10% CO2. S. pneumoniae ATCC 49619 was used to monitor quality control of CLSI methods and alternative methods. 2.7. Polymerase chain reaction and DNA sequencing A pure culture, arising from 1 colony, was used to inoculate 25 mL of THBY. After overnight incubation at 36 °C with 5% to 10% CO2, 3 mL of culture were separated into 2 microcentrifuge tubes and spun for 5 min at 20 598 × g at room temperature (Hermle Z233-M2, Germany) to pellet the cells. The supernatant was decanted, and the pellets were combined and washed with Tris–EDTA buffer (TE) followed by another centrifugation and decanting step. Cell
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lysis was achieved by adding 0.150 mL of TE, 0.025 mL of 10 mg/mL lysozyme in TE, and 0.030 mL of 2 mg/mL mutanolysin in TE to the pellet. Lysis took place during incubation for 30 min at 37 °C. DNA was extracted using MoBio UltraClean Microbial DNA Isolation Kit (MO BIO, Carlsbad, CA). The protocol was modified to extract sufficient quantities of DNA. Solution MD1 was added directly to the lysate and centrifuged to remove cell debris. The MoBio protocol was reinstituted at step 9, except that all centrifugation was at 20 598 × g. Gel electrophoresis was performed to determine if a sufficient quantity of DNA was extracted. Polymerase chain reaction (PCR) was performed using 1 μL of the DNA template in a 0.050-mL reaction volume. The QRDRs of gyrA, gyrB, parC, and parE were amplified under the following conditions: 1 denaturization cycle at 95 °C for 5 min; 30 cycles at 94 °C for 60 s, 53 °C for 60 s, 72 °C for 3 min for gyrA and parC; and 30 cycles at 92 °C for 60 s, 48 °C for 60 s, 72 °C for 2 min for gyrB and parE, each followed by 1 extension cycle at 72 °C for 10 min. A 382-bp fragment of gyrA was amplified using primers described by Pan and Fisher (1999), and a 458-bp fragment of gyrB was amplified using primers described by Munoz et al. (1995). The primers used to amplify both the 367-bp fragment of parC and the 291-bp fragment of parE were described by Pan and Fisher (1996). Gel electrophoresis was performed to determine if the desired product was amplified. PCR products were purified using a Qiaquick PCR purification kit (QIAGEN, Valencia, CA) and sequenced at the Advanced Genetic Analysis Center (St. Paul, MN). The nucleotide sequences of the parent strains were compared with published sequences for gyrA (GenBank accession no. AJ005815), gyrB (GenBank accession no. Z67740), parC, and parE (GenBank accession no. Z67739) using T-COFFEE (Notredame et al., 2000). The nucleotide sequences of the postexposure strains were compared with the sequences of the parent strains. 3. Results 3.1. Frequency of mutation Frequency of mutation values are reported for isolate 1 (wild type), isolate 2 (parC mutant), and isolate 4 (gyrA mutant). Frequency of mutation values for the other isolates studied was determined but not reported because the results were not interpretable. At 4 times the MIC, the frequency of mutation from wild-type to 1st-step parC mutant after exposure to levofloxacin was 1.5 × 10−7. The frequency of mutation from wild-type to 1st-step gyrA mutant after moxifloxacin exposure was 2.6 × 10−8. The mutation from a 1st-step parC to 2nd-step parC–gyrA occurred more frequently with moxifloxacin than with levofloxacin, 4.0 × 10−7 and b2.0 × 10−8, respectively. At 8 times the MIC, the frequency of mutation from a 1st-step gyrA mutant to a 2nd-step gyrA–parC mutant was 3.3 × 10−7 for levofloxacin and 3.0 × 10−8 for moxifloxacin (Table 2).
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Table 2 Frequencies of levofloxacin- and moxifloxacin-selected mutations in the wild-type isolate and the parC and gyrA 1st-step mutants Strain
1a 2b 4c
Levofloxacin
Moxifloxacin
Multiple of MIC
Mutation frequency
Multiple of MIC
Mutation frequency
4× 4× 8×
1.5 × 10−7 b2.0 × 10−8 3.3 × 10−7
4× 4× 8×
2.6 × 10−8 4.0 × 10−7 3.0 × 10−8
2nd-step gyrA–parC mutant strains, plus/minus an additional parE mutation, showed no change in the parC or parE amino acid sequence between pre- (parent) and postexposure isolates. However, there were some changes in gyrA, either in the nucleotide sequence, resulting in a change in the amino acid at position 81, or an additional gyrA mutation at position 101. Also, the gyrA–parC mutant developed a gyrB mutation after exposure to levofloxacin.
a
Clinical wild-type isolate kindly provided by Ron Jones, MD, JMI Laboratories. b First-step parC mutant from TRUST study, Johnson & Johnson Pharmaceutical Research. c First-step gyrA mutant created by exposing the wild-type isolate to moxifloxacin at 4× MIC.
3.2. Susceptibility testing The 1st-step parC mutant, which was still sensitive to levofloxacin and moxifloxacin, developed a secondary gyrA mutation when exposed to either fluoroquinolones, and the MIC increased 4- to 8-fold, resulting in postexposure isolates that were no longer sensitive to levofloxacin or moxifloxacin. Similarly, the 1st-step gyrA mutant that was sensitive to levofloxacin and moxifloxacin developed a secondary parC mutation after exposure to either fluoroquinolones. The MICs increased 8- to 16-fold, resulting in resistance to levofloxacin and moxifloxacin. The parC–parE mutant developed a gyrA mutation resulting in a 4- to 8-fold increase of the levofloxacin MIC and subsequent resistance. The 8- to 16-fold increase of the moxifloxacin MIC resulted in 3 of 4 isolates becoming nonsusceptible and 1 isolate remaining sensitive. The pre- (parent) and postexposure MIC results are listed in Table 1. 3.3. PCR and DNA sequencing A sufficient quantity of DNA was extracted, and sequencing of the product obtained via PCR, using the primers and conditions listed above, confirmed that the desired gene fragments were amplified. Sequence comparisons revealed the mutations listed in Table 1. In some cases, postexposure isolates chosen from the same agar dilution plate had different amino acid substitutions, and in those cases, both mutations are listed. After exposing the wild-type isolate to levofloxacin, only 1 of 2 isolates chosen for postexposure testing had developed a 1st-step parC mutation, whereas all 4 isolates exposed to moxifloxacin and chosen for postexposure testing developed a 1st-step gyrA mutation. When this 1st-step gyrA mutant was further exposed to either levofloxacin or moxifloxacin, a parC mutation developed. The isolate with a parE mutation only did not develop additional topoisomerase mutations after exposure to levofloxacin or moxifloxacin. The parent strains with a 1st-step parC mutation, plus/minus an additional parE mutation, all developed a mutation in gyrA when under pressure from either levofloxacin or moxifloxacin. The
4. Discussion The frequency of mutation calculation assumes that all colonies present on plates containing inhibitory concentrations of drug are mutants (Pan and Fisher, 1998; Pestova et al., 2000). Pan et al. states that “mutant frequencies were determined by comparing the number of colonies that grew on plates containing drug with the number colonies obtained in the absence of drug”. In our study, some of the postexposure isolates randomly chosen for MIC testing and DNA sequencing revealed only slight (2- to 4-fold) MIC increases, remaining susceptible, and additional QRDR mutations were not detected. These isolates may have mutations outside the QRDR or may have initiated active efflux allowing them to grow in the presence of inhibitory concentrations of drug. It is also possible that some of the parent isolates survived and subsequently grew as the drug concentration diminished with time (i.e., breakthrough growth). This causes speculation regarding the “all-inclusive” nature of frequency of mutation calculations. Opinions differ regarding the role of the prevalent parE Ile460Val amino acid substitution in regard to fluoroquinolone resistance (Kawamura-Sato et al., 2005; Pestova et al., 2000). This mutation was present in 3 of the isolates we studied. Notably, the isolate that possessed the Ile460Val parE mutation only and, as such, may be considered a wild type by some did not develop any further mutations when exposed to selective concentrations of levofloxacin or moxifloxacin. These results suggest that 1st-step Ile460Val parE mutants may not be able to develop a secondary mutation in gyrA or parC. Additional studies need to be done to elucidate the bona fide role of the Ile460Val parE mutation. We questioned whether the frequency of the 2nd mutation would differ depending on the fluoroquinolone used as the selecting agent. Our results indicated that moxifloxacin selected a 2nd-step mutant from a 1st-step parC mutant more frequently than levofloxacin, similar to observations by Wickman et al. (2002). Conversely, levofloxacin selected a 2nd-step mutant from a 1st-step gyrA mutant more frequently than moxifloxacin. In conclusion, our data suggest that the occurrence of a 2nd mutation will be contingent on the 1st mutation and the preferential binding site of the fluoroquinolone that drives the transformation from 1st- to 2nd-step mutant. If a binding site has been altered, the fluoroquinolone that prefers the
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altered site is less likely than a fluoroquinolone that targets a remote site to cause a 2nd mutation, and the frequency of the mutation is less. Acknowledgments The authors thank Ortho-McNeil Pharmaceuticals for providing the funding for this project. References Canton R, Morosini M, Enright MC, Morrissey I (2003) Worldwide incidence, molecular epidemiology and mutations implicated in fluoroquinolone-resistant Streptococcus pneumoniae: data from the PROTEKT surveillance programme. J Antimicrob Chemother 52:944−952. Clinical and Laboratory Standards Institute (2000) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—fifth edition. NCCLS Document M7-A5. Wayne (PA): CLSI. Clinical and Laboratory Standards Institute (2005) Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement. M100-S15. Wayne (PA): CLSI. Davies TA, Yee YC, Goldschmidt R, Bush K, Sahm DF, Evangelista A (2006) Infrequent occurrence of single mutations in topoisomerase IV and DNA gyrase genes among US levofloxacin-susceptible clinical isolates of Streptococcus pneumoniae from nine institutions (1999– 2003). J Antimicrob Chemother 57:437−442. Jorgensen JH, Weigel LM, Ferraro MJ, Swenson JM, Tenover FC (1999) Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates including those with mutations in the gyrA, parC, and parE loci. Antimicrob Agents Chemother 43:329−334.
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