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Loss of erythromycin resistance genes from strains of Streptococcus pyogenes that have developed resistance to levofloxacin
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Diagnostic Microbiology and Infectious Disease 64 (2009) 225 – 228 www.elsevier.com/locate/diagmicrobio
Loss of erythromycin resistance genes from strains of Streptococcus pyogenes that have developed resistance to levofloxacin☆ Dewan Sakhawat Billala , Muneki Hotomia , Steve S. Yanb , Daniel P. Fedorkoc , Jun Shimadaa , Keiji Fujiharaa , Noboru Yamanakaa,⁎ a Department of Otolaryngology, Wakayama Medical University, Wakayama 641-8509, Japan Food and Drug Administration, Department of Health and Human Services, Rockville, MD 20855, USA c Department of Health and Human Services, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA Received 10 June 2008; accepted 30 January 2009 b
Abstract In the past 2 to 3 decades, erythromycin resistance in Streptococcus pyogenes has been decreasing, whereas fluoroquinolone resistance (or reduction in its susceptibility) has been reported often. Although a shift of M-type prevalence and decreased pressure from macrolides have been suggested for the decrease in erythromycin resistance, we hypothesized that this might also be a result of increased antimicrobial pressure from fluoroquinolone use. Levofloxacin resistance for 4 erythromycin-resistant parent strains was induced in vitro. Their mutants became highly resistant to the fluoroquinolones but lost their erythromycin resistance trait. Erythromycin resistance was fully restored by transconjugation with respective parent strains with either mefA- or ermTR-mediated mechanisms. © 2009 Published by Elsevier Inc. Keywords: Streptococcus pyogenes; Fluoroquinolone resistance; Erythromycin resistance; Transconjugation
Reports from around the world demonstrate a steady decrease of erythromycin resistance in isolates of Streptococcus pyogenes. Resistance has decreased in Japan from 20% to 50% in 1989 to less than 10% in 1994, in Korea from 25.7% in 2003 to 4.8% in 2007, in Taiwan from 46% in 1999 to 17% in 2003, in Italy from a peak of 53% in 1997 to 20% in 2004, and in Spain from 53.6% in 2002 to 3.7% in 2004 (Fujita et al., 1994; Hsueh et al., 2006; Montagnani et al., 2006; Oliver et al., 2007; Uh et al., 2007). The authors of those studies concluded that this decrease in resistance was either due to an associated decrease in use of erythromycin or a shift in the predominant M/emm genotype(s) in circulation during the study period. However, Fujita et al. (1994) reported a decrease in the prevalence of M type 12 with a concomitant decrease in the erythromycin resistance rate ☆
Dr Dewan S. Billal is supported by Japan Society for Promotion of Science (Chiyoda, Tokyo, Japan). The opinions and information in this article are those of the authors and do not represent the views and/or policies of author Yan's affiliation. ⁎ Corresponding author. Tel.: +1-81-73-441-0651; fax: +1-81-73-4463846. E-mail address: [email protected] (N. Yamanaka). 0732-8893/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.diagmicrobio.2009.01.034
among M type 12 isolates, suggesting that other mechanisms may contribute to the decline in macrolide resistance in S. pyogenes. None of these studies report resistance rates of S. pyogenes to fluoroquinolone antibiotics. A reduction in fluoroquinolone susceptibility in S. pyogenes has been recently described with up to 10% of S. pyogenes isolates either resistant or having reduced susceptibility to fluoroquinolones (Ikebe et al., 2005; Orscheln et al., 2005). We observed the loss of erythromycin resistance in some strains of S. pyogenes after these organisms were induced to become resistant to fluoroquinolones (Billal et al., 2007). It has been demonstrated that fluoroquinolones can induce partial or total loss of a pathogenicity island in vitro in uropathogenic Escherichia coli (Soto et al., 2006). In response to acquiring resistance to vancomycin, strains of Staphylococcus aureus have been reported to lose the mecA gene and become more susceptible to methicillin or lose their β-lactamase plasmid with a corresponding large deletion in the methicillin resistance cassette SCCmec to become susceptible to β-lactam antibiotics (Adhikari et al., 2004; Reipert et al., 2003). Therefore, we hypothesized that a similar mechanism may explain the loss of erythromycin
226
D.S. Billal et al. / Diagnostic Microbiology and Infectious Disease 64 (2009) 225–228
resistance genes in the mutants of S. pyogenes. To our knowledge, there is no study regarding a concomitant reversal in susceptibility to macrolide and fluoroquinolone antimicrobial drugs. In this study, we report our investigation of fluoroquinolone selection of fluoroquinolone-resistant mutants with the concomitant loss of macrolide resistance genes from macrolide-resistant S. pyogenes parent stains in vitro. Although published data clearly show a correlation between macrolide use and/or M/emm type shifts with changes in macrolide-resistant S. pyogenes in a population, we hypothesized that the decline of macrolide resistance in S. pyogenes over the past decades could also be affected by the reversal use of macrolide and fluoroquinolone antimicrobials in the treatment of infections caused by S. pyogenes, and the fluoroquinolone induced loss of erythromycin resistance genes in S. pyogenes through unknown mechanisms. To demonstrate the hypothesis, we subjected 4 macrolide-resistant S. pyogenes isolates (2 from rhinosinusitis, 1 each from tonsillitis and pharyngitis) to in vitro selection using levofloxacin, and the genotypes and susceptibilities to levofloxacin and erythromycin of the mutants were determined. emm typing of S. pyogenes strains was performed by DNA sequencing according to the recommendation of the Division of Bacterial and Mycotic Diseases, Centers for Diseases Control and Prevention (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). In vitro fluoroquinolone resistance selection of S. pyogenes mutants was performed as described previously (Billal et al., 2007). Mutational alterations in the quinolone resistant determining regions (QRDRs) of DNA gyrase A (gyrA) and topoisomerase IV (parC) of both parent and fluoroquinolone-resistant mutants were confirmed by DNA sequence analysis as described previously (Billal et al., 2007). Transconjugation of individual mutants with corresponding parent strains was performed as described by Taylor et al. (1981). Briefly, for
filter mating methods, respective donor (parent) and recipient (mutant) cells were grown separately from a single colony in Todd–Hewitt broth supplemented with 0.5% yeast extract to a density of approximately 1 × 109 CFU/mL. The donor culture (0.5 mL) and recipient culture (1.0 mL) are mixed and collected on a nitrocellulose filter (pore size, 0.22 μm; Millipore (Billerica, MA, USA).) through gentle filtration. The filter was then placed on blood agar (BA) plates containing no antibiotics and incubated in a 5% CO2 incubator at 37 °C for 18 to 24 h. The filter was removed from the plate and placed in a sterile Petri dish. One milliliter of phosphate buffer was added onto the filter, and the cells were removed from the filter with gentle shaking. Transconjugants were selected on BA containing both levofloxacin (equivalent to 8× MIC of respective donor organism) and erythromycin (equivalent to 8× MIC of recipient organisms) at appropriate concentrations, respectively. Positive controls for the transconjugation system included all 4-parent strains used as donors and 3 different erythromycin-susceptible stored isolates as recipients. Each parent strain was transconjugated with each susceptible isolate in separate reactions. Transconjugation between paired erythromycin-susceptible and mutant strains served as the negative control. Macrolide resistance genes (ermTR and mefA) in parent, mutant, and transconjugant strains were determined by polymerase chain reaction methods described by Weber et al. (2001). MICs of all parent, mutant, and transconjugant strains to erythromycin and levofloxacin were determined by a broth microdilution method with proper quality control measurements according to recommendations by the CLSI (2006). The parent strains, mutant strains, and transconjugants were confirmed to be the same isolates by emm typing and T typing. Changes in susceptibility to levofloxacin and erythromycin as well as corresponding genotypes are shown in Table 1. All parent strains were resistant to erythromycin but
Table 1 Relationship between susceptibility to levofloxacin and erythromycin resistance genes Parent/ mutant strains
emm type
Genotype parent/ mutant
Parent-19 Mutant19-1 Mutant19-3 Mutant19-4 Parent-30 Mutant30-1 Mutant30-2 Parent-56 Mutant56-1 Mutant56-2 Mutant56-3 Parent-46 Mutant46-1 Mutant46-2 Mutant46-3
75
mefA+/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR−
12
12
12
MIC (mg/L) Erythromycin
Levofloxacin
16 0.13 0.13 0.13 N128 0.13 0.13 N128 0.13 0.13 0.13 N128 0.13 0.13 0.13
0.5 32 64 128 2 64 128 2 32 64 128 1 32 64 128
MIC (mg/L), erythromycin transconjugants
Genotype of transconjugant
N/A 16 16 16 N/A N128 N128 N/A N128 N128 N128 N/A N128 N128 N128
NA mefA+/ermB−/ermTR− mefA+/ermB−/ermTR− mefA+/ermB−/ermTR− NA mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR+ NA mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR+ NA mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR+
D.S. Billal et al. / Diagnostic Microbiology and Infectious Disease 64 (2009) 225–228
susceptible to levofloxacin. Within each of the corresponding parent-mutant sets after exposure to levofloxacin, the MIC of mutants to levofloxacin increased significantly after a few selection cycles, but all mutant strains lost their macrolide resistance genes after the first cycle of selection. All fluoroquinolone-resistant mutants were highly susceptible to erythromycin. All fluoroquinolone-resistant mutants carried point mutations in their QRDRs of gyrA and parC. Mutant strains in all sets developed a point mutation that resulted in a change from serine to tyrosine in residue 81 of gyrA. Mutants in strain 19 (emm type 75) developed a point mutation that resulted in a change from serine to alanine, and those in the other 3 strains (all emm type 12) developed a point mutation that resulted in a change from phenylalanine to valine in residue 79 of parC. There were no point mutations found in the QRDR of gyrB and parE among the mutants. Transconjugation of individual mutants with their corresponding parent strains restored the erythromycin resistance gene while retaining resistance to levofloxacin (Table 1). The frequencies of transconjugation of macrolide resistance genes were higher for mutants in strain 30 (9.2 × 10−2 to 1.81 × 10−2) than the other 3 strains (19, 46, and 56) (1 × 10−6 to 6.5 × 10−6). Strain 30 was positive for the macrolide resistance gene mefA, whereas the other 3 strains were positive for the inducible gene ermTR. Individual erythromycin resistance genes were lost in mutants, and transconjugation restored them (Table 1). Our results might indicate that macrolide-resistant S. pyogenes lost their macrolide resistance genes because of antibiotic selection pressure from levofloxacin. This observation is supported by S. pyogenes susceptibility data recently reported by Malhotra-Kumar et al. (2005a, 2005b). Their data indicate that 15% of fluoroquinolone-susceptible isolates were resistant to macrolides, but only 3.2% of fluoroquinolone-nonsusceptible isolates were macrolide resistant. It has been known for some time that fluoroquinolone antibiotics induce the SOS response in E. coli, increasing the mutation rate in these organisms (Phillips et al., 1987). It has also been observed that fluoroquinolone-resistant E. coli strains have less virulence factors than fluoroquinolone-susceptible strains (Vila et al., 2002). Soto et al. (2006) recently reported that fluoroquinolone pressure caused partial or total loss of pathogenicity islands in uropathogenic E. coli by SOSdependent and SOS-independent pathways. Exposure of E. coli to subinhibitory concentrations of ciprofloxacin induced the loss of the hly (hemolysin) and CNF-1 (cytotoxic necrotizing factor 1) genes. Concomitant loss of specific genes as a result of developing reduced susceptibility to fluoroquinolones has been observed in other bacteria. Pumbwe et al. (1996) reported that an isogenic posttherapy mutant of Pseudomonas aeruginosa was found to have complete loss of the outer membrane protein OprF. Exposure of E. coli strains to the fluoroquinolone trovafloxacin at subminimum inhibitory concentrations resulted in effects on the host cell's plasmid-borne functions that included changes in
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plasmid DNA, alteration in gene expression, and reduction in plasmid copy number (Brandi et al., 2000). Thus, possibly, acquisition of a new fluoroquinolone resistance trait may accompany a loss of a preexisting genetic trait because of adjustments in environmental fitness. Although our findings (i.e., loss of either mefA or ermTR genes) may be an isolated phenomenon, we hope this short report will prompt further exploration with additional clinical and wild-type isolates to determine the mechanisms behind the disparity of resistance to macrolides and fluoroquinolones among S. pyogenes. References Adhikari RP, Scales GC, Kobayashi K, Smith JM, Berger-Bachi B, Cook GM (2004) Vancomycin-induced deletion of the methicillin resistance gene mecA in Staphylococcus aureus. J Antimicrob Chemother 54: 360–363. Billal DS, Fedorko DP, Yan SS, Hotomi M, Fujihara K, Nelson N, Yamanaka N (2007) In vitro induction and selection of fluoroquinoloneresistant mutants of Streptococcus pyogenes strains with multiple emm types. J Antimicrob Chemother 59:28–34. Brandi L, Falconi M, Ripa S (2000) Plasmid curing effect of trovafloxacin. FEMS Microbiol Lett 184:297–302. Clinical and Laboratory Standards Institute (CLSI) (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, M7-A7. Wayne, PA: CLSI. Fujita K, Murono K, Yoshikawa M, Murai T (1994) Decline of erythromycin resistance of group A streptococci in Japan. Pediatr Infect Dis J 13: 1075–1078. Hsueh PR, Shyr JM, Wu JJ (2006) Changes in macrolide resistance among respiratory pathogens after decreased erythromycin consumption in Taiwan. Clin Microbiol Infect 12:296–298. Ikebe T, Hirasawa K, Suzuki R, Isobe J, Tanaka D, Katsukawa C, Kawahara R, Tomita M, Ogata K, Endoh M, Okuno R, Watanabe H (2005) Antimicrobial susceptibility survey of Streptococcus pyogenes isolated in Japan from patients with severe invasive group A streptococcal infections. Antimicrob Agents Chemother 49:788–790. Malhotra-Kumar S, Lammens C, Chapelle S, Mallentjer C, Weyler J, Goossens H (2005a) Clonal spread of fluoroquinolone non-susceptible Streptococcus pyogenes. J Antimicrob Chemother 55:320–325. Malhotra-Kumar S, Lammens C, Chapelle S, Wijdooghe M, Piessens J, Van Herck K, Goossens H (2005b) Macrolide- and telithromycin-resistant Streptococcus pyogenes, Belgium, 1999–2003. Emerg Infect Dis 11: 939–942. Montagnani F, Stolzuoli L, Zanchi A, Cresti S, Cellesi C (2006) Antimicrobial susceptibility of Streptococcus pyogenes and Streptococcus pneumoniae: surveillance from 1993 to 2004 in Central Italy. J Chemother 18:389–393. Oliver MA, Garcia-Delafuente C, Cano ME, Perez-Hernandez F, MartinezMartinez L, Alberti S (2007) Rapid decrease in the prevalence of macrolide-resistant group A streptococci due to the appearance of two epidemic clones in Cantabria (Spain). J Antimicrob Chemother 60: 450–452. Orscheln RC, Johnson DR, Olson SM, Presti RM, Martin JM, Kaplan EL, Storch GA (2005) Intrinsic reduced susceptibility of serotype 6 Streptococcus pyogenes to fluoroquinolone antibiotics. J Infect Dis 191: 1272–1279. Phillips I, Culebras E, Moreno F, Baquero F (1987) Induction of the SOS response by new 4-quinolones. J Antimicrob Chemother 20:631–638. Pumbwe L, Everett MJ, Hancock RE, Piddock LJ (1996) Role of gyrA mutation and loss of OprF in the multiple antibiotic resistance phenotype of Pseudomonas aeruginosa G49. FEMS Microbiol Lett 143:25–28.
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Reipert A, Ehlert K, Kast T, Bierbaum G (2003) Morphological and genetic differences in two isogenic Staphylococcus aureus strains with decreased susceptibilities to vancomycin. Antimicrob Agents Chemother 47:568–576. Soto SM, Jimenez de Anta MT, Vila J (2006) Quinolones induce partial or total loss of pathogenicity islands in uropathogenic Escherichia coli by SOS-dependent or -independent pathways, respectively. Antimicrob Agents Chemother 50:649–653. Taylor DE, De Grandis SA, Karmali MA, Fleming PC (1981) Transmissible plasmids from Campylobacter jejuni. Antimicrob Agents Chemother 19: 831–835.
Uh Y, Hwang GY, Jang IH, Cho HM, Noh SM, Kim HY, Kwon O, Yoon KJ (2007) Macrolide resistance trends in beta-hemolytic streptococci in a tertiary Korean hospital. Yonsei Med J 48:773–778. Vila J, Simon K, Ruiz J, Horcajada JP, Velasco M, Barranco M, Moreno A, Mensa J (2002) Are quinolone-resistant uropathogenic Escherichia coli less virulent? J Infect Dis 186:1039–1042. Weber P, Filipecki J, Bingen E, Fitoussi F, Goldfarb G, Chauvin JP, Reitz C, Portier H (2001) Genetic and phenotypic characterization of macrolide resistance in group A streptococci isolated from adults with pharyngo-tonsillitis in France. J Antimicrob Chemother 48: 291–294.
Diagnostic Microbiology and Infectious Disease 64 (2009) 225 – 228 www.elsevier.com/locate/diagmicrobio
Loss of erythromycin resistance genes from strains of Streptococcus pyogenes that have developed resistance to levofloxacin☆ Dewan Sakhawat Billala , Muneki Hotomia , Steve S. Yanb , Daniel P. Fedorkoc , Jun Shimadaa , Keiji Fujiharaa , Noboru Yamanakaa,⁎ a Department of Otolaryngology, Wakayama Medical University, Wakayama 641-8509, Japan Food and Drug Administration, Department of Health and Human Services, Rockville, MD 20855, USA c Department of Health and Human Services, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA Received 10 June 2008; accepted 30 January 2009 b
Abstract In the past 2 to 3 decades, erythromycin resistance in Streptococcus pyogenes has been decreasing, whereas fluoroquinolone resistance (or reduction in its susceptibility) has been reported often. Although a shift of M-type prevalence and decreased pressure from macrolides have been suggested for the decrease in erythromycin resistance, we hypothesized that this might also be a result of increased antimicrobial pressure from fluoroquinolone use. Levofloxacin resistance for 4 erythromycin-resistant parent strains was induced in vitro. Their mutants became highly resistant to the fluoroquinolones but lost their erythromycin resistance trait. Erythromycin resistance was fully restored by transconjugation with respective parent strains with either mefA- or ermTR-mediated mechanisms. © 2009 Published by Elsevier Inc. Keywords: Streptococcus pyogenes; Fluoroquinolone resistance; Erythromycin resistance; Transconjugation
Reports from around the world demonstrate a steady decrease of erythromycin resistance in isolates of Streptococcus pyogenes. Resistance has decreased in Japan from 20% to 50% in 1989 to less than 10% in 1994, in Korea from 25.7% in 2003 to 4.8% in 2007, in Taiwan from 46% in 1999 to 17% in 2003, in Italy from a peak of 53% in 1997 to 20% in 2004, and in Spain from 53.6% in 2002 to 3.7% in 2004 (Fujita et al., 1994; Hsueh et al., 2006; Montagnani et al., 2006; Oliver et al., 2007; Uh et al., 2007). The authors of those studies concluded that this decrease in resistance was either due to an associated decrease in use of erythromycin or a shift in the predominant M/emm genotype(s) in circulation during the study period. However, Fujita et al. (1994) reported a decrease in the prevalence of M type 12 with a concomitant decrease in the erythromycin resistance rate ☆
Dr Dewan S. Billal is supported by Japan Society for Promotion of Science (Chiyoda, Tokyo, Japan). The opinions and information in this article are those of the authors and do not represent the views and/or policies of author Yan's affiliation. ⁎ Corresponding author. Tel.: +1-81-73-441-0651; fax: +1-81-73-4463846. E-mail address: [email protected] (N. Yamanaka). 0732-8893/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.diagmicrobio.2009.01.034
among M type 12 isolates, suggesting that other mechanisms may contribute to the decline in macrolide resistance in S. pyogenes. None of these studies report resistance rates of S. pyogenes to fluoroquinolone antibiotics. A reduction in fluoroquinolone susceptibility in S. pyogenes has been recently described with up to 10% of S. pyogenes isolates either resistant or having reduced susceptibility to fluoroquinolones (Ikebe et al., 2005; Orscheln et al., 2005). We observed the loss of erythromycin resistance in some strains of S. pyogenes after these organisms were induced to become resistant to fluoroquinolones (Billal et al., 2007). It has been demonstrated that fluoroquinolones can induce partial or total loss of a pathogenicity island in vitro in uropathogenic Escherichia coli (Soto et al., 2006). In response to acquiring resistance to vancomycin, strains of Staphylococcus aureus have been reported to lose the mecA gene and become more susceptible to methicillin or lose their β-lactamase plasmid with a corresponding large deletion in the methicillin resistance cassette SCCmec to become susceptible to β-lactam antibiotics (Adhikari et al., 2004; Reipert et al., 2003). Therefore, we hypothesized that a similar mechanism may explain the loss of erythromycin
226
D.S. Billal et al. / Diagnostic Microbiology and Infectious Disease 64 (2009) 225–228
resistance genes in the mutants of S. pyogenes. To our knowledge, there is no study regarding a concomitant reversal in susceptibility to macrolide and fluoroquinolone antimicrobial drugs. In this study, we report our investigation of fluoroquinolone selection of fluoroquinolone-resistant mutants with the concomitant loss of macrolide resistance genes from macrolide-resistant S. pyogenes parent stains in vitro. Although published data clearly show a correlation between macrolide use and/or M/emm type shifts with changes in macrolide-resistant S. pyogenes in a population, we hypothesized that the decline of macrolide resistance in S. pyogenes over the past decades could also be affected by the reversal use of macrolide and fluoroquinolone antimicrobials in the treatment of infections caused by S. pyogenes, and the fluoroquinolone induced loss of erythromycin resistance genes in S. pyogenes through unknown mechanisms. To demonstrate the hypothesis, we subjected 4 macrolide-resistant S. pyogenes isolates (2 from rhinosinusitis, 1 each from tonsillitis and pharyngitis) to in vitro selection using levofloxacin, and the genotypes and susceptibilities to levofloxacin and erythromycin of the mutants were determined. emm typing of S. pyogenes strains was performed by DNA sequencing according to the recommendation of the Division of Bacterial and Mycotic Diseases, Centers for Diseases Control and Prevention (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). In vitro fluoroquinolone resistance selection of S. pyogenes mutants was performed as described previously (Billal et al., 2007). Mutational alterations in the quinolone resistant determining regions (QRDRs) of DNA gyrase A (gyrA) and topoisomerase IV (parC) of both parent and fluoroquinolone-resistant mutants were confirmed by DNA sequence analysis as described previously (Billal et al., 2007). Transconjugation of individual mutants with corresponding parent strains was performed as described by Taylor et al. (1981). Briefly, for
filter mating methods, respective donor (parent) and recipient (mutant) cells were grown separately from a single colony in Todd–Hewitt broth supplemented with 0.5% yeast extract to a density of approximately 1 × 109 CFU/mL. The donor culture (0.5 mL) and recipient culture (1.0 mL) are mixed and collected on a nitrocellulose filter (pore size, 0.22 μm; Millipore (Billerica, MA, USA).) through gentle filtration. The filter was then placed on blood agar (BA) plates containing no antibiotics and incubated in a 5% CO2 incubator at 37 °C for 18 to 24 h. The filter was removed from the plate and placed in a sterile Petri dish. One milliliter of phosphate buffer was added onto the filter, and the cells were removed from the filter with gentle shaking. Transconjugants were selected on BA containing both levofloxacin (equivalent to 8× MIC of respective donor organism) and erythromycin (equivalent to 8× MIC of recipient organisms) at appropriate concentrations, respectively. Positive controls for the transconjugation system included all 4-parent strains used as donors and 3 different erythromycin-susceptible stored isolates as recipients. Each parent strain was transconjugated with each susceptible isolate in separate reactions. Transconjugation between paired erythromycin-susceptible and mutant strains served as the negative control. Macrolide resistance genes (ermTR and mefA) in parent, mutant, and transconjugant strains were determined by polymerase chain reaction methods described by Weber et al. (2001). MICs of all parent, mutant, and transconjugant strains to erythromycin and levofloxacin were determined by a broth microdilution method with proper quality control measurements according to recommendations by the CLSI (2006). The parent strains, mutant strains, and transconjugants were confirmed to be the same isolates by emm typing and T typing. Changes in susceptibility to levofloxacin and erythromycin as well as corresponding genotypes are shown in Table 1. All parent strains were resistant to erythromycin but
Table 1 Relationship between susceptibility to levofloxacin and erythromycin resistance genes Parent/ mutant strains
emm type
Genotype parent/ mutant
Parent-19 Mutant19-1 Mutant19-3 Mutant19-4 Parent-30 Mutant30-1 Mutant30-2 Parent-56 Mutant56-1 Mutant56-2 Mutant56-3 Parent-46 Mutant46-1 Mutant46-2 Mutant46-3
75
mefA+/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR− mefA−/ermB−/ermTR− mefA−/ermB−/ermTR−
12
12
12
MIC (mg/L) Erythromycin
Levofloxacin
16 0.13 0.13 0.13 N128 0.13 0.13 N128 0.13 0.13 0.13 N128 0.13 0.13 0.13
0.5 32 64 128 2 64 128 2 32 64 128 1 32 64 128
MIC (mg/L), erythromycin transconjugants
Genotype of transconjugant
N/A 16 16 16 N/A N128 N128 N/A N128 N128 N128 N/A N128 N128 N128
NA mefA+/ermB−/ermTR− mefA+/ermB−/ermTR− mefA+/ermB−/ermTR− NA mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR+ NA mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR+ NA mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR+ mefA−/ermB−/ermTR+
D.S. Billal et al. / Diagnostic Microbiology and Infectious Disease 64 (2009) 225–228
susceptible to levofloxacin. Within each of the corresponding parent-mutant sets after exposure to levofloxacin, the MIC of mutants to levofloxacin increased significantly after a few selection cycles, but all mutant strains lost their macrolide resistance genes after the first cycle of selection. All fluoroquinolone-resistant mutants were highly susceptible to erythromycin. All fluoroquinolone-resistant mutants carried point mutations in their QRDRs of gyrA and parC. Mutant strains in all sets developed a point mutation that resulted in a change from serine to tyrosine in residue 81 of gyrA. Mutants in strain 19 (emm type 75) developed a point mutation that resulted in a change from serine to alanine, and those in the other 3 strains (all emm type 12) developed a point mutation that resulted in a change from phenylalanine to valine in residue 79 of parC. There were no point mutations found in the QRDR of gyrB and parE among the mutants. Transconjugation of individual mutants with their corresponding parent strains restored the erythromycin resistance gene while retaining resistance to levofloxacin (Table 1). The frequencies of transconjugation of macrolide resistance genes were higher for mutants in strain 30 (9.2 × 10−2 to 1.81 × 10−2) than the other 3 strains (19, 46, and 56) (1 × 10−6 to 6.5 × 10−6). Strain 30 was positive for the macrolide resistance gene mefA, whereas the other 3 strains were positive for the inducible gene ermTR. Individual erythromycin resistance genes were lost in mutants, and transconjugation restored them (Table 1). Our results might indicate that macrolide-resistant S. pyogenes lost their macrolide resistance genes because of antibiotic selection pressure from levofloxacin. This observation is supported by S. pyogenes susceptibility data recently reported by Malhotra-Kumar et al. (2005a, 2005b). Their data indicate that 15% of fluoroquinolone-susceptible isolates were resistant to macrolides, but only 3.2% of fluoroquinolone-nonsusceptible isolates were macrolide resistant. It has been known for some time that fluoroquinolone antibiotics induce the SOS response in E. coli, increasing the mutation rate in these organisms (Phillips et al., 1987). It has also been observed that fluoroquinolone-resistant E. coli strains have less virulence factors than fluoroquinolone-susceptible strains (Vila et al., 2002). Soto et al. (2006) recently reported that fluoroquinolone pressure caused partial or total loss of pathogenicity islands in uropathogenic E. coli by SOSdependent and SOS-independent pathways. Exposure of E. coli to subinhibitory concentrations of ciprofloxacin induced the loss of the hly (hemolysin) and CNF-1 (cytotoxic necrotizing factor 1) genes. Concomitant loss of specific genes as a result of developing reduced susceptibility to fluoroquinolones has been observed in other bacteria. Pumbwe et al. (1996) reported that an isogenic posttherapy mutant of Pseudomonas aeruginosa was found to have complete loss of the outer membrane protein OprF. Exposure of E. coli strains to the fluoroquinolone trovafloxacin at subminimum inhibitory concentrations resulted in effects on the host cell's plasmid-borne functions that included changes in
227
plasmid DNA, alteration in gene expression, and reduction in plasmid copy number (Brandi et al., 2000). Thus, possibly, acquisition of a new fluoroquinolone resistance trait may accompany a loss of a preexisting genetic trait because of adjustments in environmental fitness. Although our findings (i.e., loss of either mefA or ermTR genes) may be an isolated phenomenon, we hope this short report will prompt further exploration with additional clinical and wild-type isolates to determine the mechanisms behind the disparity of resistance to macrolides and fluoroquinolones among S. pyogenes. References Adhikari RP, Scales GC, Kobayashi K, Smith JM, Berger-Bachi B, Cook GM (2004) Vancomycin-induced deletion of the methicillin resistance gene mecA in Staphylococcus aureus. J Antimicrob Chemother 54: 360–363. Billal DS, Fedorko DP, Yan SS, Hotomi M, Fujihara K, Nelson N, Yamanaka N (2007) In vitro induction and selection of fluoroquinoloneresistant mutants of Streptococcus pyogenes strains with multiple emm types. J Antimicrob Chemother 59:28–34. Brandi L, Falconi M, Ripa S (2000) Plasmid curing effect of trovafloxacin. FEMS Microbiol Lett 184:297–302. Clinical and Laboratory Standards Institute (CLSI) (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, M7-A7. Wayne, PA: CLSI. Fujita K, Murono K, Yoshikawa M, Murai T (1994) Decline of erythromycin resistance of group A streptococci in Japan. Pediatr Infect Dis J 13: 1075–1078. Hsueh PR, Shyr JM, Wu JJ (2006) Changes in macrolide resistance among respiratory pathogens after decreased erythromycin consumption in Taiwan. Clin Microbiol Infect 12:296–298. Ikebe T, Hirasawa K, Suzuki R, Isobe J, Tanaka D, Katsukawa C, Kawahara R, Tomita M, Ogata K, Endoh M, Okuno R, Watanabe H (2005) Antimicrobial susceptibility survey of Streptococcus pyogenes isolated in Japan from patients with severe invasive group A streptococcal infections. Antimicrob Agents Chemother 49:788–790. Malhotra-Kumar S, Lammens C, Chapelle S, Mallentjer C, Weyler J, Goossens H (2005a) Clonal spread of fluoroquinolone non-susceptible Streptococcus pyogenes. J Antimicrob Chemother 55:320–325. Malhotra-Kumar S, Lammens C, Chapelle S, Wijdooghe M, Piessens J, Van Herck K, Goossens H (2005b) Macrolide- and telithromycin-resistant Streptococcus pyogenes, Belgium, 1999–2003. Emerg Infect Dis 11: 939–942. Montagnani F, Stolzuoli L, Zanchi A, Cresti S, Cellesi C (2006) Antimicrobial susceptibility of Streptococcus pyogenes and Streptococcus pneumoniae: surveillance from 1993 to 2004 in Central Italy. J Chemother 18:389–393. Oliver MA, Garcia-Delafuente C, Cano ME, Perez-Hernandez F, MartinezMartinez L, Alberti S (2007) Rapid decrease in the prevalence of macrolide-resistant group A streptococci due to the appearance of two epidemic clones in Cantabria (Spain). J Antimicrob Chemother 60: 450–452. Orscheln RC, Johnson DR, Olson SM, Presti RM, Martin JM, Kaplan EL, Storch GA (2005) Intrinsic reduced susceptibility of serotype 6 Streptococcus pyogenes to fluoroquinolone antibiotics. J Infect Dis 191: 1272–1279. Phillips I, Culebras E, Moreno F, Baquero F (1987) Induction of the SOS response by new 4-quinolones. J Antimicrob Chemother 20:631–638. Pumbwe L, Everett MJ, Hancock RE, Piddock LJ (1996) Role of gyrA mutation and loss of OprF in the multiple antibiotic resistance phenotype of Pseudomonas aeruginosa G49. FEMS Microbiol Lett 143:25–28.
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