a Departments of Pathology and Laboratory Medicine, and b Epidemiology, Albany Medical Center, Albany, NY 12208, USA
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Abstract |
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Introduction |
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ß-Lactam antibiotics that can cause an alteration in peptidoglycan synthesis can influence the expression of methicillin resistance in Staphylococcus spp. Methicillin inhibits bacteria by binding to penicillin-binding proteins (PBPs) in the cell wall and resistance occurs through the substitution of PBP 2A for PBP 2.5,6 This change is enabled by the acquisition of the foreign mec locus, which encodes PBP 2A.7 PBP 2A can take over cell wall building functions when the other PBPs are inhibited by ß-lactams.8 The mec(A) gene and its product PBP 2A are not solely responsible for resistance, and the expression of methicillin resistance is often heterogeneous in that the subpopulations of one strain will express resistance at different levels. This heterogeneity, which differs depending on the strain, appears to be associated with the interaction of mec genes with other regulatory chromosomal genes that appear to affect the level of methicillin resistance.5,912 However, the level of methicillin resistance does not correlate to the amount of PBP 2A present as a cell wall component.6,13 Accordingly, the mechanisms for regulation and expression of methicillin heteroresistance in S. aureus are complex.
The association of fluoroquinolone use and fluoroquinolone-resistant S. aureus has been widely reported.14,15 Fluoroquinolone resistance in S. aureus results primarily from mutations in genes for DNA gyrase or DNA topoisomerase IV, or regulation of active efflux pumps.16,17 Although this is different from the mechanisms controlling methicillin resistance, some analyses have indicated an association between methicillin resistance in Staphylococcus spp. and the use of ciprofloxacin.14,15,18 Others have reported the appearance of heterotropic resistances or other metabolic changes following exposure to ciprofloxacin.19 Quinolones also have the ability to affect mutational rates and error-prone SOS response in bacteria20 and therefore may genetically alter bacterial responses to selective pressures.
Over the past several years, there has been an increase in the use of fluoroquinolones in both the community and hospitals. During this time, we have also observed an increase in the incidence of MRSA.18 These data indicate that an association between fluoroquinolone use and clinically significant methicillin resistance exists. This in vitro study investigates the effect of fluoroquinolones on the expression of methicillin resistance in S. aureus.
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Materials and methods |
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S. aureus was isolated from clinical specimens submitted for routine culture to the Clinical Microbiology Laboratories at Albany Medical Center, Albany, NY, USA. S. aureus ATCC 29213 was used as an oxacillin- and fluoroquinolone-susceptible and mec(A)-negative strain.
Susceptibility tests
To determine antibiotic activity, standard disc diffusion testing was performed on all isolates.21 Quantitative activities of oxacillin and ciprofloxacin were determined either by Etest (AB Biodisk, North America, Inc., Piscataway, NJ, USA) according to the manufacturer's instructions, or by microdilution assay with 2% NaCl to enhance methicillin resistance.21 To screen for methicillin resistance, S. aureus at a concentration of c. 108 cfu/mL was incubated at 30°C for 24 h on mannitol-salt agar (BBL, Becton Dickinson Cockeysville, MD, USA) with a 1 µg oxacillin susceptibility disc. Four clinical isolates of S. aureus (A, B, C and D) were chosen for experimentation because they were susceptible to fluoroquinolones and exhibited a double zone of inhibition around the oxacillin disc, which indicates heteroresistance to oxacillin. Two fluoroquinolone- and methicillin-susceptible isolates (ATCC 29213 and clinical strain E) were not observed to exhibit a double zone of inhibition with oxacillin. These served as control strains. Ciprofloxacin (Bayer Inc., West Haven, CT, USA), levofloxacin (Ortho-McNeil, Inc., Raritan, NJ, USA), gatifloxacin (Bristol-Myers Squibb Co., Wallingford, CT, USA) and moxifloxacin (Bayer) were obtained from their respective manufacturers.
Population analysis profiling
The isolates were grown to 109 cfu/mL in tryptic soy (TS) broth (BBL) for 20 h at 37°C while shaking at 145 rpm. The TS broth contained either no antibiotic (control) or 0.5 x MIC of the fluoroquinolone for each isolate. Cultures were then plated onto TS agar with 2% NaCl, and TS agar with 2% NaCl and oxacillin 128 mg/L. Colonies were counted after 24 and 48 h at 37°C, respectively. Population analysis profiling was used to determine the resistance index, which was defined as the proportion of cfu per millilitre expressing resistance to oxacillin. This was expressed as the ratio of the number of colonies growing on agar with oxacillin to the number of colonies on antibiotic-free agar (control). For two heteroresistant strains (A and B) the assays were repeated and cultures plated onto TS agar with oxacillin ranging from 0.5 to 128 mg/L for strain A, and from 16 to 128 mg/L for strain B. Strain E served as the methicillin-susceptible control.
Genetic fingerprinting and detection of the mec(A) gene
Agarose plugs containing total cellular DNAs from isolates of S. aureus were prepared using the GenePath Group 1 Reagent kit (Bio-Rad, Hercules, CA, USA). DNAs were restricted with SmaI and subjected to pulsed-field gel electrophoresis (PFGE) in the GenePath System according to the manufacturer's instructions. S. aureus strain NCTC 8325 was included on the gel as a standard. The resulting patterns of restriction fragments (fingerprints) were compared for evidence of genetic similarity using the Bio-Rad Molecular Analyst software. The fingerprints were then transferred to a nylon Southern blot by a standard protocol, modified by extending the depurination treatment to 40 min to enhance the transfer of the large DNA fragments.22 mec(A) sequences on the blot were detected with a digoxigenin-labelled 1.3 kb PstI probe fragment from pGO158.23 Labelling, hybridization and detection of the probe were carried out with the Non-Radioactive Labeling and Detection kit [Boehringer-Mannheim (Roche), Indianapolis, IN, USA].
Growth curve
The effect of 0.5 x MIC of ciprofloxacin on bacterial growth for strain B was determined by standard broth growth curve. Briefly, 105 cfu/mL were inoculated into 50 mL of TS broth with and without ciprofloxacin 0.19 mg/L (0.5 x MIC). The cultures were incubated at 37°C for 20 h while shaking at 145 rpm. Aliquots were removed at various time points for plating onto TS agar with 2% NaCl, and TS agar with 2% NaCl plus oxacillin 128 mg/L. The number of cfu/mL was determined after 24 and 48 h at 37°C, respectively. The resistance index was calculated for each time point.
Doseresponse
To determine the effect of various concentrations of ciprofloxacin on the enhancement of oxacillin resistance, c. 5 x 105 cfu/mL was inoculated into 5 mL of TS broth in 17 x 100 mm tubes with doubling dilutions of ciprofloxacin, and one broth tube without the antibiotic. After incubation at 37°C for 20 h while shaking at 145 rpm, aliquots were plated on to TS agar with 2% NaCl, and TS agar with 2% NaCl plus oxacillin 128 mg/L. The number of cfu/mL were determined after 24 and 48 h at 37°C, respectively, and the resistance index was calculated.
Fluctuation assay
Approximately 105 cfu/mL of strain B from an overnight blood agar plate (BBL) was inoculated into fifty 17 x 100 mm tubes containing 5 mL TS broth each. Twenty-five tubes contained ciprofloxacin 0.19 mg/L (0.5 x MIC of ciprofloxacin for strain B), and 25 served as controls without added ciprofloxacin. The inoculated tubes were incubated for 20 h at 37°C while shaking at 145 rpm. Serial dilutions to determine cfu/mL were plated on TS agar with 2% NaCl and oxacillin 128 mg/L. Five tubes from each group were selected for plating onto TS agar without oxacillin to determine total cfu/mL following the incubation period. The mutation rate was calculated as described previously.19
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Results |
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The ability of heteroresistant MRSA to express subpopulations with enhanced resistance to both oxacillin and fluoroquinolones was examined, selecting colonies that grew on agar at or above their respective MICs. Colonies of strain B that grew on agar with oxacillin 128 mg/L were transferred to media containing ciprofloxacin. Of these, 100, 63 and 6% grew when transferred to agar with 0.38, 0.76 and 1.52 mg/L of ciprofloxacin, respectively (1 x, 2 x and 4 x MIC, respectively). None was detected without first being selected with oxacillin 128 mg/L. Similarly, colonies of strain B that were selected for growth on agar media containing 0.38, 0.76 and 1.52 mg/L of ciprofloxacin could be transferred to agar with oxacillin 128 mg/L at a rate of 80, 50 and 55%, respectively. Again, no growth on the oxacillin medium was detected unless strain B was first exposed to at least the MIC of ciprofloxacin. Colonies were also selected from media with oxacillin 128 mg/L and their MICs to the four fluoroquinolones were 1.5- to 3-fold higher as determined by Etest. The increase in MIC for all the fluoroquinolones occurred following exposure to oxacillin regardless of previous exposure to the fluoroquinolone. The higher resistance was also stable after five consecutive days of passage onto media without oxacillin or the fluoroquinolone.
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Discussion |
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This is similar to other reports in that the higher resistance of MRSA to fluoroquinolones is not the result of increased mutation rates. S. aureus strains that were highly susceptible to methicillin had the same mutation frequencies as MRSA.24 Our study cannot rule out completely the possibility that the fluoroquinolone may select for other types of mutations that lead to oxacillin resistance without directly mutating the oxacillin resistance genes. However, a relationship is indicated by our study, since the proportion of heteroresistant strains expressing methicillin resistance increased following exposure to fluoroquinolones, as did fluoroquinolone resistance following exposure to oxacillin. The apparent association between methicillin and fluoroquinolone resistance points to the high level of complexity of antimicrobial resistance in MRSA strains. The mechanism of interaction of fluoroquinolone exposure with methicillin resistance is unknown; however, a number of possibilities exist. Regulatory genes such as mec(I), which functions as a regulatory repressor of the mec(A) gene, could be effected by fluoroquinolone. Basal levels of mec(A) gene transcription were elevated in highly resistant MRSA that lacked or had a point mutation in the mec(I) gene.25 Fluoroquinolones might be repressing the mec(I) gene and, therefore, upregulation of the mec(A) gene is possible. The results of the fluctuation assay indicated that mutations were not occurring or, if they were, it was at a high rate. The strains grown in the presence of fluoroquinolone had approximately the same number of oxacillin-resistant colonies as those not exposed to fluoroquinolones. The representative colonies selected on oxacillin were also more resistant to inhibition by the fluoroquinolones and the resistance was across the class of fluoroquinolones used in this study. These data indicate that mec(A)-positive, heteroresistant S. aureus contains subpopulations that are resistant to oxacillin and fluoroquinolones. Selection by either antibiotic class enriches the overall population for resistance to both.
Chromosomal maps show the location of mec(A) between protein A and DNA gyrase genes.7 Since it is rare to have resistance to fluoroquinolones in methicillinsusceptible strains,17 the effect of fluoroquinolones on the selection of resistant gyrases could coincidentally influence mec(A)-mediated oxacillin resistance. This would be consistent with our observations that selection of resistant subpopulations by either fluoroquinolone or oxacillin enhances the detection of resistance for the other antimicrobial agent in mec(A)-bearing strains.
Other heterotropic associations have been reported in mec(A)-positive S. aureus. Expression of fibronectin-binding proteins was enhanced by fluoroquinolone-resistant S. aureus exposed to subinhibitory levels of ciprofloxacin.26 The susceptibilities of other structurally unrelated antibiotics were affected in MRSA by sub-MIC levels of ciprofloxacin.17 The isolation of multiply resistant variants for imipenem, fusidic acid and gentamicin increased >100-fold following exposure of MRSA to ciprofloxacin. Strain differences in the expression of resistant variants were also noted in their study. In our study, differences in the effect of the various fluoroquinolones were observed between the MRSA strains tested. Ciprofloxacin, which has the least activity against S. aureus, had the greatest effect. This effect diminished as the fluoroquinolones increased in Grampositive activity. We also noted that the resistance indices differed for the four mec(A) strains after fluoroquinolone exposure, although before exposure the resistance indices were not significantly different.
Fluoroquinolones cause an extended lag phase in MRSA that is due to the inhibition of DNA replication. As opposed to ß-lactams, which inhibit S. aureus growth for 13 h, the sub-MIC of fluoroquinolones can inhibit growth for up to 7 h and bactericidal activity can occur rapidly in 46 h.27,28 This timing is consistent with the extended 8 h lag phase in the growth of our strains in the presence of 0.5 x MIC of fluoroquinolones. During this period the more fluoroquinolone-susceptible populations would be inhibited or killed while the fluoroquinolone-resistant populations would continue to grow. The coincidence of death and growth would appear as the extended lag phase that we observed. If fluoroquinolone resistance is preferentially associated with the more oxacillin-resistant subpopulations, growth in the presence of subinhibitory levels of a fluoroquinolone would selectively enrich for the more oxacillin-resistant subpopulations. This explanation would be consistent with our observations. Therefore, the effect of the fluoroquinolone may be purely metabolic, as changes in pH, osmolarity and temperature that affect the growth rate of S. aureus enhance the detection of MRSA subpopulations. The control of the switch from heterologous to homologous expression of mec(A) is unknown29 but regulation of mec(A) transcription is correlated with expression of methicillin resistance.30 Fluoroquinolones, by delaying DNA replication, may be affecting transcription and therefore repressor protein production. The consequence of this may also cause an increase in higher level oxacillin resistance by decreased repressor levels.
An increase in MRSA carriage rate can be partly the result of the selective pressures of antimicrobial use.31,32 Other indirect factors such as antimicrobial agents that decrease bacterial competition, which allows increased colonization of MRSA in the host,33 and transmission of MRSA, may also be contributing to the increased incidence. It appears that S. aureus strains possessing mec(A) are capable of expressing heterotropic resistance to both methicillin and fluoroquinolones, which would be a selective advantage in an environment of widespread fluoroquinolone use. This study indicates that the use of fluoroquinolones with low potency for S. aureus may lead to an increased risk of colonization in individuals with high-level oxacillin-resistant strains. This is supported by the fact that countries which rarely use fluoroquinolones have a low incidence of MRSA.34 This study shows that the selection of high-level oxacillin resistance in heteroresistant S. aureus appears to be associated with fluoroquinolone exposure.
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Acknowledgements |
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Notes |
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References |
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Received 15 December 2000; returned 28 March 2001; revised 25 April 2001; accepted 21 May 2001