Non-PmrA-mediated multidrug resistance in Streptococcus pneumoniae

Ekaterina Pestovaa,*, John J. Millichapa, Farida Siddiquib, Gary A. Noskina,b and Lance R. Petersona

a Northwestern Prevention Epicenter, Division of Microbiology, Department of Pathology and b Division of Infectious Diseases, Department of Medicine, Northwestern University Medical School and Northwestern Memorial Hospital, Chicago, IL, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The PmrA multidrug transporter protein gene was inactivated in Streptococcus pneumoniae strains CP1000 (wild-type) and EBR (mutant with enhanced active multidrug efflux). While the resistance to fluoroquinolones and ethidium bromide shown by EBR was reduced to the wild-type level, neither the susceptibility to reserpine in the presence of ethidium bromide and selected fluoroquinolones, nor the ability to produce ethidium bromide-resistant mutants was eliminated in the CP1000 pmrA mutant, indicating the presence of an additional multidrug export protein(s).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Streptococcus pneumoniae is the leading cause of serious community-acquired respiratory tract infection and meningitis. Although these infections have traditionally been treated with ß-lactam or macrolide antibiotics, emerging resistance to these agents has resulted in recommenda-tions that the newer fluoroquinolones be used for therapy.1 Fluoroquinolone resistance in S. pneumoniae is a developing problem, with investigators detecting mutations in DNA gyrase and topoisomerase IV genes in clinical pneumococcal isolates.2,3 In addition, antibiotic efflux by multidrug efflux systems is being recognized as an important first step in the development of bacterial resistance to many antimicrobial agents.4,5

Thus far, PmrA is the only multidrug transport protein known to be involved in the fluoroquinolone resistance of S. pneumoniae.6 The genomes of other bacterial species encode several distinct transporters with overlapping substrate specificities that are involved in fluoroquinolone resistance.4,5,7 The objective of this study was to detect non-PmrA multidrug transporters in S. pneumoniae by inactivating pmrA and testing the knockout mutants for continued efflux of fluoroquinolones, using an archived strain of S. pneumoniae (CP1000) and a CP1000 mutant with an enhanced active efflux phenotype (EBR).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial strains

The wild-type pneumococcal strain, CP1000, was isolated before the introduction of fluoroquinolones into clinical practice. EBR is a laboratory derivative of CP1000 that overexpresses an active efflux mechanism,8 and probably carries a mutation in a regulatory locus pmrA situated outside the promoter region, since the pmrA promoter sequence in EBR was found to be unchanged (data not shown).

Antimicrobial agent susceptibility testing and growth inhibition studies

MICs of norfloxacin (Merck and Co., Inc., West Point, PA, USA), ciprofloxacin (Bayer Corporation, West Haven, CT, USA), levofloxacin (Ortho-McNeil Pharmaceuticals, Raritan, NJ, USA), moxifloxacin (Bayer Corporation), sparfloxacin (Rhone-Poulenc Rorer R-D, Vitry-sur-Seine, France) and ethidium bromide against CP1000, CP1000-pmr::cat (JJK01), EBR and EBR-pmr::cat (JJK02) were determined in duplicate by serial two-fold antibiotic dilution in Todd–Hewitt broth (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% yeast extract (THBY broth).

Growth inhibition studies using reserpine together with the same antimicrobial agents were conducted to detect operation of a reserpine-susceptible efflux system using our previously described method.9 S. pneumoniae cultures were started at a density of 1 x 106 cells/mL in THBY broth containing the respective fluoroquinolones at 0.25 x MIC, with or without the addition of reserpine 10 mg/L. Over a 12 h incubation period at 35°C, the OD550 reached by each culture was determined at various time points, and the extent of growth inhibition by each fluoroquinolone, with or without reserpine, was determined.

Disruption of pmrA in CP1000 and EBR strains

To create a disruption of pmrA, the gene (938 bp), excluding the promoter region, was amplified using primers PMRJ1 5'-TAATCTGCGCATTGCCTG-3' and PMRJ2 5'-AATCAAGGCACCGGTTCC-3', and cloned into pCR2.1 (Invitrogen, Carlsbad, CA, USA). An internal portion of the pmrA gene was replaced by a chloramphenicol resistance cassette (cat, 1047 bp) derived from pEVP3.10 In the resulting 5628 bp plasmid, cat is oriented in the opposite direction to that of pmrA. The recombinant plasmid was linearized and the DNA was used to transform both CP1000 and EBR to chloramphenicol resistance by replacement of bp 422–688 of the chromosomal pmrA gene with the cat cassette. Transformants were selected with chloramphenicol 2 mg/L, and the presence of cat in pmrA was verified by PCR after overnight incubation of transformants in THBY broth containing chloramphenicol.

Selection of mutants resistant to ethidium bromide

Ethidium bromide-resistant mutants were obtained by exposing S. pneumoniae JJK01 to ethidium bromide 2 mg/L (2 x MIC). Between 107 and 108 cells from a S. pneumoniae JJK01 culture grown in THBY broth with chloramphenicol (2 mg/L) were plated on to a top layer of THBY agar, overlaid on THBY agar with ethidium bromide (4 mg/L). A total of 1010 cells on multiple plates were screened for mutant selection. Individual clones were taken after incubation for 48 h at 37°C, grown in THBY broth with chloramphenicol 2 mg/L and tested for susceptibility to ethidium bromide and various quinolones. Two of approximately 10 such clones, designated JJK01EB2 and JJK01EB5, were arbitrarily selected for further study.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
MICs of the agents tested against EBR, CP1000, JJK01 and JJK02 are listed in the TableGo. Inactivation of pmrA in EBR, as in JJK02, reduced the MICs of compounds that are substrates of active efflux (ethidium bromide, norfloxacin and ciprofloxacin) to those of the parent strain (CP1000). Inactivation of pmrA in CP1000, as in JJK01, did not affect the MIC of any bacterial agent tested. These data suggest that in its normal state, the exporter protein PmrA does not contribute significantly to the resistance of S. pneumoniae CP1000 to the tested fluoroquinolones or ethidium bromide. However, because CP1000 demonstrated a reserpine effect (FigureGo),3 the presence of an additional export protein(s) to PmrA is indicated.


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Table. MICs of various antimicrobial agents against strains EBR, CP1000, JJK02, JJK01, JJK01EB2 and JJK01EB5
 


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Figure 1. Growth inhibition assays for strains EBR, CP1000, JJK02 and JJK01 in the presence of ethidium bromide (a) and ciprofloxacin (b) at 0.25x their respective MICs as listed in the TableGo, with and without reserpine. Circles represent growth in a drug alone, and squares represent growth in drug and reserpine. Vertical bars represent standard error for each data point for the duplicate measurements.

 
Growth inhibition experiments, with and without reserpine, conducted using CP1000, JJK01, EBR and JJK02 in medium containing ethidium bromide, ciprofloxacin or moxifloxacin, showed that the reserpine effect on efflux of ethidium bromide and ciprofloxacin (seen in CP1000 and EBR) was not completely eliminated in either of the pmrA knockout strains JJK01 and JJK02 (FigureGo). The effect is less pronounced with moxifloxacin (data not shown), in agreement with our previous data on the reduced efflux of this fluoroquinolone due to the bulkiness of the C-7 substituent.9 This finding also indicates the presence of an additional export protein(s) in CP1000 that is susceptible to inhibition by reserpine.

To test whether the inactivation of PmrA influences the ability of the cell to produce drug-resistant mutants, mutant selection was undertaken by exposing strain JJK01 to ethidium bromide 2 mg/L. Resistant mutants were recovered at a frequency of 3 x 10-8 (28 colonies per 9 x 109 cells), which is similar to the rate of mutant selection observed with readily exported fluoroquinolones such as ciprofloxacin and levofloxacin,9 indicating that the loss of PmrA does not obviously affect the cell's ability to mutate to ethidium bromide resistance.

The MICs of ethidium bromide, norfloxacin, ciprofloxacin, levofloxacin, moxifloxacin and sparfloxacin for the two mutant clones, JJK01EB2 and JJK01EB5, in the absence and presence of reserpine are shown in the TableGo. The data demonstrate a significant reserpine effect similar to the eight-fold reduction we have consistently observed for ethidium bromide against strain EBR (the diminishing effect of reserpine on ethidium bromide and fluoroquinolone resistance due to the inhibition of active efflux in CP1000 and EBR has been described3,8,9). The elevated levels of resistance to ethidium bromide and selected fluoroquinolones displayed by JJK01EB2 and JJK01EB5, with resistance profiles distinct from that of EBR, provide a strong indication for an additional resistance mechanism(s) in S. pneumoniae. The reductions in ethidium bromide, norfloxacin, levofloxacin and ciprofloxacin MICs effected by reserpine for JJK01EB2 and JJK01EB5 indicate operation of an alternative efflux mechanism(s) to PmrA in S. pneumoniae CP1000.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The presence of multiple multidrug transporters in S. pneumoniae has important clinical implications. Our investigation suggests that S. pneumoniae has options for acquisition of mutations resulting in enhanced drug efflux, similar to those identified in Escherichia coli.11 Studies have been undertaken to determine whether there is a direct correlation between specific mutations in the quinolone resistance determining regions of DNA gyrase or topoisomerase IV and antimicrobial susceptibility patterns, so that emerging resistance to fluoroquinolones can be easily detected by clinical laboratory susceptibility testing at an early stage. Unfortunately, a simple, direct correlation could not be demonstrated.12 In the case of drugs that are substrates for active efflux, it is not surprising that this connection cannot be made, since the expression of an efflux mechanism will also result in increased resistance to these agents.

The operation of multiple exporters illustrates the complexity of some microbial–antimicrobial interactions, and that a better understanding to optimize drug design and antimicrobial agent use is required. Our work provides additional support for the concept that agents that are not readily effluxed from the cell are, in principle, better chemotherapeutic agents than those that are, since agents that are not effluxed avoid the adverse consequences of export systems. As Tillotson and colleagues suggest,13 it is time to rethink our use of antimicrobial agents, with a clear focus on how best to eradicate invading pathogens that possess numerous pathways for survival.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported by the Pharmaceutical Division of Bayer Corporation, US Public Health Service Grant no. UR8/CCU515081 and by Northwestern University Medical School, Chicago, IL. This material was presented, in part, in the abstracts of the Eleventh European Congress of Clinical Microbiology and Infectious Diseases, Istanbul, Turkey, 2001 (abstract P488).


    Notes
 
* Correspondence address. Northwestern Prevention Epicenter, Galter Carriage House, Suite 701b, 215 East Chicago Avenue, Chicago, IL 60611, USA. Tel: +1-312-926-2885; Fax: +1-312-926–4139; E-mail: EPestova{at}vysis.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Bartlett, J. G., Dowell, S. F., Mandell, L. A., File, T. M., Jr, Musher, D. M. & Fine, M. J. (2000). Practice guidelines for the management of community-acquired pneumonia in adults. Clinical Infectious Diseases 31, 347–82.[Medline]

2 . Ho, P. L., Yung, R. W. H., Tsang, D. N. C., Que, T. L., Ho, M., Seto, W. H. et al. (2001). Increasing resistance of Streptococcus pneumoniae to fluoroquinolones: results of a Hong Kong multicentre study in 2000. Journal of Antimicrobial Chemotherapy48, 659–65.[Abstract/Free Full Text]

3 . Pestova, E., Millichap, J. J., Noskin, G. A. & Peterson, L. R. (2000). Intracellular targets of moxifloxacin: A comparison with other fluoroquinolones. Journal of Antimicrobial Chemotherapy 45, 583–90.[Abstract/Free Full Text]

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6 . Gill, M. J., Brenwald, N. P. & Wise, R. (1999). Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 43, 988–9.[Abstract/Free Full Text]

7 . Kaatz, G. W., Seo, S. M., O'Brien, L., Wahiduzzman, M. & Foster, T. J. (2000). Evidence for the existence of a multidrug efflux transporter distinct from NorA in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 44, 1404–6.[Abstract/Free Full Text]

8 . Baranova, N. & Neyfakh, A. A. (1997). Apparent involvement of a multidrug transporter in the fluoroquinolone resistance of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 41, 1396–8.[Abstract]

9 . Beyer, R., Pestova, E., Millichap, J. J., Stosor, V., Noskin, G. A. & Peterson, L. R. (2000). A convenient assay for estimating the possible involvement of efflux of fluoroquinolones by Streptococcus pneumoniae and Staphylococcus aureus: evidence for diminished moxifloxacin, sparfloxacin, and trovafloxacin efflux. Antimicrobial Agents and Chemotherapy 44, 798–801.[Abstract/Free Full Text]

10 . Claverlys, J. P., Dintilhac, A., Pestova, E. V., Martin, B. & Morrison, D. A. (1995). Construction and evaluation of new drug-resistance cassettes for gene disruption mutagenesis in Streptococcus pneumoniae, using an ami test platform. Gene 164, 123–8.[ISI][Medline]

11 . Jellen-Ritter, A. S. & Kern, W. V. (2001). Enhanced expression of the multidrug efflux pumps AcrAB and AcrEF associated with insertion element transposition in Escherichia coli mutants selected with a fluoroquinolone. Antimicrobial Agents and Chemotherapy 45, 1467–72.[Abstract/Free Full Text]

12 . Millichap, J. J., Pestova, E., Siddiqui, F., Noskin, G. A. & Peterson, L. R. (2001). Fluoroquinolone resistance is a poor surrogate marker for type II topoisomerase mutations in clinical isolates of Streptococcus pneumoniae. Journal of Clinical Microbiology 39, 19–21.

13 . Tillotson, G. S., Zhao, X. & Drlica, K. (2001). Fluoroquinolones as pneumococcal therapy: closing the barn door before the horse escapes? Lancet Infectious Diseases 1, 145–6.[Medline]

Received 10 May 2001; returned 22 August 2001; revised 14 November 2001; accepted 30 November 2001