Antihypertensives suppress the emergence of fluoroquinolone-resistant mutants in pneumococci: An in vitro study

International Journal of Medical Microbiology 303 (2013) 176–181

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Antihypertensives suppress the emergence of fluoroquinolone-resistant mutants in pneumococci: An in vitro study Mathias W. Pletz a,b,∗,1 , Nikolay Michaylov c,1 , Ulrike Schumacher d,1 , Mark van der Linden e,1 , Christoph B. Duesberg c,1 , Thomas Fuehner c,1 , Keith P. Klugman f,1 , Tobias Welte c,1 , Oliwia Makarewicz a,b,1 a

Center for Infectious Diseases and Infection Control, Jena University Hospital, Jena, Germany Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany c Department of Pulmonary Medicine, Hannover Medical School, Hannover, Germany d Clinical Trial Center, Jena University Hospital, Jena, Germany e German National Reference Center for Streptococci, Department of Medical Microbiology, University Hospital RWTH Aachen, Germany f Department of International Health, Emory University, Atlanta, United States b

a r t i c l e

i n f o

Article history: Received 28 December 2012 Received in revised form 1 February 2013 Accepted 3 February 2013 Keywords: Streptococcus pneumoniae Community-acquired pneumonia Fluoroquinolone Resistance Reserpine Verapamil Efflux Mutation QRDR

a b s t r a c t Background: The antihypertensives reserpine and verapamil are also inhibitors of pneumococcal efflux pumps. We addressed the following questions: (i) Do verapamil and reserpine influence the mutation ratio of pneumococci in the presence of ciprofloxacin? (ii) At which concentrations does this occur? (iii) Is this limited to isolates with efflux phenotype? Methods: 14 clinical isolates, nested in 6 genetically similar clusters, were used, 7 strains with efflux and 7 without. The mutation ratio in the presence of ciprofloxacin (3× MIC) and increasing concentrations of reserpine and verapamil was determined and the quinolone-resistance determining regions (QRDR) of selected mutants were sequenced. Analysis of the efficacy was performed using a mixed linear model, supported by descriptive statistics. Results: Reserpine and verapamil reduced the mutation ratio of QRDR in the presence of ciprofloxacin with the required concentration for a reduction ≥50% of 1 mg/l for reserpine and 50 mg/l for verapamil. The mutation prevention effect is not limited to, but is more pronounced in efflux positive phenotypes. Conclusion: Reserpine and verapamil can prevent the selection of ciprofloxacin resistant isolates by reduction of the mutation ratio, particularly in strain with an efflux phenotype. However, the required concentrations are too toxic for clinical use. © 2013 Elsevier GmbH. All rights reserved.

Introduction Streptococcus pneumoniae is the most frequent pathogen found in respiratory infections such as community-acquired pneumonia (CAP), sinusitis, otitis media and a frequent cause of bacteraemia and meningitis. In the early 1990s surveillance studies detected a sudden increase in penicillin- and macrolide-resistant pneumococci (Doern et al., 2001; Jorgensen et al., 1990; Spika et al., 1991). Macrolide resistance has been clearly demonstrated to be associated to clinical treatment failure (Daneman et al., 2006). In contrast,

∗ Corresponding author at: Universitätszentrum für Infektionsmedizin und Krankenhaushygiene, Universitätsklinikum Jena, Erlanger Alle 101, 07747 Jena, Germany. Tel.: +49 03641 9 324650; fax: +49 03641 9 324222. E-mail address: [email protected] (M.W. Pletz). 1 For the CAPNETZ study group. Please see Acknowledgements section for members of this group. 1438-4221/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ijmm.2013.02.014

for penicillin resistant pneumococci results of several studies have been contradictory and the current view of most experts is, that only strains with a penicillin MIC ≥ 4 mg/l can cause treatment failure in CAP (Yu et al., 2003). Due to the rise in penicillinand macrolide-resistant pneumococci, fluoroquinolones (FQ) have been used increasingly for the treatment of pneumococcal infections over the past decade. Numerous recently published case reports have suggested an association between FQ resistance and treatment failure (Fuller and Low, 2005; Pletz et al., 2005b, 2006a). The subunits of the FQ target enzymes DNA gyrase and topoisomerase IV are encoded by the genes gyrA/gyrB and parC/parE, respectively (Pan et al., 1996). Complete resistance occurs mainly if mutations in both enzymes are present. Strains with mutations in only one of the target enzymes frequently display a susceptible phenotype (first-step mutants) but are considered as precursors of completely resistant strains (Pletz et al., 2006c). A FQ efflux pump is mediated by the membrane ABC-transporter protein PmrA and some unknown factors (Brenwald et al., 1998,

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2003; Gill et al., 1999; Janoir et al., 1996). The insufficiently characterized efflux mechanisms are not encoded by resistance genes but are thought to be over-expressed in 8–45% of pneumococcal strains (Pletz et al., 2011). The efflux pump can be blocked by the plant alkaloid reserpine and, to a lesser degree, by verapamil (Andersen et al., 2006; Brenwald et al., 1998). Interestingly, both substances are licensed drugs for the treatment of hypertension in humans. Currently, the detection of this efflux pump is standardized on phenotypic features, i.e. an twofold increase in MIC to ciprofloxacin in the presence of reserpine at 10 mg/l (22). It has been observed previously that phenotypic ciprofloxacin resistance can be selected more frequently from isolates with an efflux phenotype (Li et al., 2002; Markham, 1999). The aim of this study was to investigate if it would be reasonable to combine efflux inhibitors with FQ in order to prevent the emergence of resistance. The following questions were addressed: (i) Do the efflux inhibitors verapamil and reserpine influence the mutation ratio within the QRDR of pneumococci in the presence of ciprofloxacin? (ii) and if so, does this occur at concentrations that are tolerated by usual clinical doses? (iii) Is this effect of preventing QRDR mutations limited to isolates with an efflux phenotype? According to the current EUCAST recommendations, ciprofloxacin should not be used for pneumococcal infections because of the risk of resistance (Leclercq et al., 2013). Nevertheless, we decided to use ciprofloxacin as test substance for a proof of principle because moxifloxacin and levofloxacin are only minor substrates of the above mentioned efflux mechanism. In addition, mutations selected by ciprofloxacin can confer resistance also to all other FQ.

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at 37 ◦ C in an atmosphere of 5% CO2 and the MIC of CIP were determined using standard agar broth doubling micro-dilution method according to the British Society for Antimicrobial Chemotherapy (Andrews, 2001). A four fold decrease in MIC of CIP in the presence of 10 ␮g/ml reserpine was interpreted as a positive efflux phenotype (Brenwald et al., 1998). Efflux positive and negative strains were pair-wise matched by genetic similarity (identical or closely related sequence type, defined as identical alleles in at least 4 out of 7 MLST-loci) and serotype. Among 186 pneumococcal isolates, we found 7 efflux positive isolates with at least one corresponding efflux negative isolates resulting in 6 pairs (Table 1). Selection of mutants and confirmatory sequencing Mutation frequencies were determined by plating suspensions containing 108 CFU onto Mueller-Hinton agar with 5% horse blood containing CIP at 3× MIC or antibiotic-free agar and the efflux inhibitors reserpine (0.01, 0.1, 1, 5 and 10 mg/l) or verapamil (10, 25, 50, 100 and 500 mg/l), respectively. In addition, a control experiment without efflux blocker was performed. After incubation at 37 ◦ C in 5% CO2 for 3 days, the colonies were counted. According to control tests, verapamil at 500 mg/l and reserpine at 10 mg/l had no impact on the growth of the tested isolates in the absence of CIP. Randomly chosen colonies (3 per experiment) of the mutants selected at 3× MIC were further analyzed by PCR and subsequently sequencing of PCR products for the presence of mutations within the parC, parE, and gyrA genes using primers and reaction condition described previously (Pan et al., 1996).

Methods Statistical methods Collection and characterization of clinical isolates Clinical isolates were collected between 2002 and 2006 from patients with community-acquired pneumonia (CAP) by the German Competence Network CAPNETZ. The study was approved by the ethical review board of each participating clinical center, and all patients included gave informed consent. A detailed description of the CAPNETZ methodology is given elsewhere (Pletz et al., 2012). Serotyping, multi-locus sequence typing (MLST) and minimal inhibitory concentration (MIC) testing were performed at the German National Reference Center for streptococci. Serotyping was performed by the standard Quellung method (Neufeld, 1902), MLST was performed as previously described using modified primers (Enright and Spratt, 1998). The strains were incubated in Mueller-Hinton medium supplemented with 5% lysed horse blood

For each single experiment, measurements were performed twice and the mean of both values was used for further evaluation. For every strain and every dose, 11 repetitions of the experiment were performed. As the primary efficacy parameter the mutation ratio, defined as the ratio of cell number under treatment (efflux inhibitor) by cell number without efflux inhibitor treatment (control experiment), was chosen. The analysis of the efficacy of reserpine/verapamil was performed using a mixed linear model, supported by descriptive statistics. The used model includes the dose of reserpine/verapamil, the presence of efflux and the interaction of dose and efflux as fixed effects as well as single experiment, strain and strain cluster with the respective nesting structure as random effects.

Table 1 Characterization of investigated isolates and parC sequences of derived mutants. None of the mutants exhibited mutation in gyrA, gyrB and parE. Strain

Serotype

Assigned cluster

MLST

MIC CIP mg/l

MIC CIP + Res in mg/l

Efflux

par C of strain

parC of derived mutants

SP16 SP44 SP28 SP58 SP35 SP91 SP73 SP217 SP219 SP26 SP10 SP234 SP11 SP239

7F 7F 7F 11A 11A 3 3 3 3 3 6B 14 23A 14

1 1 1 2 2 3 3 3 4 4 5 5 6 6

191 191 191 62 62 1377 1377 1377 180 180 1555 (7-11-1-1-9-52-1)a 557 (7-11-10-1-6-58-1)a 42 (1-8-9-9-6-4-6)b 439 (1-8-9-2-6-4-6)b

1 2 2 2 1 0.5 0.5 1 2 1 1 1 1 0.5

0.25 1 1 0.5 0.5 <0.12 <0.12 0.5 0.5 0.5 0.25 0.5 0.25 0.5

+ − − + − + + − + − + − + −

WT WT WT WT WT WT WT WT WT WT WT WT WT WT

S79F S79F WT D83G D83G S79Y S79Y WT WT WT S79F WT S79Y WT

a b

Single locus variant Triple locus variant.

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Table 2a Statistical analysis of ratio of mutation vs. control by dose of reserpine and efflux no/yes. Dose of reserpine (mg) Efflux

Statistic

0.01

0.1

1.0

5.0

10.0

Total

Yes

N Mean Standard deviation Minimum Median Maximum N Mean Standard deviation Minimum Median Maximum

99 0.84 0.53 0 0.78 5 99 0.73 0.27 0.07 0.78 1.42

99 0.78 0.67 0.16 0.71 5 99 0.71 0.28 0.02 0.74 1.62

99 0.42 0.38 0 0.33 2 99 0.54 0.49 0.04 0.44 3.34

99 0.16 0.38 0 0 1.64 99 0.20 0.22 0 0.09 0.75

99 0.03 0.11 0 0 0.82 99 0.10 0.16 0 0 0.7

495 0.45 0.56 0 0.32 5 495 0.45 0.40 0 0.44 3.34

No

Partial tests of fixed effects Effect

Numerator degrees of freedom

Denominator degrees of freedom

F value

Pr > F

Efflux Dose Efflux × Dose

1 4 4

784 784 784

6.14 588.83 2.52

0.0134 <0.0001 0.0400

Descriptive statistics included means, standard deviations, median and quartiles, minimum and maximum numbers. In addition, the rate of confirmed mutants was compared between efflux positive and efflux negative strains by McNemar’s Test for comparison of matched pairs.

Reserpine

Among 186 pneumococcal isolates, we found 7 efflux positive isolates with at least one corresponding efflux negative isolates resulting in 6 pairs (Table 1).

Whereas reserpine at 0.01 and 0.1 mg/l had only a minor effect on the mean mutation ratio, reserpine at 1.0 mg/l reduced the mean mutation ratio by ≥50% for efflux positive but not for efflux negative strains. For efflux negative strains a reserpine concentration of 5 mg/l was required for a reduction of the mutation ratio by ≥50%. The multivariate model showed a significant interaction between dose and efflux phenotype (p = 0.04) as well a significant dose effect (p < 0.0001) and a significant efflux phenotype effect (p = 0.013). A more pronounced reduction of mutation ratio was seen in the strains with efflux compared with the strains without efflux from dose 0.1 mg to dose 1.0 mg (see Fig. 1a).

Mutation ratio

Verapamil

In the presence of both – reserpine or verapamil, the mutation ratio of all strains notably decreased in concentration dependent manner (Fig. 1a and b; Tables 2a and 2b).

A reduction of ≥50% of the mean mutation ratio was observed at verapamil concentrations of 50 mg/l for both, efflux negative and positive isolates.

Results Characterization of clinical isolates

Table 2b Statistical analysis of ratio of mutation vs. control by dose of verapamil and efflux no/yes. Dose of verapamil (mg) Efflux

Statistic

10

25

50

100

500

Total

Yes

N Mean Standard deviation Minimum Median Maximum N Mean Standard deviation Minimum Median Maximum

99 1.17 1.86 0.35 0.89 19 98 0.85 0.23 0.36 0.86 1.85

99 0.93 0.59 0.19 0.81 4.2 98 0.57 0.21 0.11 0.59 0.99

99 0.59 0.40 0.02 0.53 3 98 0.36 0.15 0.06 0.34 0.76

99 0.17 0.22 0 0.11 1.84 98 0.11 0.11 0 0.09 0.49

99 0 0 0 0 0 98 0 0 0 0 0

495 0.571 0.997 0 0.489 19 490 0.38 0.349 0 0.316 1.85

No

Partial tests of fixed effects Effect

Efflux Dose Efflux × Dose

Numerator degrees of freedom

Denominator degrees of freedom

Num DF

Den DF

1 4 4

780 780 780

F value

Pr > F

41.88 1882.02 14.98

<0.0001 <0.0001 <0.0001

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(a)

179

Mutation: ratio to control

1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5

0.84 0.73

0.78 0.71 0.54 0.42

0.4 0.3 0.2 0.1 0.0

0.20

0.16 0.10

0.01

0.1

1.0

5.0

0.03

10.0

Dosis

(b)

Efflux

Mutation: ratio to control

No

Yes

4

3

2

1.17

1

0.93

0.85

0.59

0.57 0.36

0

0.11

10

25

50

0.17

100

0.00

0.00

500

Dosis Efflux

No

Yes

Fig. 1. Mean (standard deviation) mutation ratio of efflux positive (red line) vs. efflux negative isolates (blue line) by dose of (a) reserpine and (b) verpamil, total number of experiments = 990. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Beside a significant dose effect (p < 0.0001) and significant efflux effect (p < 0.0001), a significant efflux phenotype-dose interaction (p < 0.0001) was shown in the multivariate model. A more pronounced reduction of cells in the strains with efflux compared with the strains without efflux from dose 50 mg to dose 100 mg can be observed in Fig. 1b. Confirmatory sequencing In total, first step mutants with parC mutations confirmed by sequencing were derived from 10 isolates. Mutants derived from efflux positive isolates SP16, SP91, SP10, SP11, SP15 and SP186 contained substitutions at position 79 (S79F and S79Y) in ParC and showed highly reduced susceptibility to CIP both in presence and absence of efflux inhibitor (changes 4 to 12 fold). Mutants without a substitution in parC exhibited only a slight increase of CIP MIC (change of 1 to 3fold). McNemar’s Test for comparison of matched pairs revealed a trend that efflux is a risk factor for acquisition of QRDR mutations, however due to the low number of matched pairs this trend did not reach statistically significance (p = 0.0736).

Discussion Our results can be summarized as follows: (i) efflux pump inhibitors reserpine and – to a lesser degree – verapamil reduce the mutation ratio of QRDR of pneumococci in the presence of ciprofloxacin, (ii) the required concentration for a relevant reduction (≥50%) is 1 mg/l for reserpine and 50 mg/l for verapamil – concentrations probably associated with non-tolerable effects on hemodynamics in men as outlined below and (iii) the mutation prevention effect of reserpine and verapamil is not limited to, but is more pronounced in efflux positive phenotypes. For efflux positive isolates, a ≥50% reduction of mutation ratio by reserpine occurred already 1 log or approximately 3 dilution steps below the standardized concentration of 10 mg/l for testing of the efflux phenotype. Despite the limitations of our study, i.e. the usually high standard deviation of such experiments, the use of clinical, not perfectly matching isolates and the low number of isolates per group, the effect of the efflux phenotype and dosage on mutation ratio remained significant in a multivariate model including cluster and strain. We provided serotype information in

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addition to MLST date only to underline the similar genetic background of out matched pairs. We do not believe that serotype itself has an impact on our observations. The QRDR mutations detected in selected mutants are exactly the same that we (Pletz et al., 2004) and others (e.g. (Zheng et al., 2001) found in clinical isolates with confirmed FQ resistance. QRDR mutations conferring FQ resistance in streptococci can be acquired by spontaneous mutations or horizontal gene transfer (Duesberg et al., 2008; Pletz et al., 2005a, 2006b). The observed mutation prevention effect of efflux inhibitors may only apply to acquisition of spontaneous mutation. However, at least in pneumococci horizontal gene transfer plays only a minor role in regard of FQ resistance (Pletz et al., 2005a). In 2008 Garvey and Piddock have published data on relation between the multidrug and reserpine resistances of S. pneumoniae and identified reserpine resistant mutants. These authors claim that reserpine can select multidrug resistant bacteria (Garvey and Piddock, 2008). This observation is only seemingly contradictory to our results: Garvey and Piddock found, that mutants selected by resistance to reserpine alone (MIC ≥ 256 mg/l) express a highperformance efflux phenotype that is associated with increased resistance to antibiotics and dyes that are affected by efflux. We have shown that inhibition of efflux increases not only ciprofloxacin susceptibility (well known) but also inhibits the acquisition of FQ resistance conferring mutations, and that this effect is only observed in isolates with an efflux phenotype. In conclusion, a high-performance efflux system is associated with an increased likelihood for selection of ciprofloxacin resistant mutants (Garvey and Piddock, 2008) and inhibition of such an efflux reduces the likelihood for acquisition of mutations in presence of FQ (our results). Verapamil and reserpine have been licensed and used to treat hypertension in men for decades. However, for both drugs the concentration required to block the emergence of first step mutants is toxic. The recommended dosage for a standardized 70 kg male patient is 0.1–0.3 mg reserpine and 120–480 mg verapamil, resulting in plasma and or tissue levels below 0.1 mg/l for reserpine and between 0.05 and 4 mg/l for verapamil. Therefore, a combination of the tested efflux inhibitors with FQ is not feasible for clinical use. The increasing antibiotic resistance and the lack of novel compounds has intensified the research on new strategies to decrease the spread of antimicrobial resistance, e.g. (Lin et al., 2011). Fluoroquinolone resistance can be horizontally transmitted by plasmids in Gram-negative bacteria (Shaheen et al., 2013) or by recombination in streptococci (Duesberg et al., 2006; Pletz et al., 2005a, 2006b). However, in pneumococci this is not common compared to the acquisition of spontaneous mutations (Pletz et al., 2005a). Therefore, one possible strategy that would reduce the emergence of FQ resistance in pneumococci seems to be the inhibition of efflux pumps. Even if the required concentrations of reserpine and verapamil are too high for clinical use, our results may stimulate the further search for efflux pump inhibitors for possible combination with FQ in order to prevent the acquisition of FQ resistance conferring mutations. Funding The work was supported by a grant from the German Ministry of Education and Research (Bundesministerium für Bildung und Forschung), grant number 01KI1204. CAPNETZ was funded by the German Ministry of Education and Research (Bundesministerium für Bildung und Forschung), grant number 01KI07145. NM was supported by StrucMed (Hannover Medical School) and the ARGUS Foundation.

Conflict of interest Nothing to declare.

Acknowledgements CAPNETZ is a multidisciplinary approach to better understand and treat patients with community-acquired pneumonia. The network has only been made possible by the contribution of many investigators. Members of the CAPNETZ study group except the authors are S. Krüger, D. Frechen (Aachen); W. Knüppel, I. Armari (Bad Arolsen); D. Stolz (Basel); N. Suttorp, H. Schütte, A. Tessmer, P. Martus (Berlin, Charité); T. Bauer, J. Hecht (Berlin); W. Pankow, A. Lies, D. Thiemig (Berlin-Neukölln); B. Hauptmeier, S. Ewig, D. Wehde, M. Suermann (Bochum); M. Prediger, G. Zernia (Cottbus); J. Rademacher, G. Barten, L. Gosman, W. Kröner (Hannover); R. Bals (Homburg/Saar); C. Kroegel (Jena); K. Dalhoff, S. Schütz, R. Hörster, (Lübeck); G. Rohde (Maastricht); W. Petermann, H. Buschmann, R. Kröning, Y. Aydin (Paderborn); T. Schaberg, I. Hering (Rotenburg/Wümme); R. Marre, C. Schumann (Ulm); H. von Baum (Ulm, Med. Microbiology); T. Illmann, M. Wallner (Ulm); O. Burghuber, G. Rainer (Wien) and all study nurses.

References Andersen, C.L., Holland, I.B., Jacq, A., 2006. Verapamil, a Ca2+ channel inhibitor acts as a local anesthetic and induces the sigma E dependent extra-cytoplasmic stress response in E. coli. Biochim. Biophys. Acta 1758, 1587–1595. Andrews, J.M., 2001. Determination of minimum inhibitory concentrations. J. Antimicrob. Chemother. 48 (Suppl. 1), 5–16. Brenwald, N.P., Gill, M.J., Wise, R., 1998. Prevalence of a putative efflux mechanism among fluoroquinolone-resistant clinical isolates of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 42, 2032–2035. Brenwald, N.P., Appelbaum, P., Davies, T., Gill, M.J., 2003. Evidence for efflux pumps, other than PmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae. Clin. Microbiol. Infect. 9, 140–143. Daneman, N., McGeer, A., Green, K., Low, D.E., 2006. Macrolide resistance in bacteremic pneumococcal disease: implications for patient management. Clin. Infect. Dis. 43, 432–438. Doern, G.V., Heilmann, K.P., Huynh, H.K., Rhomberg, P.R., Coffman, S.L., Brueggemann, A.B., 2001. Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in the United States during 1999–2000, including a comparison of resistance rates since 1994–1995. Antimicrob. Agents Chemother. 45, 1721–1729. Duesberg, C., Malhotra-Kumar, S., Welte, T., Goossens, H., Pletz, M., 2006. Horizontal gene transfer contributes to fluoroquinolone resistance in Streptococcus pyogenes. In: Program and abstracts of the 16th European Congress of Clinical Microbiology and Infectious Diseases, pp. 1–4. Duesberg, C.B., Malhotra-Kumar, S., Goossens, H., McGee, L., Klugman, K.P., Welte, T., Pletz, M.W., 2008. Interspecies recombination occurs frequently in quinolone resistance-determining regions of clinical isolates of Streptococcus pyogenes. Antimicrob. Agents Chemother. 52, 4191–4193. Enright, M.C., Spratt, B.G., 1998. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144 (Pt 11), 3049–3060. Fuller, J.D., Low, D.E., 2005. A review of Streptococcus pneumoniae infection treatment failures associated with fluoroquinolone resistance. Clin. Infect. Dis. 41, 118–121. Garvey, M.I., Piddock, L.J., 2008. The efflux pump inhibitor reserpine selects multidrug-resistant Streptococcus pneumoniae strains that overexpress the ABC transporters PatA and PatB. Antimicrob. Agents Chemother. 52, 1677–1685. Gill, M.J., Brenwald, N.P., Wise, R., 1999. Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 43, 187–189. Janoir, C., Zeller, V., Kitzis, M.D., Moreau, N.J., Gutmann, L., 1996. High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA. Antimicrob. Agents Chemother. 40, 2760–2764. Jorgensen, J.H., Doern, G.V., Maher, L.A., Howell, A.W., Redding, J.S., 1990. Antimicrobial resistance among respiratory isolates of Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in the United States. Antimicrob. Agents Chemother. 34, 2075–2080. Leclercq, R., Canton, R., Brown, D.F., Giske, C.G., Heisig, P., Macgowan, A.P., Mouton, J.W., Nordmann, P., Rodloff, A.C., Rossolini, G.M., Soussy, C.J., Steinbakk, M., Winstanley, T.G., Kahlmeter, G., 2013. EUCAST expert rules in antimicrobial susceptibility testing. Clin. Microbiol. Infect. 19, 141–160.

M.W. Pletz et al. / International Journal of Medical Microbiology 303 (2013) 176–181 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. Lin, A., Jimenez, J., Derr, J., Vera, P., Manapat, M.L., Esvelt, K.M., Villanueva, L., Liu, D.R., Chen, I.A., 2011. Inhibition of bacterial conjugation by phage M13 and its protein g3p: quantitative analysis and model. PLoS One 6, e19991. Markham, P.N., 1999. Inhibition of the emergence of ciprofloxacin resistance in Streptococcus pneumoniae by the multidrug efflux inhibitor reserpine. Antimicrob. Agents Chemother. 43, 988–989. Neufeld, F., 1902. Über die Agglutination der Pneumokokken und über die Theorie der Agglutination. Zeitschrift für Hygiene und Infektionskrankheiten 34, 54–72. Pan, X.S., Ambler, J., Mehtar, S., Fisher, L.M., 1996. Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 40, 2321–2326. Pletz, M.W., McGee, L., Jorgensen, J., Beall, B., Facklam, R.R., Whitney, C.G., Klugman, K.P., 2004. Levofloxacin-resistant invasive Streptococcus pneumoniae in the United States: evidence for clonal spread and the impact of conjugate pneumococcal vaccine. Antimicrob. Agents Chemother. 48, 3491–3497. Pletz, M.W., McGee, L., Beall, B., Whitney, C.G., Klugman, K.P., 2005a. Interspecies recombination in type II topoisomerase genes is not a major cause of fluoroquinolone resistance in invasive Streptococcus pneumoniae isolates in the United States. Antimicrob. Agents Chemother. 49, 779–780. Pletz, M.W., McGee, L., Burkhardt, O., Lode, H., Klugman, K.P., 2005b. Ciprofloxacin treatment failure in a patient with resistant Streptococcus pneumoniae infection following prior ciprofloxacin therapy. Eur. J. Clin. Microbiol. Infect. Dis. 24, 58–60. Pletz, M.W., Fugit, R.V., McGee, L., Glasheen, J.J., Keller, D.L., Welte, T., Klugman, K.P., 2006a. Fluoroquinolone-resistant Streptococcus pneumoniae. Emerg. Infect. Dis. 12, 1462–1463. Pletz, M.W., McGee, L., Van Beneden, C.A., Petit, S., Bardsley, M., Barlow, M., Klugman, K.P., 2006b. Fluoroquinolone resistance in invasive Streptococcus pyogenes

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isolates due to spontaneous mutation and horizontal gene transfer. Antimicrob. Agents Chemother. 50, 943–948. Pletz, M.W., Shergill, A.P., McGee, L., Beall, B., Whitney, C.G., Klugman, K.P., 2006c. Prevalence of first-step mutants among levofloxacin-susceptible invasive isolates of Streptococcus pneumoniae in the United States. Antimicrob. Agents Chemother. 50, 1561–1563. Pletz, M.W., van der Linden, M., von Baum, H., Duesberg, C.B., Klugman, K.P., Welte, T., CAPNETZ study group, 2011. Low prevalence of fluoroquinolone resistant strains and resistance precursor strains in Streptococcus pneumoniae from patients with community-acquired pneumonia despite high fluoroquinolone usage. Int. J. Med. Microbiol. 301, 53–57. Pletz, M.W., von Baum, H., van der Linden, M., Rohde, G., Schutte, H., Suttorp, N., Welte, T., 2012. The burden of pneumococcal pneumonia – experience of the German competence network cAPNETZ. Pneumologie 66, 470–475. Shaheen, B.W., Nayak, R., Foley, S.L., Boothe, D.M., 2013. Chromosomal and plasmid-mediated fluoroquinolone resistance mechanisms among broad-spectrum-cephalosporin-resistant Escherichia coli isolates recovered from companion animals in the USA. J. Antimicrob. Chemother., http://dx.doi.org/10.1093/jac/dks514 [E-pub ahead of print]. Spika, J.S., Facklam, R.R., Plikaytis, B.D., Oxtoby, M.J., 1991. Antimicrobial resistance of Streptococcus pneumoniae in the United States, 1979–1987. The Pneumococcal Surveillance Working Group. J. Infect. Dis. 163, 1273–1278. Yu, V.L., Chiou, C.C., Feldman, C., Ortqvist, A., Rello, J., Morris, A.J., Baddour, L.M., Luna, C.M., Snydman, D.R., Ip, M., Ko, W.C., Chedid, M.B., Andremont, A., Klugman, K.P., 2003. An international prospective study of pneumococcal bacteremia: correlation with in vitro resistance, antibiotics administered, and clinical outcome. Clin. Infect. Dis. 37, 230–237. Zheng, X., Johnson, C., Lu, Y., Yanagihara, R., Hollingshead, S., Crain, M., Benjamin Jr., W., Waites, K.B., 2001. Clinical isolates of Streptococcus pneumoniae resistant to levofloxacin contain mutations in both gyrA and parC genes. Int. J. Antimicrob. Agents 18, 373–378.