Worldwide incidence, molecular epidemiology and mutations implicated in fluoroquinolone-resistant Streptococcus pneumoniae: data from the global PROTEKT surveillance programme

R. Canton1, M. Morosini1, M. C. Enright2 and I. Morrissey3,*

1 Hospital Ramon y Cajal, Madrid, Spain; 2 University of Bath, Bath; 3 GR Micro Ltd, 7–9 William Road, London NW1 3ER, UK

Received 30 June 2003; returned 29 July 2003; revised 8 August 2003; accepted 28 August 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To analyse the mutations and epidemiology associated with fluoroquinolone-resistant pneumococci collected as part of the PROTEKT global surveillance programme during 1999–2000.

Methods: Sixty-nine centres in 25 countries submitted a total of 3362 Streptococcus pneumoniae isolates, for which the MICs of antimicrobial agents were determined using NCCLS methodology.

Results: Levofloxacin resistance was low overall (1% worldwide), with higher rates in: Hong Kong (14.3%), South Korea (2.9%), USA (1.8%), Mexico (1.5%), Canada (1.4%) and Japan (1.3%). Levofloxacin resistance was very low or absent in European countries, and absent in Australia. Worldwide, there was a total of 35 levofloxacin-resistant isolates, of which 22 (63%) were resistant and 10 (29%) were intermediate to moxifloxacin. All levofloxacin-resistant isolates were susceptible to telithromycin (<=0.5 mg/L), linezolid (<=2 mg/L) and quinupristin/dalfopristin (<=1 mg/L). One or more mutations in the topoisomerase genes were identified in all levofloxacin-resistant isolates; most of these isolates (33/35) had a mutation in one of the DNA gyrase encoding genes (gyrA, gyrB) and one of the topoisomerase IV encoding genes (parC, parE). Eighteen (51%) isolates carried the same combination of amino acid substitutions: Ser-81->Phe in GyrA and Ser-79->Phe in ParC. Isolates displaying a levofloxacin MIC of 2–4 mg/L generally had no mutation or one mutation in either a DNA gyrase or a topoisomerase IV gene, although most mutations were in parC.

Conclusions: Most levofloxacin-resistant isolates possess two mutations (one in DNA gyrase and one in topoisomerase IV). Although multilocus sequence typing demonstrated that most of these isolates were unrelated, 12 (34%) were the Spain23F-1 clone: 10 from Hong Kong and one each from Saskatchewan, Canada and Sao Paulo, Brazil.

Keywords: multilocus sequence typing, Spain23F-1, topoisomerase mutations, fluoroquinolone resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluoroquinolones were introduced into widespread clinical practice in 1987 when ciprofloxacin became available in the major markets of Germany, the UK and the USA.1 Newer fluoroquinolone agents are used to treat community-acquired and serious infections, including those of the respiratory tract, urinary tract and intra-abdominal cavity, as well as osteomyelitis and febrile neutropenic episodes.24 However, there is increasing evidence that widespread use of these agents has increased resistance to the whole family of fluoroquinolones in several species of bacteria.57

In Streptococcus pneumoniae, resistance to fluoroquinolones has remained rare despite the escalating levels of resistance to penicillins and macrolides.811 But recent reports from Hong Kong, the USA, Canada, Spain and Hungary have identified strains of S. pneumoniae that are resistant to levofloxacin (MIC >= 8 mg/L) or ciprofloxacin (MIC >= 4 mg/L) and demonstrate increased MICs of gatifloxacin and moxifloxacin.1216

Surveillance studies are important tools in identifying and monitoring antimicrobial resistance. Studies should be sensitive enough to detect changes at a local level, and ideally should be equipped to investigate the resistance mechanisms involved. The PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study is a longitudinal, global, multicentre surveillance programme aimed at monitoring levels of antimicrobial resistance worldwide in the most common respiratory tract pathogens, and investigating the prevalent resistance mechanisms in macrolide- and fluoroquinolone-resistant isolates.17

This paper presents data collected worldwide during 1999–2000 on the susceptibility of S. pneumoniae isolates to the fluoroquinolones mainly used to treat respiratory tract infections. All levofloxacin-intermediate (MIC 4 mg/L) and -resistant (MIC >= 8 mg/L) pneumococci were molecularly typed. Moreover, mutations in the genes that encode the DNA gyrase and topoisomerase IV enzymes in these strains and those strains displaying a levofloxacin MIC of 2 mg/L were also characterized.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collecting centres

During 1999–2000, 69 centres in 25 countries participated and submitted isolates: Argentina (2), Australia (2), Austria (1), Belgium (1), Brazil (7), Canada (7), France (4), Germany (7), Hong Kong (1), Hungary (1), Indonesia (1), Ireland (1), Italy (2), Japan (6), Mexico (4), the Netherlands (1), Poland (1), Portugal (2), South Korea (2), Spain (2), Sweden (1), Switzerland (2), Turkey (1), the UK (2), and the USA (8).

Bacterial isolates

Centres were requested to collect a minimum of 60 isolates of S. pneumoniae from patients with community-acquired respiratory tract infections (RTIs), or from hospitalized patients within 48 h of admission. These infections included acute bacterial exacerbation of chronic bronchitis (or acute bacterial exacerbation of chronic obstructive pulmonary disease), pharyngitis, pneumonia, otitis media and sinusitis. Sources for isolation of S. pneumoniae were cultures from blood, sputum, bronchoalveolar lavage, middle ear fluid, nasopharyngeal swab or aspirate and sinus aspirate. Isolates from patients with nosocomial RTI or cystic fibrosis were excluded. Additional information on the design and methodology of the PROTEKT surveillance programme has been published previously.17

Antimicrobial activity testing

MICs were determined at a central laboratory (GR Micro Ltd, London, UK) for a panel of new and established antimicrobial agents. The NCCLS broth microdilution method was used, with lyophilized microtitre plates (Sensititre, Trek Diagnostic Systems Ltd, East Grinstead, UK) and an inoculum of 3–7 x 104 cfu in 100 µL medium.17

Fluoroquinolone resistance mechanism detection

Isolates with levofloxacin MICs of 2, 4 or 8 mg/L were subcultured on blood agar, and the DNA was extracted using a High Pure PCR Template kit (Roche, Lewes, UK) according to the manufacturer’s instructions. All the DNA topoisomerase genes (gyrA, gyrB, parC and parE) were amplified and sequenced using a method described previously.18

The gene fragments were re-assembled to form complete genes using Seqman software (DNASTAR, Madison, Wisconsin, USA). These nucleotide sequences were translated into amino acid sequences and compared—using MegAlign software (DNASTAR) —with those of the wild-type S. pneumoniae R6 gene sequence.

Multilocus sequence typing

Multilocus sequence typing (MLST), described previously,19 was performed on the levofloxacin-resistant isolates to determine the epidemiological relatedness of fluoroquinolone-resistant pneumococci.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During the period 1999–2000, a total of 3362 isolates of S. pneumoniae were collected worldwide: 1521 (45.2%) from Europe, 687 (20.4%) from North America, 518 (15.4%) from Latin America, 515 (15.3%) from The Far East and 121 (3.6%) from Australasia.

The MIC50 and MIC90 values for levofloxacin were consistently 1 mg/L for all countries, with the exception of an MIC50 of <=0.5 mg/L in Turkey and an MIC90 of 16 mg/L in Hong Kong (Table 1). The MIC50 and MIC90 values of ciprofloxacin were 1 mg/L and 2 mg/L, respectively, except in Indonesia (MIC50 2 mg/L), Hungary and Turkey (MIC90 1 mg/L) and Hong Kong (MIC90 >= 64 mg/L). Moxifloxacin MIC90 values were four- to eight-fold lower than those of levofloxacin, with all countries other than Hong Kong (MIC90 4 mg/L) recording MIC90 values of 0.25 or 0.12 mg/L. Overall, 35 isolates (1.0%) of S. pneumoniae were resistant to levofloxacin (MIC >= 8 mg/L) and 22 (0.7%) to moxifloxacin (MIC >= 4 mg/L); however, resistance was much higher in Hong Kong (both 14.3%). Resistance to the fluoroquinolones was generally very low in Europe, and absent in Australia (Table 1).


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Table 1. Comparison of the fluoroquinolone MIC data and resistance in isolates of S. pneumoniae from PROTEKT 1999–2000 
 
Of the 35 levofloxacin-resistant isolates, 22 (63%) were resistant and 10 (29%) were intermediate (MIC 2 mg/L) to moxifloxacin. Penicillin resistance (MIC >= 2 mg/L) among these isolates was 51% (18/35), 6% (2/35) were intermediate (MIC 0.12–1 mg/L) and 43% (15/35) were susceptible (MIC <= 0.06 mg/L). Resistance to erythromycin (MIC >= 1 mg/L) was high (77%, 27/35), and high-level resistance to erythromycin (MICs >= 64 mg/L) was displayed by most of the isolates from Canada (80%, 4/5) and the Far East (89%, 16/18). All levofloxacin-resistant isolates were susceptible to the newer antibacterials: telithromycin (MIC, range 0.008–0.5 mg/L; mode 0.03 mg/L), linezolid (MIC, range 0.5–2 mg/L; mode 1 mg/L) and quinupristin/dalfopristin (MIC, range 0.25–1 mg/L; mode 0.5 mg/L), (Table 2). Sources of the levofloxacin-resistant isolates were sputum (25), bronchoalveolar lavage (2), blood (2), sinus (3), throat (1) and unspecified (2). The underlying disease of patients associated with fluoroquinolone-resistant isolates was pneumonia (12), acute exacerbation of chronic bronchitis (4), acute exacerbation of chronic obstructive pulmonary disease (2) and unspecified (17).


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Table 2. MICs of standard and newer antimicrobial agents to the 35 levofloxacin-resistant S. pneumoniae from PROTEKT 1999–2000
 
Analysis of the amino acid substitutions in the 35 levofloxacin-resistant isolates is shown in Table 3. One or more mutations in the type II topoisomerase genes (gyrA, gyrB, parC and parE) were identified in all 35 isolates. Most isolates (n = 33, 94%) were found to possess a mutation in one of the DNA gyrase encoding genes (gyrA, gyrB) and in one of the topoisomerase IV encoding genes (parC, parE). Most of these mutations are well established, but those that previously have not been described in the quinolone resistance-determining region (QRDR) of the genes are shown in italics in Table 3. The QRDR for each gene was taken as that described by Pan et al. (1996).20 Eighteen (51%) isolates, including 9/10 from Hong Kong, carried both Ser-81->Phe in GyrA and Ser-79->Phe in ParC.


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Table 3. Identification of the mutations in the type II topoisomerase genes in the 35 levofloxacin-resistant strains of S. pneumoniae collected from PROTEKT 1999–2000
 
The two levofloxacin-resistant isolates that did not contain two type II topoisomerase gene mutations were from South Korea. One isolate contained only one mutation, a recognized modification in ParC (Asp-83->Tyr); the other isolate was found to have no mutation in the QRDR of any of the four genes, although a novel mutation in gyrB producing Ala-297->Ser was identified. It is possible that fluoroquinolone efflux may be contributing to resistance in these isolates, but this aspect was not investigated.

Two isolates recovered from Connecticut, USA and Hong Kong showed the highest level of levofloxacin resistance (MIC 32 mg/L) that could be attributed to three type II topoisomerase alterations. However, six other isolates [three in the USA (one each in Alabama, Connecticut, and California) and three in Canada (one in Saskatchewan and two in Edmonton)] also possessed three mutations, and a further two isolates (one in Ohio, USA and one in Edmonton, Canada) possessed four mutations, yet none of these possessed a levofloxacin MIC as high as 32 mg/L.

One further isolate of note was from Osaka, Japan, which showed resistance to levofloxacin (but not to moxifloxacin) as a consequence of mutations in parE and gyrB instead of the conventional parC and gyrA.

Several mutations outside the QRDR were identified in each gene; these probably have no effect on fluoroquinolone resistance but are worthy of mention. There were 11 different alterations in GyrA: isolate 1 from the USA had Ala-191->Val, isolates 27 and 29 from South Korea both had Ser-418->Thr and isolate 27 also had Ile-711->Val, isolate 31 from Japan had Ser-414->Tyr and isolate 34 from Germany had Ile-238->Thr. There were a multitude of alterations in isolates 12 and 14 from Mexico; Val-239->Ala, Arg-361->His, Ala-396->Ser, Glu-570->Asp and Leu-747->Phe were found in both isolates and Ala-653->Ser was also found in isolate 12. Six alterations outside the QRDR occurred in GyrB: Arg-571->Cys in isolate 1 from the USA, Glu-217->Gln in isolates 9, 10 and 11 from Canada, Lys-169->Thr in isolate 14 from Mexico, Asp-160->Glu and Thr-329->Ala in isolates 27 and 29 from South Korea, Ala-297->Ser in isolates 26 from South Korea and 32 from Japan. Six different alterations were also found in ParC, these were: from the USA, Ala-394->Thr in isolate 3, Thr-780->Ile in isolate 5, Asp-822->Tyr in isolate 6; Leu-435->Val and Gln-436->Leu in isolate 12 from Mexico and Lys-303->Glu in isolate 35 from Switzerland. Five alterations outside the QRDR occurred in ParE: Asp-252->Asn and Leu-308->Ile in isolate 1 from the USA, Arg-380->His in isolate 11 from Canada, and Asp-217->Asn and Leu-235->Phe in isolates 12 and 14 from Mexico.

Nine of the 10 isolates of S. pneumoniae with a levofloxacin MIC of 4 mg/L (intermediate resistance) were examined to identify amino acid changes in the topoisomerase genes, and these data are presented in Table 4. One isolate was unavailable for analysis. The remaining nine isolates originated from Japan (six isolates), the USA, Canada and Switzerland (one isolate each). All of these isolates were susceptible to moxifloxacin. Three isolates possessed Ser-79 alterations in ParC only and three isolates possessed Ser-81 alterations in GyrA only. One isolate possessed two substitutions (Asp-435->Glu in GyrB and Ser-79->Phe in ParC) and two isolates had ‘wild-type’ topoisomerase subunit proteins.


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Table 4. Identification of the mutations in the type II topoisomerase subunits in the PROTEKT 1999–2000 isolates of S. pneumoniae with MICs to levofloxacin of 2–4 mg/La
 
Forty isolates with an MIC of 2 mg/L for levofloxacin were also examined and most (n = 26, 65%) had ‘wild-type’ topoisomerase subunit proteins. A single mutation in parC was found in 12 isolates and a single mutation in gyrA was identified in two isolates.

Overall, there were five single gyrA mutants with a levofloxacin MIC of 2 or 4 mg/L. Interestingly, four of the five mutants possessed a moxifloxacin MIC of 1 mg/L, which is four times higher than the MIC90 of moxifloxacin for pneumococci with levofloxacin MICs of 2 or 4 mg/L (0.25 mg/L), which may reflect the target preference of DNA gyrase for moxifloxacin.

The sequence types determined by MLST are shown in Table 5. The country that submitted the largest number of levofloxacin-resistant isolates was Hong Kong, and MLST demonstrated that all 10 isolates were the same Spain23F-1 clone. Interestingly, this clone was also identified in Saskatchewan, Canada and Sao Paulo, Brazil. MLST also showed that the three resistant isolates from Edmonton, Canada, were from the same clone (the Netherlands serotype 23), which also occurred in Zurich, Switzerland. Six isolates produced MLST profiles that had not been reported previously.


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Table 5. Multilocus sequence testing in the levofloxacin-resistant isolates of S. pneumoniae from PROTEKT
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ciprofloxacin has been used widely for 15 years and the newer fluoroquinolones, such as levofloxacin and moxifloxacin, which have increased activity against S. pneumoniae, have been used to treat RTIs, including community-acquired pneumonia, since 1997. Although there have been a few reports of S. pneumoniae resistance to ciprofloxacin, and clinical failure,2124 the fluoroquinolones have remained highly effective against this organism. Surveillance studies throughout the 1990s found resistance to be very low (<1%)25,26 and most studies continue to do so.2729 It was only in 1999 that isolated reports of increased resistance to fluoroquinolones occurred and began to give cause for concern.7,30,31

Our data support the finding that fluoroquinolone resistance is generally low, but that there are pockets of increased resistance among S. pneumoniae across the world. It should be noted, however, that the fluoroquinolone-resistant isolates in our study were contributed by only one or two centres in each country, and the result therefore may not be representative of the situation in individual countries. The overall levofloxacin resistance rate was 1.0%, with 17 countries showing no levofloxacin-resistant S. pneumoniae. Increased prevalence of fluoroquinolone resistance was found in the USA (1.8%), Canada (1.4%), South Korea (2.9%), Japan (1.3%) and Hong Kong (14.3%). However, this increase is probably only significant in Hong Kong where the levofloxacin MIC90 was 16 mg/L compared with 1 mg/L in all other countries. The MLST data show that in Hong Kong this resistance was due predominantly to the spread of the virulent Spain23F-1 clone (as described previously by Ho et al.12,32,33 and Morrissey et al.18) with Ser-81->Phe and Ser-79->Phe substitutions in GyrA and ParC, respectively. However, the highest level of resistance was observed with the Spain23F-1 clone that possessed a Ser-81->Tyr substitution in GyrA and an additional Asp-453->Asn substitution in ParE. In this case and as previously suggested,11 it is likely that resistance was facilitated by the ability of this clone to disseminate. Interestingly, the same clone was found in Saskatchewan, Canada and Sao Paulo, Brazil. In Canada, this clone possessed an additional Pro-424->Ser substitution in ParE and in Brazil the ParC substitution was not at Ser-79 but at Asp-83. Clonal dominance in levofloxacin-resistant S. pneumoniae was also observed in Edmonton, Canada. This contrasts with a recent study in Canada where PFGE analysis showed 34 fluoroquinolone-resistant pneumococci to be unrelated.34 The difference may well be due to the methodology used because MLST is better able to group isolates based on ancestral lineage than PFGE. Interestingly, Zhanel et al.35 did not find fluoroquinolone resistance in Edmonton and it is possible therefore that the clonal dominance we describe in Edmonton is a more recent phenomenon. In Edmonton, the clone was the Netherlands serotype 23 strain, which was resistant to erythromycin but not penicillin. This clone was also found in Zurich, Switzerland, but on this occasion it was not resistant to erythromycin. Elsewhere, levofloxacin resistance appears to have been due to mutations in a diverse population of clones. We await further data from the PROTEKT study to see if, in future years, fluoroquinolone resistance increases and whether any increase is due to clonal spread or mutations in ‘random’ isolates.

Resistance to levofloxacin regularly confers resistance to other quinolones. In this study, 63% and 29% of levofloxacin-resistant isolates were also resistant and intermediate to moxifloxacin, respectively. Co-resistance to penicillin or macrolides was also prevalent (51% and 77%, respectively) and almost half the isolates were resistant to all three classes of antimicrobials. However, alternative agents such as telithromycin, linezolid and quinupristin/dalfopristin appeared to maintain activity against these multiresistant pneumococci. Of these, telithromycin was the most potent antimicrobial, with all the levofloxacin-resistant isolates susceptible to telithromycin (MIC 0.008–0.5 mg/L).

Of the newer agents tested, telithromycin is the only compound to have been licensed for use in community-acquired RTIs and may therefore be an effective alternative to the older agents, particularly in regions of high antibacterial resistance among S. pneumoniae.

Genotyping of the 35 levofloxacin-resistant isolates of S. pneumoniae generally supported the theory that resistance to levofloxacin and other newer fluoroquinolones requires at least two mutation events, one in DNA gyrase and one in topoisomerase IV.36,37 There were, however, eight isolates that did not fit this pattern, at least not with recognized amino acid substitutions. Further research in this area may prove that some of these unreported mutations contribute to fluoroquinolone resistance. Some of the amino acid changes lie close to recognized mutations in the QRDRs and are most likely to be significant. Specifically, these are in GyrB: Ala-297->Ser, Asp-435->Val and Glu-486->Gly; in ParC: Asp-78->Ala and Lys-136->Thr; and in ParE: Asn-405->Thr, Pro-424->Ser and Pro-454->Ser.

Investigation of the levofloxacin isolates with an MIC of 2 or 4 mg/L showed a different pattern from that seen in the levofloxacin-resistant isolates. These intermediate or marginally susceptible isolates had either one mutation or no mutations, and the proportion of single mutations was higher in the levofloxacin-intermediate group than in the -susceptible group. This supports the theory that one mutation will increase the MIC of levofloxacin and other fluoroquinolones but not sufficiently to cause resistance, in most cases.36,37 However, it must be noted that two isolates resistant to levofloxacin (but intermediate to moxifloxacin) were found to have a single topoisomerase mutation only. It is possible that changes in non-topoisomerase gene(s) are responsible for resistance and these factors remain to be investigated. Likewise, isolates with no mutations identified in the DNA gyrase or topoisomerase IV genes but with an MIC >= 2 mg/L may have some mutations that confer enhanced efflux activity, but not to the extent of allowing complete resistance to these agents. The development of these mutations will be tracked during the course of the PROTEKT study.

It has been suggested in two separate studies that fluoroquinolone resistance is associated with chronic bronchitis/obstructive pulmonary disease.38,39 Although our data for patient diagnosis were incomplete (data was only available for 18 out of 35 isolates), it would appear that other disease states such as community-acquired pneumonia (12 out of 17 isolates with diagnosis data) can also be associated with fluoroquinolone-resistant S. pneumoniae. To complicate the situation further, nine out of the 12 community-acquired pneumonia patients were all from Hong Kong. As discussed above, fluoroquinolone resistance in Hong Kong was dominated by a single clone. Therefore, as fluoroquinolone resistance is still quite rare at present it is difficult to make broad conclusions regarding the type of patients that are associated with fluoroquinolone-resistant pneumococci.

In conclusion, resistance to fluoroquinolones among S. pneumoniae remains below 1% in many countries, although higher levels were found in North America, Mexico and the Far East, especially Hong Kong. Most levofloxacin-resistant isolates possess two mutations (one in DNA gyrase and one in topoisomerase IV), whereas isolates with an MIC of 2 or 4 mg/L to levofloxacin have either no mutations or just one mutation. These first mutations can be in either DNA gyrase or topoisomerase IV; however, most were in topoisomerase IV, in the parC gene.


    Acknowledgements
 
We would like to thank the centres that participate in the PROTEKT study for the supply of isolates, the GR Micro PROTEKT team for MIC determinations, Sarah Bakker and Sylvie Buckridge for the sequencing work and Aventis for financial support.


    Footnotes
 
* Corresponding author. Tel: +44-20-7388-7320; Fax: +44-20-7388-7324; E-mail: i.morrissey{at}grmicro.co.uk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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