Circulation of international clones of levofloxacin non-susceptible Streptococcus pneumoniae in Taiwan

ORIGINAL ARTICLE

BACTERIOLOGY

Circulation of international clones of levofloxacin non-susceptible Streptococcus pneumoniae in Taiwan Y.-C. Hsieh1, L.-Y. Chang2, Y.-C. Huang3, H.-C. Lin4, L.-M. Huang2 and P.-R. Hsueh5 1) Division of Paediatric Infectious Diseases, Department of Paediatrics, Chang Gung Children’s Hospital, Chang Gung University College of Medicine, Taoyuan, 2) Department of Paediatrics, National Taiwan University Hospital, Taipei, 3) Division of Infectious Diseases, Department of Paediatrics, Chang Gung Memorial Hospital-Kaohsiung Medical Centre, Chang Gung University College of Medicine, Kaohsiung, 4) Department of Paediatrics, China Medical University Hospital, Taichung and 5) Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan

Abstract Levofloxacin susceptibility testing was carried out for a total of 2539 Streptococcus pneumoniae isolates obtained from January 2001 to February 2008 at the National Taiwan University Hospital (NTUH) and a further 228 pneumococcal isolates obtained from January 2004 to December 2006 at three other hospitals in different geographical areas in Taiwan. Levofloxacin non-susceptible S. pneumoniae isolates were subsequently analysed for serotype and molecular epidemiology. Rates of levofloxacin non-susceptibility of S. pneumoniae increased significantly from 1.2% in 2001 to 4.2% in 2007 at NTUH. A total of 30 isolates of levofloxacin non-susceptible S. pneumoniae isolates (MIC ‡ 4 mg/L) were available for evaluation of serotype, antimicrobial susceptibility, nucleotide sequence of the quinolone resistance-determining regions of parC, gyrA, parE and gyrB, reserpine effect on quinolone susceptibility and multilocus sequence type. Among these isolates, seven (23.3%) were from children, and two (6.7%; one from a 3- and one from a 93-year-old patient) were from blood. One levofloxacin-resistant isolate (MIC = 8 mg/L) was recovered from a previously healthy child with bacteraemic necrotizing pneumonia complicated by empyema and a haemolytic-uraemic syndrome. All isolates except two had Ser79 and/or Asp83 changes in ParC, and/or Ser81 or Glu85 changes in GyrA. An efflux phenotype concerning levofloxacin was detected in only one (3.3%) isolate. A novel clone (ST3642), genetically related to Spain9V-3 and belonging to serotype 11A, was identified. Dissemination of clonal complexes related to Spain23F-1, Taiwan19F-14, Spain9V-3 and Taiwan23F-15 has contributed to levofloxacin non-susceptibility among these S. pneumoniae isolates from Taiwan. Keywords: Clonal spread, levofloxacin-non-susceptible, Streptococcus pneumoniae, Taiwan Original Submission: 27 December 2008; Revised Submission: 13 March 2009; Accepted: 5 April 2009 Editor: J.-L. Mainardi Article published online: 23 September 2009 Clin Microbiol Infect 2010; 16: 973–978 10.1111/j.1469-0691.2009.02951.x Corresponding author and reprint requests: P-R. Hsueh and L-M Huang, Departments of Laboratory Medicine and Internal Medicine, Paediatrics National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei 100, Taiwan E-mails: [email protected]; [email protected]

Introduction Levofloxacin, a newer fluoroquinolone with increased activity against Streptococcus pneumoniae, has been an effective antimicrobial agent in treating adults with community-acquired pneumonia. However, significant increases in the incidence of levofloxacin resistance have been reported over the past

decade in some countries, such as Hong Kong, the USA, Canada, Spain and South Africa [1–5]. Levofloxacin is not approved for use in children. The implementation of the pneumococcal 7-valent conjugate vaccine for children, beginning in 2000 in the USA, has successfully reduced the incidence of invasive pneumococcal disease and acute otitis media caused by S. pneumoniae with vaccine serotypes, and has decreased the spread of multidrug-resistant S. pneumoniae [6–8]. Nevertheless, multidrug-resistant S. pneumoniae with non-vaccine serotypes has emerged [9,10]. These findings suggest the need for more prudent use of fluoroquinolones in order to maintain their effectiveness as a defence against S. pneumoniae infection. This report describes the serotypes, antimicrobial susceptibility and

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molecular epidemiology of levofloxacin non-susceptible S. pneumoniae isolates collected between 2001 and 2008 in Taiwan.

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presence of reserpine (10 mg/L; Sigma, St Louis, MO, USA) [14]. Serotyping

Materials and Methods Bacterial isolates

In order to analyse the trend of levofloxacin non-susceptibility among S. pneumoniae isolates, we collected non-duplicate (one isolate per patient) clinical isolates recovered from various clinical specimens (blood, pleural effusion, cerebrospinal fluid, ascites, sputum, and other respiratory secretions) of patients treated at National Taiwan University Hospital (NTUH) from 2001 to 2008.These isolates were identified by conventional biochemical methods [11]. Susceptibility results of S. pneumoniae were determined by the disk diffusion method [12]. In addition, during the period 2004–2006, a total of 228 S. pneumoniae isolates were prospectively collected from three other hospitals in Taiwan: 36 from China Medical College Hospital, Taichung; 18 from Buddist Tzu Chi General Hospital, Hualien; and 174 from Chang-Gung Memorial Hospital, Kaohsiung [13].

The serotypes of isolates were determined using the capsular swelling method (Quellung reaction). All antisera were obtained from the Statens Seruminstitut (Copenhagen, Denmark). PCR and DNA sequence analysis

Bacterial genomic DNA was prepared with the QIAGEN Genomic DNA system (QIAGEN, Valencia, CA, USA). PCR reactions to detect mutations in the quinolone resistancedetermining regions (QRDRs) were performed using previously described primers [15]. Multilocus sequencing typing

Multilocus sequencing typing (MLST) was carried out as previously described [16]. New MLST profiles were submitted to the curator at http://spneumoniae.mlst.net for new ST numbers. A clonal complex was defined as previously described [17]. Statistical analysis

Standard powders of fluoroquinolones were obtained from their manufacturers as follows: levofloxacin (Daiichi Pharmaceuticals, Tokyo, Japan); ofloxacin (Daiichi Pharmaceuticals); gatifloxacin (Bristol-Myers Squibb, Princeton, NJ, USA); moxifloxacin and ciprofloxacin (Bayer Co., Leverkusen, Germany); trovafloxacin (Pfizer, New York, NY, USA); and gemifloxacin (GlaxoSmithKline, Greenford, UK). The MICs of levofloxacin were determined by the broth microdilution method for levofloxacin non-susceptible isolates. The results were interpreted according to the guidelines established by the CLSI [12]. Streptococcus pneumoniae ATCC 49619 was used as a quality control. A total of 56 isolates, (50 isolates from the NTUH, 47 isolates from 2001–2007 and three isolates from 2008, and six isolates from the three other hospitals) with levofloxacin MICs ‡ 4 mg/L were identified. Thirty isolates, including 24 isolates from the NTUH and six isolates from the three other hospitals, were available for MIC determination of ofloxacin, ciprofloxacin, gatifloxacin, trovafloxacin, moxifloxacin and gemifloxacin by the broth microdilution method (in-house panels) [11,12].

Trends of rates of levofloxacin non-susceptibility over time were evaluated using the Cochran–Armitage Trend Test. A p value < 0.05 was considered statistically significant.

Results Of the 2239 S. pneumoniae isolates collected at NTUH from 2001–2007, 47 (2.1%) were non-susceptible to levofloxacin (Fig. 1). There was a significant increasing trend in Levofloxacin-nonsusceptible isolates (%)

Antimicrobial susceptibility testing

5 4.2 4

*

3

2.5

2.3

2005

2006

2.1 2 1.2

1.4

1.2

1

0

2001

2002

2003

2004

2007

(n = 332) (n = 345) (n = 239) (n = 331) (n = 360) (n = 345) (n = 287)

FIG. 1. Trend of non-susceptibility to levofloxacin among StreptococReserpine efflux assay

cus pneumoniae isolates collected from 2001–2007 at National Tai-

The efflux phenotype was defined as a fourfold or greater reduction in the MICs of various quinolones tested in the

wan University Hospital. *A significant increasing trend (p <0.05) from 2001–2007.

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Hsieh et al.

Clones of levofloxacin non-susceptible Streptococcus pneumoniae

levofloxacin non-susceptible rates among S. pneumoniae isolates at NTUH (p <0.05) (Fig. 1). A 3.5-fold increase in rates from 1.2% in 2001 to 4.2% in 2007 was noted. Among the 30 isolates of levofloxacin non-susceptible S. pneumoniae (MICs of ‡ 4 mg/L) available for study, 24 (80%) were collected from NTUH and seven (23.3%) were recovered from children (Table 1). The majority of levofloxacin non-susceptible isolates (n = 28, 93.3%) were from respiratory tract secretions. The mean age of these patients was 62 years with a median of 77 years (range 3–93 years). Two isolates (6.7%) were recovered from blood; one (isolate 3) from a 3-year-old boy, and the other (isolate 15) from a 93-year-old female patient. The previously healthy boy developed pneumococcal pneumonia complicated with necrotizing pneumonia, empyema and haemolytic-uraemic syndrome. His history was negative for day-care centre attendance and for family members with a diagnosis of tuberculosis (TB). However, his history of antibiotic use could not be determined. The adult patient had a history of chronic obstructive airway disease and developed pneumococcal pneumonia with septic shock. The following seven serotypes were found among the levofloxacin non-susceptible isolates: 19F (n = 11, 36.7%), 23F (n = 9, 30%), 14 (n = 4, 13.3%), 6B (n = 2, 6.7%), 9V (n = 1, 3.3%), 11A (n = 3, 10%). Spread of international clones or clones related to international clones has been observed. Spain23F-1 (ST81) was the largest clone, which included serotype 23F (n = 6) and serotype 19F (n = 4). Spain23F-1-related clones ST2891 (n = 2) and ST83 (n = 1) were also found. Taiwan19F-14 (ST236) was the second largest clone, which included serotype 19F (n = 4) and serotype 14 (n = 1). Spain9V-3 (ST156)-related clones ST3642 (n = 3), ST1569 (n = 1) and ST166 (n = 1) constituted the third largest. ST 3642 was a novel clone consisting of three isolates of serotype 11A. Among the isolates with a levofloxacin MIC of 64 mg/L, three (75%, 3/4) belonged to ST3271 (double locus variant of Taiwan23F15(ST242)). Interestingly, each of these three isolates had a different serotype (serotypes 23F, 19F and 14) and was recovered in a different year (2003, 2006 and 2008). These findings indicate that ST3271 isolates with high-level levofloxacin resistance circulated in the community. Gemifloxacin had the highest level of antipneumococcal activity. The MICs of gemifloxacin were 16- to 128-fold lower than those of levofloxacin. Only one of the levofloxacin non-susceptible isolates had a gemifloxacin MIC which exceeded 1 mg/L: isolate 18 with a gemifloxacin MIC of 4 mg/L. According to the CLSI criteria, 60% (n = 18) of the 30 isolates were non-susceptible to gemifloxacin and trovafloxacin, 63.3% (n = 19) were non-susceptible to

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moxifloxacin, 93.3% (n = 28) were non-susceptible to gatifloxacin and 100% (n = 30) were non-susceptible to ofloxacin. Sequencing of the QRDRs of ParC, GyrA, ParE and GyrB revealed seven types of amino acid substitution in ParC, four types of amino acid substitution in GyrA, four types of amino acid substitution in ParE and one type of amino acid substitution in GyrB (Table 1). Most of the isolates with levofloxacin MICs from 4 to 8 mg/L had amino acid changes involved in resistance, including Ser79 or Asp83 alterations in ParC, or Ser81 or Glu85 alterations in GyrA. Isolates 16 and 29 were found not to have a significant amino acid change but a I460V polymorphism in ParE. Efflux phenotype was not detected in isolate 29. Isolate 16 had the same I460V polymorphism as isolate 29, but had an efflux phenotype. Most of the isolates with levofloxacin MICs from 16 to 64 mg/L had both Ser79 and/ or Asp83 changes in ParC, and Ser81 or Glu85 changes in GyrA. Isolates 20, 21 and 22 only had S79Y changes in ParC and the I460V polymorphism in ParE; these isolates had no amino acid changes in GyrA and GyrB and lacked the efflux phenotype. Only some isolates of Spain23F-1 or Taiwan19F-14 shared the identical amino acid changes or polymorphisms at the QRDRs. Two isolates of Spain23F-1 carried K137N in ParC, E85K in GyrA, and I460V, D435N in ParE. Three isolates of Spain23F-1 carried S79F, K137N in ParC, S81F in GyrA, I460V in ParE. Two isolates of Taiwan19F carried S79F in ParC, and I460V in ParE. Amino acid changes or polymorphism in other isolates of Spain23F-1 or Taiwan19F-14 were heterogeneous. By contrast, three isolates belonging to ST3642 and three high-level levofloxacin-resistant isolates (MICs of 64 mg/L) belonging to ST3271 shared identical amino acid changes and polymorphisms at the QRDRs. An efflux phenotype concerning levofloxacin was detected in only one of the 30 levofloxacin non-susceptible isolates. Among the 30 levofloxacin non-susceptible isolates, an efflux mechanism contributing to ciprofloxacin resistance was detected in 14 isolates, to gemifloxacin resistance in three isolates, and to moxifloxacin resistance in one isolate.

Discussion Taiwan is a country with a high prevalence of b-lactam and macrolide-resistant S. pneumoniae [11]. Nevertheless, the most recent previous studies from Taiwan found the overall prevalence rate of levofloxacin resistance in S. pneumoniae to be only c. 2% [11,13]. During the 8-year period of the present study, levofloxacin non-susceptibility among S. pneumoniae isolates increased over time at NTUH, and thus a

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9V 19F 23F 14 14 6B 6B 19F

23/NTUHc,d 24/NTUH 25/NTUH 26/NTUH 27/NTUH 28/CMCH 29/CMCHd 30/NTUHd

ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 973–978 81 81 83 (81SLV) 81 2891 (81SLV) 2891 (81SLV) 81 81 81 81 81 81 81 236 236 236 236 236 1569 (156SLV) 3642 (156DLV) 3642 (156DLV) 3642 (156DLV) (81SLV) 166 (156SLV) 3271 (242DLV) 3271 (242DLV) 3271 (242DLV) 2652 (9DLV) 90 1106 76

ST

156 242 242 242 70

81 81 81 81 81 81 81 81 81 81 81 81 81 236 236 236 236 236 156 156 156 156

Clonal complex

79 86 58 78 Sputum 6 6 75

6 6 77 80 80 75 87 70 93 80 89 87 84 7 3 72 85 4 79 77 77 80

Age (years)

Sputum Sputum Sputum Sputum S79Y Nasal pus Nasal pus Sputum

Nasal pus Nasal pus Sputum Sputum Sputum Sputum Sputum Sputum Blood Sputum Sputum Sputum Sputum Throat Blood Sputum Sputum Nasal pus Sputum Sputum Sputum Sputum

Source

N68S, D83Y S79F, D83Y S79F, D83Y S79F, D83Y S81F – – –

– – D83Y – – – S79F S79F S79F S79F S79F S79F, D91N S79Y S79F S79F – S79Y S79F, D83N S79Y S79Y S79Y S79Y

parCa,b

S81F S81F S81F S81F – S81F – S81F

E85K E85K S81F S81Y E85G E85G S81F S81F S81F S81F S81Y S81F S81F – – – S81F E85K S81F – – –

gyrAa,b

– – – – – D435N – D435N

D435N D435N – D435N, R447C D435H D435H – – – – – – – – – – – – – – – –

parEa,b

– E474K E474K E474K 16 – – –

– – – – – – – – – – – – – E474K E474K – – – – – – –

gyrBa,b

16 64 64 64 4 8 4 8

8 8 8 8 8 8 8 16 16 16 16 16 32 4 8 8e 32 64 16 16 16 16

LVX LVX

MIC (mg/L)

8 32 32 32 2 4 2 4

4 2 2 4 2 4 8 4 8 8 8 8 16 1 2 0.5 8 16 8 8 8 8

GATI

4 8 4 4 32 1 1 1

1 2e 1 1 1 1 2 2 2 2 2 2 4 0.25 0.25 1 4 8 4 2 2 4

MOX

32 128 128 64 4 8 8 16

8 16 16 16 16 16 16 16 16 16 16 32 32 8 8 4 32 64 32 16 16 32

OFX

4 16 16 16 32e 0.25 0.25 0.5

0.5 0.25 1 1 0.5 0.5 4 2 4 2 2 4 4 0.5 1 0.5 8 8 4 4 4 4

TRO

64e 64 64 64e 0.25 4 2 4

4e 4e 16e 8 4e 4e 16e 32e 16e 16e 32 32 128e 8 8 4 64 64e 32 32 16 32

CIP

0.06 0.06 0.06

1 0.5 0.5 0.5

0.06 0.06 0.12 0.12e 0.06 0.06 0.25 0.25 0.25 0.25 0.25 0.25 1e 0.06 0.06 0.06 0.5 4e 0.5 0.5 0.25 0.5

GEMI

a

LVX, levofloxacin; GATI, gatifloxacin; MOX, moxifloxacin; OFX, ofloxacin; TRO, trovafloxacin; CIP, ciprofloxacin; GEMI, gemifloxacin; SLV, single locus variant; DLV, double locus variant; CGMH, Chang-Gung Memorial Hospital; NTUH, National Taiwan University Hospital; All amino acid positions refer to S. pneumoniae sequences [28,29]. b No amino acid change in the quinolone resistance-determining regions compared with R6. c Isolate had K137N change in ParC that appears not to contribute to fluoroquinolone resistance [25]. d Isolate had I460V change in GyrA that appears not to contribute to fluoroquinolone resistance [26]. e Efflux phenotype (four-fold or greater reduction in MICs of fluoroquinolones in the presence of reserpine).

England14-9 Spain6B-2

Taiwan23F-15

Spain9V-3

Taiwan19F-14

23F 23F 23F 23F 23F 19F 19F 19F 19F 19F 23F 23F 23F 19F 19F 19F 14 19F 14 11A 11A 11A

1/CGMHc,d 2/CGMHc,d 3/NTUHd 4/NTUHc,d 5/NTUHc,d 6/NTUHd,e 7/NTUHc,d 8/NTUHd 9/NTUHc,d 10/NTUHc,d 11/NTUHc,d 12/NTUHd 13/NTUHc,d 14/NTUHd 15/NTUHd 16/NTUHd 17/NTUHd 18/CGMHd 19/CGMHc,d 20/NTUHc,d 21/NTUHc,d 22/NTUHc,d

Spain23F-1

Serotype

Isolate/ hospital

Related international clones

TABLE 1. Characteristics of 30 Streptococcus pneumoniae isolates non-susceptible to levofloxacin, collected from 2001 to 2008 in Taiwan

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Clones of levofloxacin non-susceptible Streptococcus pneumoniae

total of 30 isolates not susceptible to levofloxacin were collected for analysis. Reported risk factors for levofloxacin resistance were nosocomial infection, age in conjunction with ‡ 75 years chronic obstructive airway diseases, residence in a nursing home, and prior fluoroquinolone treatment [18]. In the study, we identified an invasive isolate of levofloxacin-resistant S. pneumoniae from a previously healthy child. Levofloxacin resistance was rarely seen in children because fluoroquinolones are not recommended for use in paediatric patients. There have been few reports on invasive disease caused by levofloxacin non-susceptible S. pneumoniae in children [5,19,20]. Most cases of invasive disease caused by levofloxacin non-susceptible S. pneumoniae have been in human immunodeficiency virus-seropositive children with a history of TB treatment (some patients received fluoroquinolones in the regimen), who usually had associated nosocomial infections [5]. Taiwan is an area of high disease burden, with a high incidence of drug-resistant TB [21]. Whether the use of fluoroquinolones in the treatment of TB in adults has increased the potential for spread of levofloxacin non-susceptible S. pneumoniae in children remains to be studied. Reports on the clonal spread of fluoroquinolone-resistant S. pneumoniae are not as abundant as those on penicillinresistant strains. Clonal dissemination attributable to fluoroquinolone resistance has been reported in Hong Kong, the USA and Spain [1,3,4]. In Canada and Korea, the genetic relationship among fluoroquinolone-resistant pneumococcal isolates was heterogeneous [14,22], which indicated that fluoroquinolone resistance arose via independent mutations. In Spain, fluoroquinolone resistance was found to have been acquired by pre-existing clones through point mutation or recombination with viridians streptococci [23]. In this study, we found that dissemination of the international clones Spain23F-1, Taiwan19F-14 and Taiwan23F-15 contributed to levofloxacin resistance in S. pneumoniae in Taiwan. The heterogeneity of amino acid substitutions in the QRDRs among isolates belonging to Spain23F-1 and Taiwan19F-14 clones suggests that these organisms disseminate not only by clonal spread but also by independent selection due to antimicrobial pressure. Interestingly, dissemination of clonal complexes related to Spain23F-1, Taiwan19F-14 and Taiwan23F-15 was also responsible for the spread of high b-lactam resistance in Taiwan [13]. These international clones might have some biological advantage that enables them to build up various antimicrobial resistances. On the other hand, we found that ST3271 (double locus variant of Taiwan23F-15), with high-level resistant strains, circulated in the community in different years but were not increasing in frequency; these appear to have more of a random occurrence than clonal dissemination.

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Fluoroquinolone resistance in S. pneumoniae is mediated by mutations in the QRDRs of DNA gyrase and/or topoisomerase IV, or by decreasing intracellular drug accumulation through active efflux [24]. Decreased fluoroquinolone susceptibility in S. pneumoniae occurs in a stepwise fashion, which is also dependent on the selecting agents [25]. Generally, amino acid changes at ParC/S79 or GyrA/S81 confer fluoroquinolone resistance, whereas amino acid changes at ParC/K137 and ParE/I460 do not [26]. In the present study, all but two of the isolates had Ser79 and/or Asp83 alterations in ParC, and/or Ser81 or Glu85 alterations in GyrA. The efflux pump had the greatest effect on ciprofloxacin MICs. Reserpine-inhibited efflux was found not only in isolates with low-level ciprofloxacin resistance but also in isolates with high-level ciprofloxacin resistance. Isolate 29 had a levofloxacin MIC of 4 mg/L, but only one amino acid alteration was found: I460V polymorphism in parE. I460V in parE has not been found to contribute to fluoroquinolone resistance in previous studies [26]. The MICs of levofloxacin in isolates 20, 21 and 22 were the same as in isolate 19. However, isolate 19 had an additional amino acid change of S81F in GyrA, which has been shown to contribute to increased levofloxacin MICs in numerous previous studies [26]. Based on the above two observations of isolates with increased levofloxacin MICs lacking resistance-associated mutations, we hypothesize that genes other than topoisomerase genes may be involved in levofloxacin resistance in pneumococci. Four isolates with high-level levofloxacin resistance (MIC = 64 mg/L) in this study had both Ser79 and Asp83 changes in ParC and Ser81 or Glu85 changes in GyrA. In a previous study, E85K in GyrA was found only in S. pneumoniae isolates with high-level ciprofloxacin resistance [27]. With regard to levofloxacin resistance, isolate 18 harbouring E85K in GyrA had high-level levofloxacin resistance. However, isolates 1 and 2 also had E85K in GyrA but the MICs of levofloxacin (8 mg/L) revealed only low-level resistance. Our results indicate that E85K in GyrA contributes to highlevel levofloxacin resistance in situations where there is a relevant previous mutation in ParC. This study documents a significant increasing trend of levofloxacin non-susceptibility among S. pneumoniae isolates at NTUH. These results may not represent the situation in the whole of Taiwan however, and continued surveillance of levofloxacin resistance in S. pneumoniae is needed.

Transparency Declaration This work was supported by grants from the National Science Council, Taiwan and Wyeth-Ayerst (Asia) Ltd, Taiwan

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branch. The authors declare that they have no conflicting interests in relation to this work.

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