Abstract

Objectives: To determine the relationship between New Zealand isolates of antibiotic-resistant Streptococcus pneumoniae and internationally widespread resistant clones.

Methods: Fifty-nine isolates representing both invasive and non-invasive pneumococci from multiple locations, serotypes and years were analysed by multilocus sequence typing.

Results: Major international clones, including Spain23F-1, France9V-3 and Taiwan19F-14, were found to be present in New Zealand. A one-allele variant of the Taiwan19F-14 clone (aroE 15→4, ST 271) was particularly prominent.

Conclusions: Antibiotic-resistant pneumococci have not evolved de novo in New Zealand, but were introduced to the country during the early 1990s.

Introduction

Streptococcus pneumoniae is an important cause of community-acquired infection and its increase in resistance to antibiotics has complicated therapeutic options worldwide. The increase in prevalence of resistant pneumococci is often attributed to the clonal dissemination of one or more particular pneumococcal strains. Often these strains arise in countries where antibiotic use is not well regulated, and subsequently spread worldwide. Molecular epidemiological methods, particularly multilocus sequence typing (MLST), are well suited to tracking the clonal spread of such strains.

New Zealand, as with many other countries, saw a rapid increase in the prevalence of penicillin non-susceptible S. pneumoniae in the late 1990s. In order to explain this observed increase, we sought to determine whether a resistant strain(s) emerged de novo in New Zealand, or if a resistant clone(s) from overseas, which subsequently radiated into New Zealand, was responsible.

Materials and methods

Pneumococcal isolates were obtained from those submitted to the central communicable disease laboratory, Institute of Environmental Science and Research (ESR), Porirua. Fifty-nine isolates were obtained from 12 cities throughout New Zealand (Figure 1) during 1992–2000. Representatives of major antibiotic-resistant clones (as assessed by PFGE; data not shown) were included from hospitals and laboratories throughout New Zealand. Thirty-nine isolates (66%) were included from the North Island and 20 (34%) from the South Island. The two major contributing centres were Auckland and Christchurch, with 20 (34%) and 15 (25%) isolates from each, respectively. Forty-three (73%) of the pneumococcal isolates included were from invasive disease; 37 (63%) from blood and six (10%) from other sterile sites. Non-invasive isolates (n=16, 27%) typical of major clones circulating in New Zealand (as determined by PFGE; data not shown) were also characterized. A number of isolates were selected retrospectively in order to establish when major clones were first recovered in New Zealand.

Penicillin and cefotaxime MICs were determined using Etest (AB Biodisk, Solna, Sweden), whereas resistance to other antibiotics (erythromycin, chloramphenicol, tetracycline, co-trimoxazole and vancomycin) was determined by disc diffusion (Oxoid, Basingstoke, UK) in accordance with NCCLS standard guidelines.1 Serotyping was performed by the Streptococcus Reference Laboratory (ESR Porirua, New Zealand), using the capsular reaction test (Neufeld test) and interpreted using the Danish system of nomenclature. MLST was performed as described by Enright & Spratt.2 DNA sequencing was performed using an ABI Prism 310 automated sequencer (Applied Biosystems) and BigDye terminator mix (Applied Biosystems). SmaI macrorestriction profiles were generated and compared using PFGE, as described previously.3 Amplification of the genes pbp2b and pbp2x  was performed using PCR, as described previously.3

Results and discussion

Twenty-nine (49%) of the 59 New Zealand isolates could be assigned to one of five major antibiotic-resistant clones defined by the Pneumococcal Molecular Epidemiology Network (PMEN).4 The clones identified were Spain23F-1 (ST 81, n=13), Spain6B-2 (ST 90, n=2), France9V-3 (ST 156, n=8), England14-9 (ST 9, n=1) and Taiwan19F-14 (ST 236, n=5). A further 18 isolates (31%) were single-allele derivatives of three of the PMEN major antibiotic-resistant clones: Spain23F-1 (spi 4→6, ST 83, n=1), France9V-3 (ddl 1→14, ST 162, n=3) and Taiwan19F-14 clones (two variants: ddl 26→1, ST 237, n=1; and aroE 15→4, ST 271, n=13). In the current study, one-allele variants were considered to be members of the same clonal group. Data are summarized in Table 1.

Not surprisingly, two of the major drug-resistant pneumococcal clones identified in New Zealand were determined to be members of the Spain23F-1 clone and the France9V-3 clone. Previous MLST studies have shown the widespread global distribution of these clones.5,6 The first member of the Spain23F-1 clonal group identified in this study was isolated from a blood culture in Auckland during early 1994. Twelve of the 14 members of this clone were from invasive specimens. Within the Spain23F-1 clonal lineage, pbp2b and pbp2x  restriction fragment length polymorphism (RFLP) profiles were conserved, as were the corresponding MICs of both penicillin and cefotaxime. A notable exception was observed, however, in the three Spain23F-1 clonal group isolates expressing the 19F capsular type. In two of the three 19F isolates, the MIC was at least one doubling dilution higher for both β-lactams than those isolates expressing the 23F capsule.

All 11 isolates from the France9V-3 clonal group were recovered from blood cultures. All members of this group with ST 156 were isolated during 1995 or thereafter, and exhibited reduced susceptibility to β-lactams. However, three single-allele variants (ST 162, ddl 1→14) were isolated during or prior to 1995 and were all susceptible to penicillin. The pbp2b gene has been shown to be physically linked to the ddl gene, and co-transfer has been demonstrated previously.7 This is supported by the acquisition of β-lactam resistance also being coupled to a change in the pbp RFLP patterns. None of the New Zealand examples of the France9V-3 clone was found to be multidrug-resistant, although one isolate did express resistance to co-trimoxazole as well as penicillin.

In contrast to the Spain23F-1 clone, which had been prevalent in Spain for at least a decade before its intercontinental spread was first documented,8 the Taiwan19F-14 clone has emerged relatively recently, but seems equally adept at global dissemination. Nineteen isolates of the Taiwan19F-14 clonal group were noted, all of which were serotype 19F with the exception of a single serotype 14 isolate. Curiously, the single-allele variant (ST 271, aroE 15→4) of the Taiwan19F-14 clone was the most prominent in the current study; of 19 isolates, 13 (68%) were ST 271. This particular variant has previously been described as the major Korean 19F clone (www.mlst.net). Five isolates were from invasive sites (three ST 271 and two ST 236).

The New Zealand Taiwan19F-14 clonal group is resistant to erythromycin, co-trimoxazole and tetracycline and is near uniformly susceptible to chloramphenicol and vancomycin. Two isolates recovered during 1999–2000, however, had acquired resistance to chloramphenicol. Levels of β-lactam resistance are variable among this group. In the case of the five ST 236 isolates, the penicillin and cefotaxime MICs were consistently 2 and 1 mg/L (one instance of a cefotaxime MIC of 0.5 mg/L), respectively; however, among the ST 271 isolates MICs were in the range 2–8 mg/L and 1–32 mg/L of penicillin and cefotaxime, respectively. The observed MIC variability did not correlate with changes in their respective pbp RFLP profiles (data not shown).

The Taiwan19F-14 clone has emerged only recently. It was first formally identified when multiply antibiotic-resistant S. pneumoniae from Taiwanese hospitals were examined by MLST, showing this clone was present in Taiwan at least as early as 1993.6 In 1995, two serotype 19F isolates were recovered from blood cultures in a London hospital that had the same allelic profile.6 This is the same year that the Taiwan19F-14 clone was observed in New Zealand and coincides with a period of increased prevalence of penicillin-resistant S. pneumoniae throughout the country. The Taiwan19F-14 clone was identified in the USA as early as 1997–1998.9 Therefore it seems likely that the Taiwan19F-14 strain did in fact originate in Taiwan, or elsewhere in Asia, and began to disseminate globally as early as 1995.

The remaining 12 isolates yielded allelic profiles that matched eight previously defined STs, but do not belong to any of the currently described PMEN clones. These were predominantly invasive, non-resistant isolates, and were representative of multiple serotypes: 9N (ST 66, n=2), 14 (ST 124, n=2; ST 129, n=1; and ST 346, n=1), 19A (ST 199, n=2; and ST 876, n=1), 19F (ST 146, n=1) and 23F (ST 36, n=2). In each instance, these sequence types corresponded to isolates from invasive disease recovered elsewhere in the world. In particular, the ST 124 isolates and one single-allele variant (ddl 14→5, ST 129) are members of an important clone associated with invasive disease and have been recovered throughout the world, including Sweden, Denmark, Norway, Finland, the UK, Australia and Canada. Another invasive clone, ST 199, and a single-allele variant (spi 17→6, ST 876), has been recovered previously from invasive disease in the UK. The inclusion of antibiotic-susceptible invasive clones in the PMEN database will better allow the distribution of these strains to be monitored worldwide.

Antibiotic-resistant S. pneumoniae in New Zealand can be attributed mainly to the global spread of international resistant clones; in particular the well-described Spain23F-1 and France9V-3 clones, as well as the more recently described Taiwan19F-14 clone. The use of MLST and the PMEN-described clones4 allow effective worldwide surveillance of important pneumococcal clones.

Present address: US Naval Medical Research Unit-3, Cairo, Egypt.

Figure 1.

Distribution and serotype (number of isolates) of pneumococcal isolates throughout New Zealand.

Figure 1.

Distribution and serotype (number of isolates) of pneumococcal isolates throughout New Zealand.

Table 1.

Distribution of MLST sequence types for 59 New Zealand isolates matching pre-existing allelic profiles in the database

    MIC (mg/L)
 
  Allelic profiles
 
      
Year of isolation ST Serotype n penicillin cefotaxime Resistance profilea aroE gdh gki recP spi xpt ddl 
Spain23F-1              
    1994–2000 81 23F 1–4 E5, S3/4, T2/4, C 
    1996–2000 81 19F 2–4 1–8 E, S2/2, T2/2, C 
    1997 81 14 E, S, T, C 
    1997 83 23F E, S, C 
Spain6B-2              
    1993–96 90 6B E, S, T, C 
France9V-3              
    1995–98 156 9V 0.5–1 S1/3 11 10 
    1998 156 14 – 11 10 
    1994–95 162 9V 0.016 0.016 – 11 10 14 
England14-9              
    1992 14 0.016 0.016 
Taiwan19F-14              
    1997–98 236 19F 0.5–1 E, S, T 15 16 19 15 20 26 
    1998 237 19F 0.5 16 E, S, T 15 16 19 15 20 
    1995–2000 271 19F 12 2–8 1–32 E, S, T, C2 16 19 15 20 26 
    1999 271 14 E, S, T 16 19 15 20 26 
Non-PMEN clones              
    1993–97 36 23F 0.016–2 0.016–1 C1 
    1993–95 66 9N 0.03 0.016 – 
    1994–95 124 14  0.016 – 14 11 14 
    1994 129 14 0.016 0.016 14 11 
    1994 146 19F 0.016 0.016 – 15 14 
    1992–93 199 19A 0.25 0.06 13 14 17 14 
    1995 346 14 0.5 0.12 – 10 41 47 16 14 
    1995 876 19A 0.12 0.06 13 14 14 
    MIC (mg/L)
 
  Allelic profiles
 
      
Year of isolation ST Serotype n penicillin cefotaxime Resistance profilea aroE gdh gki recP spi xpt ddl 
Spain23F-1              
    1994–2000 81 23F 1–4 E5, S3/4, T2/4, C 
    1996–2000 81 19F 2–4 1–8 E, S2/2, T2/2, C 
    1997 81 14 E, S, T, C 
    1997 83 23F E, S, C 
Spain6B-2              
    1993–96 90 6B E, S, T, C 
France9V-3              
    1995–98 156 9V 0.5–1 S1/3 11 10 
    1998 156 14 – 11 10 
    1994–95 162 9V 0.016 0.016 – 11 10 14 
England14-9              
    1992 14 0.016 0.016 
Taiwan19F-14              
    1997–98 236 19F 0.5–1 E, S, T 15 16 19 15 20 26 
    1998 237 19F 0.5 16 E, S, T 15 16 19 15 20 
    1995–2000 271 19F 12 2–8 1–32 E, S, T, C2 16 19 15 20 26 
    1999 271 14 E, S, T 16 19 15 20 26 
Non-PMEN clones              
    1993–97 36 23F 0.016–2 0.016–1 C1 
    1993–95 66 9N 0.03 0.016 – 
    1994–95 124 14  0.016 – 14 11 14 
    1994 129 14 0.016 0.016 14 11 
    1994 146 19F 0.016 0.016 – 15 14 
    1992–93 199 19A 0.25 0.06 13 14 17 14 
    1995 346 14 0.5 0.12 – 10 41 47 16 14 
    1995 876 19A 0.12 0.06 13 14 14 
a

Predominant resistance profile; E, erythromycin; S, co-trimoxazole; T, tetracycline; C, chloramphenicol. Subscripts indicate number of isolates resistant to given agent, if not all resistant. Co-trimoxazole resistance and tetracycline resistance were not tested in all isolates; in these instances the subscript denotes resistant isolates as a fraction of those tested.

We thank Diana Martin and Maggie Brett for providing pneumococcal isolates, and Helen Heffernan and Lucinda Hall for their comments on the manuscript. D.C.B. was the recipient of a PhD scholarship from the Institute of Environmental Science and Research, New Zealand, which is gratefully acknowledged. We acknowledge the use of the pneumococcal MLST database which is located at Imperial College London and is funded by the Wellcome Trust.

References

1.
National Committee for Clinical Laboratory Standards. (
2003
). Performance Standards for Antimicrobial Disc Susceptibility Tests—Eighth Edition: Approved Standard M2-A8. NCCLS, Wayne, PA, USA.
2.
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
,
3049
–60.
3.
Bean, D. C., Ikram, R. B. & Klena, J. D. (
2004
). Molecular characterization of penicillin non-susceptible Streptococcus pneumoniae in Christchurch, New Zealand.
Journal of Antimicrobial Chemotherapy
 
54
,
122
–9.
4.
McGee, L., McDougal, L., Zhou, J. et al. (
2001
). Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network.
Journal of Clinical Microbiology
 
39
,
2565
–71.
5.
Zhou, J., Enright, M. C. & Spratt, B. G. (
2000
). Identification of the major Spanish clones of penicillin-resistant pneumococci via the Internet using multilocus sequence typing.
Journal of Clinical Microbiology
 
38
,
977
–86.
6.
Shi, Z. Y., Enright, M. C., Wilkinson, P. et al. (
1998
). Identification of three major clones of multiply antibiotic-resistant Streptococcus pneumoniae in Taiwanese hospitals by multilocus sequence typing.
Journal of Clinical Microbiology
 
36
,
3514
–9.
7.
Enright, M. C. & Spratt, B. G. (
1999
). Extensive variation in the ddl gene of penicillin-resistant Streptococcus pneumoniae results from a hitchhiking effect driven by the penicillin-binding protein 2b gene.
Molecular Biology and Evolution
 
16
,
1687
–95.
8.
Munoz, R., Coffey, T. J., Daniels, M. et al. (
1991
). Intercontinental spread of a multiresistant clone of serotype 23F Streptococcus pneumoniae.
Journal of Infectious Diseases
 
164
,
302
–6.
9.
Richter, S. S., Heilmann, K. P., Coffman, S. L. et al. (
2002
). The molecular epidemiology of penicillin-resistant Streptococcus pneumoniae in the United States, 1994–2000.
Clinical Infectious Diseases
 
34
,
330
–9.