Molecular characterization of penicillin non-susceptible Streptococcus pneumoniae in Christchurch, New Zealand

David C. Bean1,*, Rosemary B. Ikram2 and John D. Klena3

1 Centre for Infectious Disease, Barts and The London School of Medicine and Dentistry, Queen Mary College, University of London, London E1 2AD, UK; 2 Medlab South Ltd., Christchurch, New Zealand; 3 School of Molecular Biosciences, Washington State University, Pullman, Washington, USA

Received 5 January 2004; returned 5 March 2004; revised 5 April 2004; accepted 10 April 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: To determine the epidemiological relationship between non-invasive penicillin non-susceptible Streptococcus pneumoniae isolates collected in the Christchurch community between 1997 and 2001.

Methods: One hundred and ninety-seven pneumococcal isolates were examined by macrorestriction profile analysis of SmaI-digested genomic DNA separated by PFGE and restriction fragment length polymorphism analysis of penicillin binding protein genes.

Results: Four major clonal lineages were identified, the largest and most homogenous containing 95 (48.2%) of the isolates, the bulk of which (93.7%), had identical macrorestriction patterns. Members of this clonal group were multidrug-resistant and exhibited high resistance to third-generation cephalosporins, with MICs ≥8.0 mg/L not uncommon (23.1%). Two of the clonal groups, each containing 24 (12.2%) isolates, appeared indistinguishable from the globally widespread Spain23F-1 and France9V-3 strains, respectively. The fourth (12.7% of isolates) multidrug-resistant clone possessed intermediate penicillin susceptibility (MIC 0.12 mg/L).

Conclusions: This study shows that several distinct penicillin-resistant pneumococcal clones are present in the Christchurch community, most of which appear to have been imported into New Zealand.

Keywords: pneumococci , PFGE , cephalosporins , epidemiology


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Streptococcus pneumoniae is an important cause of community-acquired respiratory infections, including sinusitis, otitis media and pneumonia as well as serious invasive infections such as septicaemia and meningitis. The ability to effectively treat pneumococcal infection has been compromised in recent years due to the acquisition of antibiotic resistance, particularly to ß-lactam drugs.1 Pneumococcal resistance to ß-lactams has been attributed to alterations of the penicillin-binding proteins (PBPs) which reduce their affinity.2 The first pneumococcal isolate resistant to penicillin was reported in 1967 from a patient in Australia,3 and resistant pneumococci have subsequently increased in prevalence worldwide. In some countries, extremely high rates of penicillin-resistant pneumococci (MIC ≥0.12 mg/L) have recently been reported, for example 79.7% in Korea,4 65.6% in Spain,5 and 72% in Taiwan.6

During the 1990s, New Zealand also reported an increase in antibiotic resistance among S. pneumoniae.7 Reduced susceptibility to penicillin (MIC ≥0.12 mg/L) increased from 0.2% in 1992 to 24% in 1996 among community isolates from Auckland8 and from 1.9% in 1995 to 9.9% in 1997 among invasive isolates nationwide.9 A study of community pneumococcal isolates from four centres during 1997 found the prevalence of high-level penicillin resistance (MIC ≥2 mg/L) to be 10% in Christchurch.10 By comparison, the reported incidence in the other major New Zealand centres was 6%, 5% and 0% in Auckland, Hamilton and Wellington, respectively, suggesting Christchurch has among the highest prevalence in New Zealand.

Through routine surveillance we noted that pneumococci with reduced susceptibility to penicillin had steadily increased in frequency in the Christchurch community, from 3% in late 1994 to 30% in early 1999. This rapid increase in prevalence of resistant pneumococci mirrored the situation seen in some other countries such as Iceland, where resistant pneumococci, which had not been observed before 1988, reached a prevalence of 17% by 1992.11 Likewise in France, resistant pneumococci were rare (≤1%) until 1987,12 but increased steadily thereafter to reach 25% prevalence in 1993.13 A similar observation was made in Hong Kong where pneumococci with reduced penicillin susceptibility were first reported in 1993, and by 1995 they had increased in prevalence to 28.9%.14

When such dramatic increases in resistance rates occur during a relatively short time period, detailed molecular analysis of the pneumococcal isolates often reveals that a significant proportion of the resistant population are clonally related. In Iceland, 57 serotype 6B isolates were shown to be indistinguishable from a Spanish 6B clone by both SmaI macrorestriction profile analysis (MRP-SmaI) determined by PFGE and multilocus enzyme electrophoresis (MLEE).15 In France, 65% of resistant isolates were found to belong to serotype 23F, and were genetically related as assessed by MRP-SmaI.13 In Hong Kong, molecular analysis by MRP-SmaI of 105 resistant isolates showed that 74% comprised a major clonal group that was indistinguishable from the globally widespread Spanish serotype 23F clone. However, in Hong Kong, this clonal lineage was found to express capsular types 23F, 19F and 14 suggesting that capsular switching had occurred in this clone.16

Based on these overseas observations, we hypothesized that the recent rapid increase in pneumococcal penicillin resistance in Christchurch was due to the dissemination of an existing resistant clone. To address this hypothesis, we examined the population structure of penicillin non-susceptible pneumococci collected in Christchurch between 1997 and 2001.


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

Clinical pneumococcal isolates used in this study were obtained from Medlab South, a community medical laboratory that provides routine diagnostic testing for medical practitioners in Christchurch (population 400 000). This laboratory is used by about half the general practitioners in the region. Isolates were routinely cultivated on tryptic soya agar (TSA) supplemented with 5% defibrinated sheep's blood (Life Technologies, Auckland, New Zealand) at 37°C in 5% CO2. Pneumococci were isolated from clinical specimens and identified using standard techniques including characteristic colony morphology, {alpha}-haemolysis on TSA 5% sheep's blood agar, optochin sensitivity and bile solubility. Repeat isolates from the same patient were excluded.

Antibiotic susceptibility testing

Isolates were tested for penicillin non-susceptibility with a 1 µg oxacillin disc. Isolates with reduced susceptibility were tested against five other antibiotics by the disc diffusion method; erythromycin (15 µg) tetracycline (30 µg), co-trimoxazole (25 µg), chloramphenicol (30 µg) and vancomycin (30 µg) (Oxoid, Basingstoke, UK). Tests were carried out and interpreted according to the recommendations of the NCCLS.17 S. pneumoniae ATCC 49619 was used as a susceptible control strain. Penicillin and cefotaxime MICs were determined using Etest strips (AB-Biodisk, Solna, Sweden). All pneumococcal isolates with reduced susceptibility to penicillin (MIC ≥0.12 mg/L) were collected during two sampling periods: August 1997 through to May 1999 (163 isolates) and March 2001 through to July 2001 (34 isolates).

Pulsed-field gel electrophoresis

PFGE was carried out as described by Hall et al.18 with minor modifications. Overnight growth was suspended in 3 mL of PETT IV (10 mM Tris–HCl pH 7.6, 1 M NaCl) buffer to give turbidity equivalent to a 3.0 McFarland Standard. The cells were collected by centrifugation, resuspended in 125 µL of PETT IV buffer and mixed with an equal volume of 1.6% pulsed-field certified agarose (Bio-Rad, Hercules, CA, USA). The molten agarose was injected into plug moulds, and once solidified, incubated overnight in 2 mL of ESP buffer (0.5 M EDTA pH 8.0, 0.5% N-lauroyl sarcosine) containing proteinase K (final concentration 0.5 mg/mL) at 50°C. Following cell lysis, plugs were transferred to sterile 50 mL Falcon tubes and were washed three times in TE buffer (10 mM Tris–HCl pH 7.6, 1 mM EDTA), overnight at 4°C. Embedded DNA was digested using either of the restriction enzymes SmaI or ApaI (Roche, Mannheim, Germany) in 150 mL volumes using 20 U of enzyme in accordance with the manufacturer's instructions. PFGE was carried out in a 1% agarose gel (pulsed-field certified agarose; Bio-Rad, Hercules, CA, USA) in 0.5 x TBE buffer (50 mM Tris–HCl, 50 mM boric acid, 0.1 mM EDTA). Concatamers of lambda DNA (PFG marker; New England Biolabs, Beverly, MA, USA) were used as a size standard. Electrophoresis was carried out on a CHEF DRIII system (Bio-Rad). Electrophoretic conditions were: running temperature 14°C, gradient 6.0 V/cm, run time 22 h, initial switch time 5 s, final switch time 35 s and a linear ramping factor. Gels were stained with ethidium bromide (0.5 mg/L) post-electrophoresis and visualized on a UV trans-illuminator.

MRP-SmaI analysis

Banding patterns were compared visually and were interpreted as described by Tenover et al.19 with modifications. Tenover et al. suggested that patterns differing in up to three bands from an index pattern can be considered highly related, and those differing in up to six bands considered possibly related. As the current investigation was spread temporally over 5 years, it was decided that all ‘possibly related’ profiles were to be considered as belonging to the same macro-restriction group. Each group was numbered arbitrarily as new profiles were observed. Within each group, unique profiles were considered distinct subgroupings, and distinguished with an uppercase letter, e.g. 1A, 1B, 1C, etc. Without the ability to discern an index case, types are considered to belong to a given group if they differ by no more than six bands from the most similar member of the cluster, as suggested by Hall.20 Pneumococci were characterized and compared with two predominant international clones defined by the Pneumococcal Molecular Epidemiology Network (PMEN):21 ATCC 700669 (Spain23F-1) and ATCC 700671 (France9V-3).

PCR amplification of pbp genes

PCR amplification of the pbp genes was carried out using the previously described primers; Pn2Bup 5'-GAT CCT CTA AAT GAT TCT CAG GTG G-3' and Pn2Bdown 5'-CAA TTA GCT TAG CAA TAG GTG TTG G-3' for pbp2b,22 and pbp2x-up 5'-CGT GGG ACT ATT TAT GAC CGA AAT GG-3' and pbp2x-dn 5'-AAT TCC AGC ACT GAT GGA AAT AAA CAT ATT A-3' for pbp2x.23 Total cellular DNA extraction was carried out using the rapid guanidine thiocyanate procedure described by Pitcher et al.24 Amplification was carried out in 50 mL volumes, each reaction consisted of 1 µg of template DNA; the appropriate primers at a final concentration of 2 µM per reaction; each dNTP at a final concentration of 200 µM; MgCl2 at a concentration of 1.5 mM; and 2.5 U of Taq DNA polymerase (Roche) in the reaction buffer provided. Reaction mixtures were overlaid with two drops of mineral oil and amplification was carried out in a Corbett Research FTS 320 thermal cycler. Amplification consisted of an initial denaturation of 1 min at 94°C, 35 cycles of the following three-step cycle; template denaturation for 15 s at 94°C, primer annealing for 45 s at 55°C and template elongation for 1 min at 72°C. A final extension step of 5 min at 72°C was included.

Restriction fragment length polymorphism (RFLP) analysis of pbp genes

PCR-amplified pbp genes were subjected to restriction endonuclease digestion using either DdeI or HinfI (Roche). Restriction digests were carried out in final volume of 15 µL and incubated at 37°C for 3 h. RFLP patterns were visualized after electrophoresis through 3.0% agarose gels (ultraPURE, Life Technologies, Auckland, New Zealand) at 6 V/cm for 2 h.

Serotyping

Serotyping was carried out by the New Zealand Reference Laboratory (Porirua, Wellington) using the capsular reaction test (Neufeld test) and the Danish system of nomenclature. Serotype data were not available for all isolates.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The age of the patients from whom the isolates were recovered ranged from 1 week to 91 years. Ninety-one isolates (46.1%) were recovered from patients 2 years or younger, and 33 (16.8%) were recovered from patients 60 years or older. Isolates were recovered from 101 (51.2%) females, 94 (47.7%) males, with the sex of two patients unknown. The most prominent sites of isolation were ears (29.4%), eyes (28.4%) and sputa (20.3%), accounting for 78.1% of the total number of isolates. The remaining 21.9% came from sites associated with asymptomatic carriage of S. pneumoniae, notably from throat and nasal swabs. None of the isolates was from invasive specimens.

Antimicrobial susceptibilities

The criterion for inclusion in this study was a penicillin MIC of 0.12 mg/L or greater. The highest penicillin MIC observed was 8.0 mg/L, and the MIC50 and MIC90 were 2 and 4 mg/L, respectively. Cefotaxime MICs ranged from 0.06 to 16 mg/L, with MIC50 and MIC90 values of 1.0 and 8.0 mg/L, respectively. In addition to penicillin resistance, 180 (91.4%) isolates were resistant to co-trimoxazole, 156 (79.2%) were resistant to erythromycin, 148 (75.1%) were resistant to tetracycline and 32 (16.2%) resistant to chloramphenicol. No resistance to vancomycin was observed. Multidrug resistance, defined as being resistant to three non-related antimicrobial agents (including ß-lactams), was observed in 158 (80.2%) isolates. The most frequently observed multi-resistant phenotype consisted of resistance to co-trimoxazole, erythromycin, tetracycline, as well as reduced penicillin susceptibility. This phenotype was noted in 119 (60.4%) of the isolates.

Clustering by PFGE generated macrorestriction profiles

The 197 pneumococcal isolates could be divided into 26 groups by MRP-SmaI analysis of restriction fragments separated by PFGE. The four largest groups (Groups 1, 2, 3 and 4) accounted for 168 (85.2%) of the isolates (48.2%, 12.2%, 12.2% and 12.7% for each group, respectively). Groups 1, 2 and 3 comprised pneumococcal isolates with intermediate to high penicillin resistance (MIC range 0.5–8.0 mg/L), whereas members of MRP group 4 were strictly of intermediate penicillin susceptibility (MIC 0.12 mg/L) although multiply resistant to other classes of antibiotics (Table 1). The remaining 29 isolates had penicillin MICs varying between 0.12 and 4 mg/L.


View this table:
[in this window]
[in a new window]
 
Table 1. Distribution of SmaI MRP subgroups, and their associated serotypes and antibiotic resistance profiles, among the four major MRP groups of penicillin-resistant pneumococci in Christchurch

 
The most prominent group (MRP group 1) contained 95 isolates (48.2%), and comprised five different MRP subtypes (Figure 1a). Of these, 89 had SmaI profiles that were indistinguishable (type 1A) and were considered clonal. To further assess the relatedness of this group, a subset of 42 isolates were digested with a second restriction endonuclease (ApaI) and analysed by PFGE. Each of the isolates yielded indistinguishable MRPs (data not shown). Each of the MRP group 1 isolates analysed had identical antimicrobial resistance patterns being resistant to co-trimoxazole, erythromycin, and tetracycline, with reduced susceptibility to ß-lactams. MICs of penicillin and cefotaxime were in the range 1–8 mg/L and 0.5–16 mg/L, respectively. All isolates of this group were susceptible to chloramphenicol. Fourteen of the isolates belonging to this group were serogrouped and determined to belong to serogroup 19, four of the 14 were typed further as 19F.



View larger version (140K):
[in this window]
[in a new window]
 
Figure 1. Comparison of PFGE separated SmaI-generated restriction fragments among major groups of Christchurch penicillin non-susceptible isolates of S. pneumoniae. Lambda ladder molecular size markers (shown in kbp) are indicated (M). (a) Group 1 isolates; lanes 1–5: Types 1A–1E; (b) Group 2 isolates; lanes 1: ATCC 700669 (Spain23F-1), 2–4: Types 2A–2C, 5–8: Types 2E–2H; (c) Group 3 isolates; lanes 1: ATCC 700671 (France9V-3), 2–6: Types 3A–3E; (d) Group 4 isolates, lanes 1–8: Types 4A–4H.

 
Macrorestriction group 2 comprised 24 (12.2%) isolates. The group was more genetically heterogeneous than MRP group 1, evident by the identification of a total of 13 different subtypes, each containing from one to eight isolates. Subtypes arose due to slight but distinct changes in mobility of individual bands around 200–250 kb. Isolates within MRP group 2 had a range of penicillin and cefotaxime MICs of 0.5–4.0 mg/L and 0.25–2.0 mg/L, respectively. All isolates were multi-resistant, typically exhibiting resistance to co-trimoxazole, erythromycin and chloramphenicol, with some isolates also resistant to tetracycline. Ten representative isolates from MRP group 2 were serotyped, and expressed capsular type 23F. MRP group 2 isolates were related to the PMEN-defined Spain23F-1 clone (ATCC 700669), being most similar to profile 2E (Figure 1b).

Macrorestriction group 3 consisted of 24 isolates (12.2%) and five subtypes. Each subtype contained between four and six isolates. Penicillin and cefotaxime MICs for this group were in the range 0.5–4.0 mg/L and 0.5–2.0 mg/L, respectively. This group was typically not multidrug-resistant, although 18 isolates (75%) were resistant to co-trimoxazole as well as penicillin. Only two members of this group were serotyped, and were determined to be one of each serotype 9 and 14. MRP group 3 isolates were related to a representative of the France9V-3 clone (ATCC 700671), the most similar profile being 3C (Figure 1c).

Macrorestriction group 4 contained 25 isolates (12.7%) distributed among 10 subtypes (Figure 1d). Each subtype contained between one and eight isolates. A degree of heterogeneity was exhibited between MRPs, however, most of the diversity observed could be attributed to a single SmaI fragment, which varied in size between 100 and 150 kb. MRP group 4 isolates were all intermediately resistant to penicillin (MICs 0.12–0.25 mg/L), but all were multidrug-resistant. All MRP group 4 isolates were resistant to co-trimoxazole and erythromycin. Most (96%) were also resistant to tetracycline. Only two group 4 isolates were serotyped, and both of these were found to be non-typeable.

Macrorestriction profiles 5–25 contained the remaining 29 isolates and represented unique profiles that differed significantly (six bands or greater) from each of the major groups, and from each other. Of these 21 MRP groups, most (81%) contained only one isolate representative of that profile (Table 2). Four MRP groups (5, 9, 10 and 12) could be differentiated into two subgroups, the largest of which (9A) contained three isolates. Many of the isolates were also resistant to antibiotics besides penicillin; resistance to co-trimoxazole, tetracycline, erythromycin and chloramphenicol was noted in 20, 17, 16 and seven isolates (69%, 59%, 55%, 24%), respectively. A notable multidrug-resistant cluster, MRP group 10, contained four isolates in three subgroups and each of these four isolates was serotyped as 19F. These isolates had penicillin and cefotaxime MICs of 2–4 mg/L and 1–4 mg/L, respectively. They also expressed resistance to all of the non-ß-lactam drugs tested, except vancomycin.


View this table:
[in this window]
[in a new window]
 
Table 2. Distribution of SmaI MRP subgroups, and their associated serotypes and antibiotic resistance profiles, among the 21 minor MRP groups of penicillin-resistant pneumococci in Christchurch

 
Analysis of pbp gene RFLP patterns

Twelve different RFLP profiles of the pbp2b gene were observed (Figure 2a). The three most frequently occurring profiles (A, B and H) were associated with 98 (49.7%), 55 (28.0%) and 26 (13.2%) isolates, respectively. Twelve different RFLP profiles of the pbp2x gene were observed (Figure 2b). The three most frequently occurring profiles (A, B and F), were associated with 86 (43.7%), 67 (34.0%) and 27 (13.7%) isolates, respectively.



View larger version (87K):
[in this window]
[in a new window]
 
Figure 2. Comparison of RFLP profiles of pbp genes. Molecular size marker (M) indicated in base pairs. (a) PCR amplified pbp2b gene digested with DdeI; lanes 1–10 correspond to RFLP profiles A–J, respectively; (b) PCR amplified pbp2x gene digested with HinfI; lanes 1–11 correspond to RFLP profiles A–K, respectively.

 
The association of pbp RFLP types with MRPs and their associated ß-lactam MICs is shown in Tables 1 and 2. MRP group 1 was almost exclusively associated with the pbp2b/2x RFLP profile A/A, and this combination was associated with resistance to both penicillin (MIC50/90 4/4 mg/L; range 1.0–8.0 mg/L) and cefotaxime (MIC50/90 2/8 mg/L; range 0.5–16 mg/L). MRP groups 2 and 3 were associated with the pbp2b/2xRFLP profile B/B. It has been noted previously that the Spain23F-1 and France9V-3 clones (MRP groups 2 and 3, respectively) have identical RFLP profiles for pbp1a, 2b and 2x genes,21 suggesting a common origin of resistance within these two clones. The pbp2b/2x RFLP profile B/B was noted in 52 isolates (including MRP groups 8, 10 and 20), and was associated with penicillin resistance (MIC50/90 2/4 mg/L; range 0.5–4.0 mg/L) and intermediate cefotaxime susceptibility (MIC50/90 1/2 mg/L; range 0.5–4.0 mg/L). The MRP group 4 produced pbp RFLP profile H/F, which was consistently associated with intermediate penicillin susceptibility (MIC 0.12 mg/L) and susceptibility to cefotaxime. Among the 21 non-clonal MRP groups, considerable variation was observed within the pbp2b/2x RFLP patterns indicating further the genetic heterogeneity among these isolates.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The isolation frequency of penicillin non-susceptible pneumococci has increased significantly in many parts of the world during the 1990s.25 The application of molecular techniques (such as MRP) has introduced a means of effectively discriminating between clonally related isolates, and genetically unrelated isolates sharing a common phenotype (e.g. serotype). When applied to rapidly emerging resistant pneumococcal populations, clonal lineages are often found to comprise a large proportion of the population.16,18

One of the most successful multi-resistant pneumococcal clones throughout the world was first identified in Spain during the early 1980s and subsequently designated the Spain23F-1 clone according to PMEN nomenclature.21 Given the global distribution of the Spain23F-1 clone, it was unsurprising to detect this clone in New Zealand. However, the Christchurch variants of the Spain23F-1 clone differ slightly from its previously described antibiotic susceptibility profile. The more common profile observed included resistance to erythromycin as well as co-trimoxazole and chloramphenicol, with or without resistance to tetracycline (48% and 44%, respectively). This resistance pattern has been associated with the Spain23F-1 clone previously; in Hong Kong, 76 of 105 isolates were shown to be related to the Spain23F-1 clone by MRP-SmaI/ApaI, yet 93% were resistant to erythromycin.16 This illustrates the further evolution of this highly successful clone in response to macrolide usage, to which very high rates of resistance have been reported in some parts of the world.26

Another successful clone, the France9V-3 clone, has also demonstrated its capacity to disseminate globally, and establish itself in regions geographically widespread. Consequently, it was not surprising to observe the France9V-3 clone in New Zealand in approximately equal abundance to the Spanish clone. Although only two isolates of the New Zealand France9V-3 variant were serotyped, one was found to be serotype 14 suggesting exchange of genes for capsule biosynthesis had occurred. The acquisition of serotype 14 by the France9V-3 clone has been shown previously in Uruguay,27 and was later confirmed by Coffey et al.28

Isolates belonging to PFGE group 1 were notably associated with high levels of resistance to cefotaxime; 26 of 95 (27.3%) of the isolates had a cefotaxime MIC ≥4 mg/L. In 1997–98, a significant increase in prevalence of pneumococcal resistance to cefotaxime (MIC ≥2 mg/L) was described in New Zealand. Prevalence increased from 5.4% in 1996 to 25.4% during the first 6 months of 1997.29 During this time, 216 cefotaxime-resistant (MICs ≥2 mg/L) pneumococcal isolates were submitted to the New Zealand antibiotic resistance reference laboratory.30 Of these, 113 (52%) had cefotaxime MICs of ≥4 mg/L (the revised non-meningitis cefotaxime resistance breakpoint in the 2002 NCCLS guidelines) and most (112/113) belonged to serotype 19F. DNA MRP analysis also suggested that these isolates were clonal.30 This was the first report of the epidemic multiresistant serotype 19F clone that is now prolific in New Zealand. The MRPs described in 1997 were indistinguishable from the MRP group 1 penicillin-resistant isolates from Christchurch described in this study (data not shown).

This study shows that the major New Zealand clone (MRP group 1) is homogenetic as assessed by MRP analysis. Other globally widespread pneumococcal clones identified in New Zealand during this study (e.g. Spain23F-1) have small, but significant changes in their MRP profiles due to evolutionary divergence. The high level of homogeneity observed in the New Zealand 19F clone suggests that it arrived in New Zealand recently and therefore dissemination of this clone would also have been recent. Comparison of the MRP 1 with the published macro-restriction profile of the Taiwan19F-14 clone21 shows many similarities with the predominant New Zealand 19F clone.

MRP group 4 was also multidrug-resistant (resistant to erythromycin, co-trimoxazole and tetracycline), yet it had only intermediate resistance to penicillin (MIC 0.12 mg/L) and was susceptible to cefotaxime (MIC range 0.06–0.12 mg/L). Of the 25 isolates belonging to this group, 23 (92%) were isolated from eye swabs. This is considerably more frequent than non-group 4 isolates of which 19% were isolated from eyes (P ≤ 0.001, {chi}2 test). Two of the MRP group 4 isolates were submitted for serotyping and were found to be non-typeable. Non-typeable pneumococci have previously been implicated in outbreaks of conjunctivitis31 and molecular analysis of such outbreaks often shows the non-typeable pneumococci to be genetically indistinguishable.32,33 Consequently, isolates of MRP group 4 are likely to be responsible for many cases of pneumococcal conjunctivitis in Christchurch.

Overall, although four clonal groups have been shown to be present in Christchurch, the most predominant appears to be a New Zealand variant of the Taiwan19F-14 clone, which accounted for nearly half the isolates characterized in this study. Assuming this to be the case, it would seem there is an intimate link in the movement of antibiotic resistance between New Zealand and countries of South-East Asia. This is likely to be influenced by increased tourism and other movements of population in this area of the world. This observation is significant, as future surveillance strategies must therefore consider Asia as a likely candidate for the introduction of novel resistance mechanisms in New Zealand. However, it should also be noted that the New Zealand variant of the Taiwan19F-14 clone appears to have characteristics which make them unique from their parental lineage; notably high level macrolide resistance mediated by the erm(B) gene,34 and its high-level resistance to cephalosporins.30

The surveillance of antibiotic-resistant pneumococci in New Zealand should be continued, with due attention to the mechanisms of resistance. This will enable the rapid identification of new resistant clones and novel resistance genotypes, and better allow the therapeutic consequences to be anticipated. However, for long-term extrapolation of resistance trends in New Zealand, antimicrobial resistance surveys in neighbouring countries within the Asia/Pacific region must be considered, as these are the most likely reservoirs for the importation of antibiotic-resistant pathogens into New Zealand.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Lucinda Hall and Scott Godfrey for their critical comments on the manuscript. This work was supported in part by a grant from SmithKline Beecham. DCB was the recipient of a PhD scholarship from the Institute of Environmental and Scientific Research, which is gratefully acknowledged.


    Footnotes
 
* Corresponding author. Tel: +44-20-7377-7000, extension 3228; Fax: +44-20-7377-7259; Email: d.c.bean{at}qmul.ac.uk


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Appelbaum, P. C. (2002). Resistance among Streptococcus pneumoniae: implications for drug selection. Clinical Infectious Diseases 34, 1613–20.[CrossRef][ISI][Medline]

2 . Hakenbeck, R., Kaminski, K., Konig, A. et al. (1999). Penicillin-binding proteins in ß-lactam-resistant Streptococcus pneumoniae. Microbial Drug Resistance 5, 91–9.[ISI][Medline]

3 . Hansman, D. & Bullen, M. M. (1967). A resistant pneumococcus. Lancet 2, 264–5.[CrossRef][ISI]

4 . Song, J. H., Lee, N. Y., Ichiyama, S. et al. (1999). Spread of drug-resistant Streptococcus pneumoniae in Asian countries: Asian Network for Surveillance of Resistant Pathogens (ANSORP) study. Clinical Infectious Diseases 28, 1206–11.[ISI][Medline]

5 . Oteo, J., Alos, J. & Gomez-Garces, J. (2001). Antimicrobial resistance of Streptococcus pneumoniae isolates in 1999 and 2000 in Madrid, Spain: a multicentre surveillance study. Journal of Antimicrobial Chemotherapy 47, 215–8.[Abstract/Free Full Text]

6 . Chiou, C. C. C., Liu, Y. C., Huang, T. S. et al. (1998). Extremely high prevalence of nasopharyngeal carriage of penicillin-resistant Streptococcus pneumoniae among children in Kaohsiung, Taiwan. Journal of Clinical Microbiology 36, 1933–7.[Abstract/Free Full Text]

7 . Brett, M. & Ellis-Pegler, R. (2001). Surveillance of antimicrobial resistance in New Zealand. New Zealand Public Health Report 8, 17–21.

8 . Riley, D., MacCulloch, D. & Morris, A. J. (1997). Resistant Streptococcus pneumoniae in the community. New Zealand Medical Journal 110, 107–8.

9 . Brett, M. S. & Martin, D. R. (1999). A significant increase in antimicrobial resistance among pneumococci causing invasive disease in New Zealand. New Zealand Medical Journal 112, 113–5.[ISI][Medline]

10 . Brett, W., Masters, P. J., Lang, S. D. et al. (1999). Antibiotic susceptibility of Streptococcus pneumoniae in New Zealand. New Zealand Medical Journal 112, 74–8.[ISI][Medline]

11 . Kristinsson, K. G., Hjalmarsdottir, M. A. & Steingrimsson, O. (1992). Increasing penicillin resistance in pneumococci in Iceland. Lancet 339, 1606–7.[CrossRef][ISI][Medline]

12 . Geslin, P., Buu-Hoi, A., Fremaux, A. et al. (1992). Antimicrobial resistance in Streptococcus pneumoniae: an epidemiological survey in France, 1970–1990. Clinical Infectious Diseases 15, 95–8.[ISI][Medline]

13 . Ferroni, A., Nguyen, L., Gehanno, P. et al. (1996). Clonal distribution of penicillin-resistant Streptococcus pneumoniae 23F in France. Journal of Clinical Microbiology 34, 2707–12.[Abstract]

14 . Kam, K. M., Luey, K. Y., Fung, S. M. et al. (1995). Emergence of multiple-antibiotic-resistant Streptococcus pneumoniae in Hong Kong. Antimicrobial Agents and Chemotherapy 39, 2667–70.[Abstract]

15 . Soares, S., Kristinsson, K. G., Musser, J. M. et al. (1993). Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980s. Journal of Infectious Diseases 168, 158–63.[ISI][Medline]

16 . Ip, M., Lyon, D. J., Yung, R. W. H. et al. (1999). Evidence of clonal dissemination of multidrug-resistant Streptococcus pneumoniae in Hong Kong. Journal of Clinical Microbiology 37, 2834–9.[Abstract/Free Full Text]

17 . National Committee for Clinical Laboratory Standards (2002). Performance Standards for Antimicrobial Susceptibility Testing—12th Informational Supplement. NCCLS, Wayne, PA, USA.

18 . Hall, L. M. C., Whiley, R. A., Duke, B. et al. (1996). Genetic relatedness within and between serotypes of Streptococcus pneumoniae from the United Kingdom: analysis of multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and antimicrobial resistance patterns. Journal of Clinical Microbiology 34, 853–9.[Abstract]

19 . Tenover, F. C., Arbeit, R. D., Goering, R. V. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis—criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 2233–9.[Free Full Text]

20 . Hall, L. M. C. (1998). Application of molecular typing to the epidemiology of Streptococcus pneumoniae. Journal of Clinical Pathology 51, 270–4.[Abstract]

21 . 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.[Abstract/Free Full Text]

22 . Dowson, C. G., Hutchison, A. & Spratt, B. G. (1989). Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae. Molecular Microbiology 3, 95–102.[ISI][Medline]

23 . 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.[ISI][Medline]

24 . Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Letters in Applied Microbiology 8, 151–6.[ISI]

25 . Felmingham, D. & Gruneberg, R. N. (2000). The Alexander Project 1996–1997: latest susceptibility data from this international study of bacterial pathogens from community-acquired lower respiratory tract infections. Journal of Antimicrobial Chemotherapy 45, 191–203.[Abstract/Free Full Text]

26 . Hsueh, P. R., Teng, L. J., Lee, L. N. et al. (1999). Extremely high incidence of macrolide and trimethoprim-sulfamethoxazole resistance among clinical isolates of Streptococcus pneumoniae in Taiwan. Journal of Clinical Microbiology 37, 897–901.[Abstract/Free Full Text]

27 . Camou, T., Hortal, M. & Tomasz, A. (1998). The apparent importation of penicillin-resistant capsular type 14 Spanish/French clone of Streptococcus pneumoniae into Uruguay in the early 1990s. Microbial Drug Resistance 4, 219–24.[ISI][Medline]

28 . Coffey, T. J., Daniels, M., Enright, M. C. et al. (1999). Serotype 14 variants of the Spanish penicillin-resistant serotype 9V clone of Streptococcus pneumoniae arose by large recombinational replacements of the cpsA-pbp1a region. Microbiology 145, 2023–31.[Abstract]

29 . Raymond, N. J. & Grimwood, K. (1998). Antibiotic-resistant pneumococci. New Zealand Medical Journal 111, 87–9.[ISI][Medline]

30 . Brett, M. S. (2001). Emergence of a high-level cefotaxime-resistant Streptococcus pneumoniae strain in New Zealand. Journal of Medical Microbiology 50, 173–6.[Abstract/Free Full Text]

31 . Shayegani, M., Parsons, L. M., Gibbons, W. E., Jr et al. (1982). Characterization of nontypable Streptococcus pneumoniae like organisms isolated from outbreaks of conjunctivitis. Journal of Clinical Microbiology 16, 8–14.[ISI][Medline]

32 . Martin, M., Turco, J. H., Zegans, M. E. et al. (2003). An outbreak of conjunctivitis due to atypical Streptococcus pneumoniae. New England Journal of Medicine 348, 1112–21.[Abstract/Free Full Text]

33 . Ertugrul, N., Rodriguez-Barradas, M. C., Musher, D. M. et al. (1997). BOX-polymerase chain reaction-based DNA analysis of nonserotypeable Streptococcus pneumoniae implicated in outbreaks of conjunctivitis. Journal of Infectious Diseases 176, 1401–5.[ISI][Medline]

34 . Bean, D. C. & Klena, J. D. (2002). Prevalence of erm (B) and mef (A) erythromycin resistance determinants in isolates of Streptococcus pneumoniae from New Zealand. Journal of Antimicrobial Chemotherapy 50, 597–9.[Abstract/Free Full Text]