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1 Infection Research Group, Level 9, Glasgow Dental Hospital, 378 Sauchiehall Street, Glasgow G2 3JZ, UK
2 Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, UK
3 Scottish Meningococcus and Pneumococcus Reference Laboratory, Stobhill Hospital, Glasgow, UK
4 Department of Biological Sciences, University of Warwick, UK
Correspondence
A. J. Smith
a.smith{at}dental.gla.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Portsmouth City PCT, Finchdean House, Milton Road, Portsmouth, UK.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), antibiotic resistance in this organism has continued to emerge. Levels of antibiotic resistance have expanded to include a wider range of antimicrobials, and an increased number of affected geographical locations (Klugman, 1990; Schreiber & Jacobs, 1995). DNA-sequencing techniques have led to the identification of pneumococcal antimicrobial-resistant clones. This phenomenon is due to the acquisition of resistance genes within specific clonal lineages (Munoz et al., 1992). In particular, clones originating from Spain, South Africa, Hungary and the USA have achieved a global distribution (Coffey et al., 1996; McDougal et al., 1995; Smith & Klugman, 1997). The understanding of the epidemiology of the emergence and spread of resistance within the pneumococcal population has been facilitated by the Pneumococcal Molecular Epidemiology Network (PMEN) (McGee et al., 2001). The PMEN system takes advantage of multilocus sequence typing (MLST), a DNA-sequence-based typing method, which uses data from a number of housekeeping genes to provide a sequence type (ST). MLST data are highly discriminatory and electronically portable between laboratories via the internet, making the technique ideal for investigating the clonal spread of antibiotic-resistant pneumococci (Enright & Spratt, 1998a). Criteria for inclusion of a clone in the PMEN database include widespread geographical distribution, persistence over a number of years, and resistance to one or more antibiotics (McGee et al., 2001).
Previous work (Jefferies et al., 2003) using MLST and serotyping has constructed a phylogenetic framework for a collection of pneumococci isolated in Scotland. We describe an analysis of these isolates in terms of their antimicrobial susceptibility, ST and relationship to the globally significant PMEN clones.
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METHODS |
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); they included rare serotypes, and isolates well distributed with respect to geographical location within Scotland, type of disease caused, and age of patient. Isolates were not preselected according to antimicrobial resistance.
Serotyping.
Isolates were serotyped by co-agglutination, as described by Smart (1986).
MLST.
MLST (Enright & Spratt, 1998a) was performed on Streptococcus pneumoniae strains using a semi-automated protocol (Jefferies et al., 2003, 2004). Single-locus variants (SLVs) and double-locus variants (DLVs) are thought to be sufficiently related to be considered members of the same clonal group of the parental ST clone [based upon related sequence types (BURST), http://eburst.mlst.net/].
Antimicrobial susceptibility testing.
MICs for Scottish and PMEN isolates were determined using Etest strips (AB Biodisk), in accordance with the manufacturer's instructions. Breakpoints for susceptibility were those given by the British Society for Antimicrobial Chemotherapy (http://www.bsac.org.uk; penicillin resistance 
2 mg l–1, intermediate resistance 0·12–1·0 mg l–1; erythromycin resistance >1 mg l–1; clindamycin resistance >1 mg l–1; ceftriaxone resistance 2 mg l–1).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The isolates were assigned to 109 STs. The mean age of patients was 45 years (range <1 month to 103 years). The range of penicillin susceptibility was 0·004–2 µg ml–1, with the majority of isolates penicillin susceptible (MIC <0·12 µg ml–1, n=214). One isolate, an SLV of the Taiwan19F-14 clone, was resistant to ceftriaxone, and the collection contained just two isolates with a ceftriaxone MIC of 2 µg ml–1. There were 26 erythromycin-resistant isolates, and these were found in STs 9, 90, 143, 156, 180, 814, 860, 271, 285, 320, 377 and 1000.
Isolates with identical STs to PMEN clones
There were 16 isolates with STs identical to three PMEN clones (Spain9V-3, England14-9 and Utah35B-24) (Table 1). Although members of the Spain9V-3 clone are penicillin resistant, and erythromycin sensitive, all of the seven isolates in our collection with an identical ST to the Spain9V-3 PMEN clone had lower penicillin MICs than the corresponding type strain. One of these isolates was an erythromycin-resistant, penicillin-intermediate variant of the Spain9V-3 clone. Three of the isolates related to the Spain9V-3 clone had different capsular types (serotypes 8 and 14, and a non-typable isolate). In contrast, the eight isolates with an ST identical to the erythomycin-resistant England14-9 clone were erythromycin resistant, and had an identical serotype to the type strain. The remaining isolate, which had an identical ST and serotype to the penicillin-resistant Utah35B-24 clone, was an erythromycin resistant, but penicillin intermediate, variant of this clone.
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) revealed seven SLV groups related to seven PMEN clones, and four DLV groups related to four PMEN clones. For 14 of the 15 SLVs of the PMEN clones, the penicillin MIC was less than the corresponding PMEN type strain (intermediate resistance).
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). Of interest was the observation that the SLVs occurred at the same loci for a particular clonal group; for example, the ddl locus for SLVs of Utah35B-24 and Spain9V-3, and gki for SLVs of Spain6B-2. DLVs were found for four clonal groups (Spain9V-3, Tennessee23F-4, Poland23F-16 and Columbia23F-26). One isolate (ST 176 03-1562) was a DLV of the Poland23F-16 and Columbia23F-26 clones. The DLV isolates related to both the Poland23F-16 clone and the Columbia23F-26 clone, and a DLV related to the Tennessee23F-4 clone demonstrated capsule switching. The two DLV clonal groups related to pencillin-resistant PMEN clones (ST 163 and 176) had lower penicillin MICs than the corresponding PMEN type strain; both of these isolates had different ddl loci compared with the corresponding PMEN type strain. For the erythromycin-resistant PMEN clone (Tennessee23F-4), the DLV isolates in our collection were erythromycin-sensitive variants, and all varied from the type strain at the gki and recP loci.
STs of antibiotic-resistant isolates not related to epidemic clones
Analysis of our collection of 252 pneumococci for the presence of resistance to penicillin or erythromycin revealed six isolates not found in the PMEN database (Table 3). Two of these isolates were resistant to penicillin, erythromycin and clindamycin, and belong to a multi-drug-resistant clone isolated in Australia, China and Hong Kong (ST 320). There were four isolates that were erythromycin-resistant variants of previously described clones. All these clones are not assigned to the PMEN database. In addition, we noted a serotype 6 isolate with high-level erythromycin resistance belonging to ST 814; to our knowledge, this is the first description of this ST.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The incidence of invasive pneumococcal disease in Scotland is similar to that reported in most studies from the USA, Sweden, and other parts of the UK, with serotypes/groups 14, 23, 9, 6 and 19 predominating among pneumococci that cause invasive disease (Kyaw et al., 2000, 2002; Clarke et al., 2003). Of interest is the relatively low prevalence of high-level penicillin resistance among pneumococci in Scotland; over the period 1992–1999, only two isolates were reported with MICs 2 mg l–1 (0·2 % of isolates) (Kyaw et al., 2000). More recent data for the year 2000 found no isolate (n=360) with a penicillin MIC >2 mg l–1 (Clarke et al., 2003). Levels of erythromycin resistance were higher, at 10 % over the period 1994–1999, rising to 13 % in 2000 (Kyaw et al., 2000; Clarke et al., 2003).
Our collection contained a small number of isolates identical to the Spain9V-3 clone, but some of these displayed different capsular types (non-typable, 8 and 14), and had lower penicillin MICs than the corresponding PMEN type strain; the strains were tested using Etest methodology. Of interest was the sequence changes in the D-alanine, D-alanine ligase (ddl) locus of SLV and DLV variants of the Spain9V-3, Poland23F-16, Utah35B-24 and Columbia23F-26 PMEN clones. The ddl gene is known to be closely linked to the penicillin-binding protein pbp2b gene, particular alleles of which confer resistance to 
-lactam antibiotics. These changes may account for the differing susceptibilities of these isolates to -lactam agents (Beall et al., 2002). The pbp2b genes may have been acquired as part of a hitchhiking effect, along with the DNA fragments acquired during alterations in the ddl locus (Enright & Spratt, 1998b).
Most of the Scottish erythromycin-resistant isolates related to the PMEN clones were related to the England14-9 clone, which displays the most frequently identified serotype in Scotland (Kyaw et al., 2000). This clone displays the drug efflux erythromycin-resistance pattern (MIC 2–12·0 mg l–1), which is thought to be due to carriage of the mefA gene (Amezaga et al., 2002). Of the erythromycin-resistant isolates not related to the PMEN clones, all demonstrated the high-level erythromycin-resistance phenotype (MLSB).
A unique feature of the global spread of drug-resistant pneumococci during the last four decades is the emergence of relatively few genetic lineages that have achieved unusually wide geographic spread. The mechanisms of dissemination and successful establishment of these clones are unknown. At present, the incidence of penicillin- and multi-drug-resistant pneumococci is relatively low in Scotland. Our collection of pneumococci from Scotland, which was not selected on the basis of antimicrobial susceptibility, has shown that isolates with an identical genotype to PMEN clones are circulating in the Scottish population; however, many of these isolates do not display the same phenotypic traits of high-level antimicrobial resistance or serotype as the parental clone. Further work is required to elucidate whether future changes in the antibiotic resistance pattern of Scottish pneumococci are driven by the clonal expansion of PMEN clones, and/or horizontal recombination of locally induced resistance genes.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Beall, B., McEllistrem, M. C., Gertz, R. E., Boxrud, D. J., Besser, J. M., Harrison, L. H., Jorgensen, J. H. & Whitney, C. G., Active Bacterial Core Surveillance/Emerging Infections Program Network, (2002). Emergence of a novel penicillin-nonsusceptible, invasive serotype 35B clone of Streptococcus pneumoniae within the United States. J Infect Dis 186, 118–122.[CrossRef][Medline]
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Denham, B. C. & Clarke, S. C. (2005). Serotype incidence and antibiotic susceptibility of Streptococcus pneumoniae causing invasive disease in Scotland, 1999–2002. J Med Microbiol 54, 327–331.
Enright, M. C. & Spratt, B. G. (1998a). A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144, 3049–3060.[Abstract]
Enright, M. C. & Spratt, B. G. (1998b). Extensive variation in the ddl gene of penicillin-resistant Streptococcus pneumoniae results from a hitchhiking effect driven by the pencillin-binding protein 2b gene. Mol Biol Evol 16, 1687–1695.
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McGee, L., McDougal, L., Zhou, J. & 8 other authors (2001). Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network. J Clin Microbiol 39, 2565–2571.
Munoz, R., Musser, M., Crain, M., Briles, D. E., Marton, A., Parkinson, A. J., Sorensen, U. & Tomasz, A. (1992). Geographic distribution of penicillin-resistant clones of Streptococcus pneumoniae: characterised by penicillin-binding protein profile, surface protein A typing and multi locus enzyme analysis. Clin Infect Dis 15, 112–118.[Medline]
Schreiber, J. & Jacobs, M. (1995). Antibiotic-resistant pneumococci. Pediatr Clin North Am 42, 519–537.[Medline]
Smart, L. E. (1986). Serotyping of Streptococcus pneumoniae strains by coagglutination. J Clin Pathol 39, 328–331.
Smith, A. M. & Klugman, K. P. (1997). Three predominant clones identified within penicillin-resistant South African isolates of Streptococcus pneumoniae. Microb Drug Resist 3, 385–389.[Medline]
Received 9 August 2005;
revised 2 September 2005;
accepted 5 September 2005.
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