J Med Microbiol 53 (2004), 1097-1099; DOI: 10.1099/jmm.0.45733-0
© 2004 Society for General Microbiology
ISSN 0022-2615
Genetic relatedness of antibiotic-resistant pneumococci isolated during case clusters
S C Clarke1,2,
D Lawrie1 and
M A Diggle1
1Scottish Meningococcus and Pneumococcus Reference Laboratory, Department of Microbiology, Stobhill Hospital, Balornock Road, Glasgow G21 3UW, UK 2Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK
Correspondence S. C. Clarke stuartcclarke{at}hotmail.com
Received May 5, 2004
Accepted July 23, 2004
Multilocus sequence typing of Streptococcus pneumoniae associated with two case clusters of disease is reported here for the first time. Isolates from the first cluster were serotype 19F, resistant to penicillin and erythromycin, and were characterized as ST 320. Isolates from the second cluster were serogroup 4, resistant to ciprofloxacin, and were characterized as ST 206. Therefore, the isolates from these clusters were antibiotic-resistant, of serotypes infrequently isolated, and of uncommon sequence types.
Abbreviations: MLST, multilocus sequence typing; ST, sequence type.
The pneumococcus remains an important cause of pneumonia, meningitis and septicaemia (Obaro & Adegbola, 2002). Concerns have been raised in recent years regarding an increase in the number of strains which have intermediate or complete resistance to certain antibiotics, particularly penicillin (Heath & Breathnach, 2002). Antibiotic-resistant pneumococci are mostly isolated from hospital-acquired infection or those individuals with chronic disease (Heath & Breathnach, 2002), and, in some countries, more than 20 % are intermediate or fully resistant to penicillin (Bradley & Scheld, 1997; Butler et al., 1996). However, the identification of antibiotic-resistant strains is important and the use of molecular typing methods is crucial to understanding their spread (Enright & Spratt, 1998). Here, we describe the isolation of antibiotic-resistant pneumococci in Scotland from two clusters of disease and the application of multilocus sequence typing (MLST) for clonal identification.
Isolates of pneumococci from invasive and non-invasive disease are received by the Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) from diagnostic microbiology laboratories throughout Scotland. The characterization of these pneumococci includes serotyping, determination of antibiotic MICs and MLST. During a typical year, around 900 isolates are processed from a population of approximately 5 million. The methods used for serotyping by coagglutination and the determination of antibiotic MICs by E-test are as previously described (Kyaw et al., 2000, 2002b). Pneumococci were considered resistant to pencillin if the MIC was 
2.0 mg l–1, resistant to erythromycin if the MIC was 1.0 mg l–1, resistant to ciprofloxacin if the MIC 4 mg l–1 and resistant to cefotaxime if the MIC was 2.0 mg l–1. MLST was also performed as previously described using automated methodology (Jefferies et al., 2003).
The first cluster involved the isolation of Streptococcus pneumoniae from two patients with respiratory infection in the same town. The isolates were characterized by the SMPRL as being of serotype 19F. Antibiotic susceptibility testing indicated resistance against penicillin and erythromycin (Table 1). The isolates were, however, susceptible to cefotaxime. MLST analysis showed them to be of sequence type (ST) 320. Serogroup 19 was the fourth most common in Scotland in 1999 (Kyaw et al., 2000), but the antibiotic susceptibility pattern and ST are uncommon. As of 1 July 2004, there were eight ST 320 isolates represented on the MLST database (http://spneumoniae.mlst.net). These were from Australia (n = 1), Norway (n = 1), China (n = 1), Hong Kong (n = 3) and Korea (n = 2). Five were reported as serotype 19F, two as serotype 19A and one as serogroup 19; all were multiply resistant (Table 1). Importantly, ST 320 differs from the internationally disseminated multi-resistant clone Taiwan 19F-14 by two alleles and is a member of a large complex containing some 18 different STs (McGee et al., 2001). The complex assignment is based on five or more alleles being shared compared with the existing central ST.
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Table 1. Characteristics of the ST 320 isolates from cluster one and those represented on the MLST database NR, Not reported; R, resistant; PG, penicillin; EM, erythromycin; CI, ciprofloxacin; TC, tetracycline; CT, cefotaxime; CL, chloramphenicol. All MICs are quoted in mg l–1.
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The second cluster involved the isolation of S. pneumoniae from the blood of four different patients attending the same hospital ward. All four isolates were characterized as serogroup 4 and were resistant to ciprofloxacin but susceptible to penicillin and erythromycin (Table 2). Serogroup 4 was the fifth most common in Scotland in 1999 (Kyaw et al., 2000). Due to the ward cluster and their resistance to ciprofloxacin, the isolates were processed for MLST to determine the strain similarity. All four isolates were of ST 206. As of 1 July 2004, there were only two STs represented on the MLST database, both of which were reported from the UK in 1996 and 1997. There were no antibiotic susceptibility data for these two isolates. ST 206 is part of a clonal complex containing four other STs, all but one of which were serogroup 4. ST 206 is a single locus variant of ST 205 which has been reported from the UK, but also from Sweden, Denmark, Australia and Canada. Interestingly, all STs within this complex on the MLST database were reported as being susceptible to pencillin and erythromycin, although ciprofloxacin susceptibility was not reported for these isolates.
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Table 2. Characteristics of the serotype 4 ST 206 isolates from cluster two and those represented on the MLST database NR, Not reported; PG, penicillin; EM, erythromycin; CI, ciprofloxacin. All MICs are quoted in mg l–1. MICs for tetracycline, cefotaxime and chloramphenicol were not reported.
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Although the serogroup 19 strains were non-invasive, the isolation of such strains in the UK highlights the possibility for the emergence of antibiotic-resistant invasive pneumococci in the UK (Kyaw et al., 2002a; Ziglam & Finch, 2002). Pneumococci are highly competent bacteria and can acquire antibiotic-resistance markers from other pneumococci or other bacterial species occupying the same ecological niche (Gillespie, 2001). The isolation of these strains from more than one patient for each clonal type also indicates the possibility for these strains to spread. Moreover, the isolation of serogroup 4 pneumococci from four patients on the same ward suggests that the bacteria could have been nosocomally acquired. The public health importance of such case clusters will depend on the wider application of MLST coupled with the epidemiological validation of plausible opportunities for transmission. Additional emphasis should also be made concerning the submission of MLST data to the website. The MLST websites provide a useful global epidemiological tool and an obvious weakness is the dependency of results being submitted in a timely and accurate manner by clinical diagnostic and reference laboratories in order that conclusions may be drawn by the scientific community.
Antibiotic resistance in pneumococci has become a global problem. The introduction of new pneumococcal conjugate vaccines is aimed at controlling the main disease-causing pneumococcal serotypes and should also help limit the spread of antibiotic-resistant clones within these serotypes (Cartwright, 2002; Obaro & Adegbola, 2002). However, adequate surveillance measures must also take place and MLST is an example of a laboratory-based, nucleotide sequencing method that can identify and track clones associated with certain serotypes and antibiotic resistance (Cartwright, 2002; Enright & Spratt, 1998; Sa-Leao et al., 2001). Therefore, for the first time, MLST of antibiotic-resistant pneumococci associated with clusters of disease has been reported. This paper therefore highlights the importance of serotyping pneumococci, performing molecular typing analyses such as MLST, and undertaking routine surveillance of pneumococcal strains for antibiotic resistance. The use of these methods in combination can lead to improved surveillance of pneumococcal infection, which is essential for determining public health priorities.
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ACKNOWLEDGEMENTS
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This publication made use of the Multi Locus Sequence Typing Website (www.mlst.net) developed by Dr Man-Suen Chan and David Aanensen and funded by the Wellcome Trust. Funding for the robot liquid handling systems and DNA sequencers was provided by the Meningitis Association (Scotland) and the National Services Division of the Scottish Executive.
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