Characterisation of macrolide-non-susceptible Streptococcus pneumoniae colonising children attending day-care centres in Athens, Greece during 2000 and 2003

ORIGINAL ARTICLE

10.1111/j.1469-0691.2006.01555.x

Characterisation of macrolide-non-susceptible Streptococcus pneumoniae colonising children attending day-care centres in Athens, Greece during 2000 and 2003 M. Souli, K. Volonakis, A. Kapaskelis, I. Galani, V. Grammelis, R. Vorou, M. Tsivra, Z. Chryssouli, D. Katsala and H. Giamarellou Fourth Department of Internal Medicine, Athens University School of Medicine, University General Hospital Attikon, Chaidari, Greece

ABSTRACT Nasopharyngeal Streptococcus pneumoniae isolates colonising young children are representative of isolates causing clinical disease. This study determined the frequency of macrolide-non-susceptible pneumococci, as well as their phenotypic and genotypic characteristics, among pneumococci collected during two cross-sectional surveillance studies of the nasopharynx of 2847 children attending day-care centres in the Athens metropolitan area during 2000 and 2003. In total, 227 macrolide-non-susceptible pneumococcal isolates were studied. Increases in macrolide non-susceptibility, from 23% to 30.3% (p <0.05), and in macrolide and penicillin co-resistance, from 3.4% to 48.6% (p <0.001), were identified during the study period. The M resistance phenotype, associated with the presence of the mef(A) ⁄ (E) gene, predominated in both surveys, and isolates carrying both mef(A) ⁄ (E) and erm(AM) were identified, for the first time in Greece, among the isolates from the 2003 survey. Pulsed-field gel electrophoresis analysis of the isolates from the 2000 survey indicated the spread of a macrolide- and penicillin-resistant clone among day-care centres. The serogroups identified most commonly in the study were 19F, 6A, 6B, 14 and 23F, suggesting that the theoretical protection of the seven-valent conjugate vaccine against macrolide-non-susceptible isolates was c. 85% during both study periods. Keywords

Children, colonisation, day-care centres, macrolide resistance, pneumococci, surveillance

Original Submission: 9 December 2005;

Revised Submission: 26 April 2006; Accepted: 16 June 2006

Clin Microbiol Infect 2007; 13: 70–77

INTRODUCTION Macrolides have been used widely for empirical treatment of community-acquired respiratory tract infections because of their efficacy against both Streptococcus pneumoniae and atypical respiratory pathogens. However, increasing use of macrolides has been associated with an increase in macrolide resistance [1]. Two main macrolide resistance mechanisms have been identified in pneumococci: energy-dependent efflux, associated with the presence of mef genes [2], and target site modification, associated with the erm gene family [3]. Mechanisms encountered less Corresponding author and reprint requests: H. Giamarellou, 4th Department of Internal Medicine, University General Hospital Attikon, 1 Rimini Street, 124 62 Chaidari, Greece E-mail: [email protected]

frequently involve mutations of ribosomal proteins or 23S rRNA genes [4]. There is now emerging evidence that macrolide resistance in S. pneumoniae results in clinical failure in cases of both otitis media and pneumonia [5–9]. The nasopharynx of colonised individuals is the main reservoir of pneumococci in the community. Most young children become carriers at some point, and are responsible for spreading pneumococci to other children, especially in the crowded environment of a day-care centre. Although only a small percentage of colonised children will develop an invasive infection, pneumococcal nasopharyngeal isolates reflect the strains circulating currently in a particular community and may enable the capsular types causing invasive disease to be predicted [10,11].

 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

Souli et al. Macrolide-non-susceptible S. pneumoniae 71

The aims of the present study were to determine the prevalence of macrolide non-susceptibility, and the phenotypic and genotypic characteristics of macrolide-non-susceptible S. pneumoniae, among isolates colonising the nasopharynx of healthy children in the Athens metropolitan area, Greece. Data from the present study were expected to contribute to an understanding of the epidemiology of macrolide resistance in this community, and to guide empirical treatment of common community-acquired pneumococcal infections. MATERIALS AND METHODS

was used for quality control as a reference strain. All of the erythromycin-non-susceptible strains were characterised further. Serotyping Serotyping was performed with the Quellung reaction using the 12 pooled antisera contained in the Pneumotest panel, as well as selected factor sera (Statens Serum Institut, Copenhagen, Denmark). Macrolide resistance phenotype The macrolide resistance phenotype was determined by the triple disk-diffusion method [13]. An MLSB phenotype was defined as resistance to erythromycin, clindamycin and quinupristin (streptogramin B), and an M phenotype was defined as resistance to erythromycin alone.

Study population The S. pneumoniae isolates were collected during two crosssectional surveillance studies conducted in the greater Athens metropolitan area during the first trimester of 2000 and 2003. Nasopharyngeal samples were obtained from healthy children, aged 1–6 years, who attended day-care centres located in various parts of the Athens metropolitan area. Only children whose parents had signed a consent form were included in the study. Trained physicians obtained perinasal specimens of nasopharyngeal secretions using a cotton-tipped flexible aluminium wire swab (Medical Wire & Equipment, Corsham, UK). Specimens were plated immediately on Mueller–Hinton II agar (Becton Dickinson, Sparks, MD, USA) supplemented with sheep blood 5% v ⁄ v. The plates were enclosed in Gas Pak CO2 Pouches (Becton Dickinson) and sent to the microbiology laboratory for incubation. Bacteria S. pneumoniae isolates were identified on the basis of colony morphology, appearance following Gram’s stain, susceptibility to optochin (5-lg disk; Becton Dickinson), solubility in bile and a latex agglutination test (Slidex pneumo-kit; bioMe´rieux, Marcy L’Etoile, France). Confirmed pneumococci were stored in Todd Hewitt broth (Becton Dickinson) supplemented with Bacteriological agar 0.3% w ⁄ v (Gibco BRL, Paisley, UK) and glycerine 3% v ⁄ v, and were kept at )80C until required for further testing. Susceptibility testing MICs of benzylpenicillin, ampicillin, erythromycin, cefuroxime, ceftriaxone, cefepime, moxifloxacin, linezolid, vancomycin and quinupristin–dalfopristin were determined by Etest (AB Biodisk, Solna, Sweden) on Mueller–Hinton II agar supplemented with sheep blood 5% v ⁄ v. Results were read after incubation for 20–24 h in CO2 5% v ⁄ v and were interpreted according to the manufacturer’s instructions. Results falling between serial two-fold dilutions were rounded-up to the next highest concentration. Telithromycin (Aventis, Vitry, France) MICs were determined by the agardilution method in ambient air, and results were interpreted according to CLSI criteria [12]. S. pneumoniae ATCC 49619

Pulsed-field gel electrophoresis (PFGE) The erythromycin-non-susceptible isolates from the 2000 survey were subjected to molecular typing by PFGE [14]. A PFGE-based clonal group was defined as a group of isolates with genetically-related PFGE patterns, as described by Tenover et al. [15]. Detection of macrolide resistance mechanisms by DNA amplification Genomic DNA isolation was performed with a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s guidelines. To ensure genomic DNA integrity for all isolates studied, the 16S)23S spacer region of S. pneumoniae (337 bp) was amplified by PCR [16]. The isolates were screened by multiplex PCR for rRNA erm methylases and for genes coding for inactivating enzymes or macrolide efflux pumps with primers for erm(A), erm(B), erm(C), msr(A) ⁄ msr(B), mph(A), mph(B), ere(A), ere(B) and mef(A) ⁄ (E) as described by Sutcliffe et al. [17], and for mph(C) as described by Tait-Kamradt et al. [18]. Discrimination between subtypes mef(A) and mef(E), which are 90% identical at the nucleotide level, was not attempted, and ‘mef(A) ⁄ (E)’ was used to designate the presence of the mef gene in the isolates of this study. The erm(AM) gene was not amplified by the set of primers used for the amplification of the erm(B) gene (despite 99% identity at the nucleotide level) because of A to G and G to A transitions at the site of annealing of the forward primer. The presence of erm(AM) was determined by PCR with gene-specific internal primers (5¢-TTGGAACAGGTAAAGGGCATT and 5¢-TTGGCGTGTTTCATTGCTTG). S. pneumoniae BM4200 [19] and genomic DNA from S. pneumoniae 02J1175 [20] were used as positive controls for PCR amplification of erm(AM) and mef(A) ⁄ (E), respectively. All primers were synthesised by MWG-Biotech (Ebersbery, Germany). Statistical analysis The data were analysed by the chi-square test with Yates’ correction and by Fisher’s exact test, as appropriate. Twotailed p values £0.05 were considered to be significant.

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72 Clinical Microbiology and Infection, Volume 13 Number 1, January 2007

RESULTS

Table 2. Antimicrobial susceptibilities of 74 macrolidenon-susceptible Streptococcus pneumoniae isolates with different resistance genotypes from the year 2000 survey

Bacteria and their susceptibilities The results in Table 1 summarise the study population and the isolates of S. pneumoniae that were recovered. The day-care centres that were chosen to participate in the study were representative of various socio-economic parts of the Athens metropolitan area, and 25 centres were common to both study periods. Of 89 macrolide-non-susceptible isolates (erythromycin MIC ‡2 mg ⁄ L) recovered during 2000, 31 (34.8%) were also non-susceptible to penicillin, and three isolates were resistant to macrolides, penicillin and cefepime. All 31 macrolide- and penicillin-non-susceptible isolates were susceptible to moxifloxacin, telithromycin, linezolid, vancomycin and quinupristin–dalfopristin. Seventy-four of the 89 macrolide-non-susceptible isolates from 2000 were available for further phenotypic and genotypic characterisation (see below). Of the 138 macrolide-non-susceptible isolates recovered during 2003, 106 (76.8%) were also non-susceptible to penicillin, 67 (48.6%) were resistant to three antimicrobial agents (macrolides, penicillin and cefuroxime), and ten were resistant to more than three antimicrobial agents (macrolides, penicillin, cefuroxime and ceftriaxone). Table 1 also summarises the genotypic analysis results for the erythromycin-non-susceptible isolates from both study periods. All isolates possessing only a mef(A) ⁄ (E) resistance determinant displayed the M phenotype, whereas all isolates that were PCR-positive for erm(AM) had the constitutive MLSB phenotype. The three isolates that harboured both the erm(AM) and the mef(A) ⁄ (E) genes were assigned to the constitutive MLSB phenotype. The antimicrobial susceptibilities of the S. pneumoniae isolates with different resistance genotypes are shown in Tables 2 and 3. The MICs Table 1. Summary of the demographics and susceptibilities of the Streptococcus pneumoniae isolates investigated during the 2000 and 2003 surveys

Participating day-care centres (n) Children enrolled (n) No. of isolates recovered Carriage rate (%) Isolates tested (n) Macrolide-non-susceptible isolates n (%) mef(A) ⁄ (E)-positive isolates n (%) erm(AM)-positive isolates n (%) mef(A) ⁄ (E)- and erm(AM)-positive isolates n (%)

Year 2000

Year 2003

49 1451 461 31.8 387 89 (23) 40 (54) 34 (46) 0

36 1396 485 34.7 456 138 (30.3) 87 (63) 48 (34.8) 3 (2.2)

Genotype

Antibiotic

MIC range (mg ⁄ L)

MIC50 (mg ⁄ L)

MIC90 (mg ⁄ L)

Susceptiblea (%)

erm(AM) (n = 34)

Erythromycin Penicillin Ampicillin Ceftriaxone Cefepime Erythromycin Penicillin Ampicillin Ceftriaxone Cefepime

256 to >256 0.004–0.38 <0.016–1 <0.002–0.38 <0.002–1 2–48 0.008–2 <0.016–1.5 0.006–1 0.012–1.5

>256 0.012 <0.016 0.008 0.023 16 0.023 0.016 0.023 0.047

>256 0.125 0.125 0.125 0.38 32 1 1 0.5 1

0 79.4 100 100a 100a 0 50 92.5 100 95

mef(A) ⁄ (E) (n = 40)

a Susceptibility (mg ⁄ L) was defined according to the recommendations supplied with Etests as: erythromycin £1; penicillin £0.06; ampicillin £2; ceftriaxone (for nonmeningitis isolates) £1; and cefepime (for non-meningitis isolates) £1.

Table 3. Antimicrobial susceptibilities of 138 macrolidenon-susceptible Streptococcus pneumoniae isolates with different resistance genotypes from the year 2003 survey Genotypea

Antibiotic

MIC range (mg ⁄ L)

MIC50 (mg ⁄ L)

MIC90 (mg ⁄ L)

Susceptibleb (%)

erm(AM) (n = 48)

Erythromycin Penicillin Cefuroxime Ceftriaxone Moxifloxacin Telithromycin Linezolid Erythromycin Penicillin Cefuroxime Ceftriaxone Moxifloxacin Telithromycin Linezolid

24 to >256 0.008–4 <0.016–8 0.008–1 0.016–0.38 0.008–0.5 0.047–2 2–64 0.016–8 <0.016–16 0.012–4 0.064–0.38 0.008–0.5 0.5–2

>256 0.032 0.125 0.032 0.125 0.03 0.75 16 1.5 4 0.5 0.125 0.125 1

>256 2 4 0.75 0.25 0.125 1 32 4 8 1 0.19 0.125 1.5

0 50 66.7 100 100 100 100 0 9.2 21.8 90.8 100 100 100

mef(A) ⁄ (E) (n = 87)

a

Susceptibilities of three isolates harbouring both erm(AM) and mef(A) ⁄ (E) are described in Results. Susceptibility (mg ⁄ L) was defined according to the recommendations supplied with Etests as: erythromycin £1; penicillin £0.06; cefuroxime sodium £0.5; ceftriaxone (for non-meningitis isolates) £1; moxifloxacin £1; and linezolid £2. b

of erythromycin, penicillin, cefuroxime and ceftriaxone for the three isolates with the dual resistance mechanism were 64–>256, 0.064–4, 0.25–8 and 0.125–2 mg ⁄ L, respectively. All three of these isolates were susceptible to moxifloxacin, telithromycin and linezolid. In both surveys, the majority of isolates that were non-susceptible to both macrolides and penicillin belonged to the mef(A) ⁄ (E) genotype (74% in 2000; 74.5% in 2003). The same was observed for isolates that were nonsusceptible to more than three antibiotics (100% in 2000; 73% in 2003). No other macrolide resistance genes were detected. PFGE The PFGE results for the 74 macrolide-non-susceptible isolates from the year 2000 survey are

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Souli et al. Macrolide-non-susceptible S. pneumoniae 73

shown in Table 4. Major PFGE clones were defined arbitrarily as clusters containing more than five isolates and were represented by PFGE patterns 1–5. In total, 38 (51.3%) of all isolates belonged to these five predominant clones. The 13 indistinguishable isolates with PFGE pattern 1 were from five day-care centres, whereas the nine isolates with PFGE pattern 2 (serotype 19F) were identified in nine different day-care centres with no apparent epidemiological link. Only one or two PFGE clones were recognised in ten of 14 day-care centres where two or more macrolidenon-susceptible pneumococcal isolates were identified, indicating an extensive spread of certain clones within day-care centres. Serotyping The serotype distribution of the isolates studied is shown in Table 5. In the year 2000 survey, the proportion of the recovered serotypes included in, or partially covered by, the seven-valent conjugate pneumococcal vaccine was 85% (Table 6), whereas 98.7% of isolates were covered by the 23-valent polysaccharide vaccine. Within

Table 4. Pulsed-field gel electrophoresis (PFGE) clones detected among 74 macrolide-non-susceptible isolates from the year 2000 survey, and the correlation of PFGE clones with serotypes and macrolide resistance determinants PFGE clone (no. of isolates)

Macrolide resistance determinant

Serotype(s) (n)

No. of DCCs with the same clone

1 (13) 2,2A (9) 3 (6) 4 (5) 5 (5) 6 (4) 7 (3) 8 (3) 9 (2) 10 (2) 11 (2) 12, 12A (2) 13, 13A (2) 14 (1) 15 (1) 16 (1) 17 (1) 18 (1) 19 (1) 20 (1) 21 (1) 22 (1) 23 (1) 24 (1) 25 (1) 26 (1) 27 (1) 28 (1) NT (1)

mef(A) ⁄ (E) mef(A) ⁄ (E) mef(A) ⁄ (E) erm(AM) erm(AM) erm(AM) erm(AM) mef(A) ⁄ (E) erm(AM) mef(A) ⁄ (E) mef(A) ⁄ (E) mef(A) ⁄ (E) erm(AM) erm(AM) erm(AM) erm(AM) mef(A) ⁄ (E) erm(AM) erm(AM) erm(AM) erm(AM) erm(AM) erm(AM) erm(AM) mef(A) ⁄ (E) mef(A) ⁄ (E) erm(AM) erm(AM) erm(AM)

14 19F 23F 19A 6B (4), 6A (1) 6A (2), 6B (2) 15C (2), 15B (1) 6A (2), 6B (1) 6B 15A (1), 15C (1) 19F 23F 6B 11A 23F 19A 10A 6A 18A ⁄ B 11A 19A 23F 6B 15B 14 6A 24A 6B 3

5 9 1 1 1 1 1 2 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

DDC, day-care centre; NT, non-typeable.

Table 5. Serotype distribution of macrolide-non-susceptible Streptococcus pneumoniae isolates from 2000 and 2003 No. of isolates by year Serotype ⁄ serogroup

2000

2003

14 6B 19F 23F 6A 19A 15C 15B 15A 18A ⁄ B Othera Non-typeableb

14 13 11 10 7 7 3 2 1 1 5 0

15 21 37 13 26 2 1 0 1 3 15 4

a Includes serogroup ⁄ serotypes 1, 3, 7, 8, 10A, 11A, 12, 20, 22, 23B, 24A, 33, and 13 ⁄ 28. b Omni serum-negative.

Table 6. Distribution of macrolide-non-susceptible Streptococcus pneumoniae isolates according to serotype 2000

Serotype Seven-valent included 14 6B 19F 23F 18C 9V 4 Partially covered 6A 19A 18A ⁄ B 23A ⁄ B 9N Non-vaccinea serotypes

n (%)

2003 Cumulative percentage

n (%)

Cumulative percentage

14 13 11 10 0 0 0

(18.9) (17.6) (14.9) (13.5)

19 37 52 65

15 21 37 13 0 0 0

(10.9) (15.2) (26.8) (9.4)

11 26 53 62

7 7 1 0 0 11

(9.5) (9.5) (1.3)

74 84 85

(18.8) (1.5) (2.2) (0.7)

81 83 85 86

(14.9)

100

26 2 3 1 0 20

(14.5)

100

a Serotypes ⁄ serogroups other than those included in or partially covered by (crossreactive with) the seven-valent conjugate vaccine.

the subgroup of macrolide- and penicillin-nonsusceptible isolates, 96.3% belonged to serotypes related to (included in or partially covered by) the seven-valent vaccine, with the most common being 19F, 6A and 6B. Serotypes 19F and 6A were the most prevalent serotypes among macrolide- and penicillin-nonsusceptible isolates from the 2003 survey. Protection by the seven-valent conjugate pneumococcal vaccine was an estimated 85.8% for this subgroup of isolates, and 89.7% for isolates resistant to more than three antibiotics. Overall protection against the macrolide-non-susceptible isolates from 2003 was 86% and 96.4% by the sevenvalent and the 23-valent vaccines, respectively.

 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 13, 70–77

74 Clinical Microbiology and Infection, Volume 13 Number 1, January 2007 Non-vaccine serogroups ⁄ serotypes identified were (in descending order) 15C, 15B, 11A, 15A, 10A, 24A and 3 for the year 2000, and 8, 1, 20, 3, 7, 12, 15A, 15C, 22, 33 and 13 ⁄ 28 for the year 2003. In the entire collection of isolates, serotypes 14, 19F and 6A were encountered more frequently among isolates showing the M resistance phenotype, whereas serotypes 6B, 23F and 19A were associated more often with the MLSB resistance phenotype. No significant differences were observed between the two study periods with respect to serotype distribution of isolates exhibiting the same phenotype (data not shown). DISCUSSION This study identified a statistically significant increase in the frequency of macrolide-non-susceptible pneumococci colonising the nasopharynx of healthy children in the Athens metropolitan area, from 23% in 2000 to 30.3% in 2003 (p <0.05). Also detected was a statistically significant increase in the frequency of the macrolide- and penicillin-non-susceptible subgroup of isolates (from 34.8% to 76.8%, p <0.001), whereas the proportion of macrolide-resistant and penicillinsusceptible isolates decreased from 65.2% in 2000 to 30.2% in 2003 (p <0.001). The increase in macrolide and penicillin co-resistance (from 3.4% to 48.6%, p <0.001) was one of the most alarming findings of the study. On the basis of these findings, the Hellenic Society for Infectious Diseases and the Hellenic Society for Chemotherapy issued guidelines for the treatment of community-acquired pneumonia in 2005, which recommended that monotherapy with macrolides should be restricted to a specific patient subgroup (i.e., those without co-morbidities, without riskfactors for drug-resistant S. pneumoniae, and without severe illness). Increased use of macrolides has been identified as one of several risk-factors for increasing rates of macrolide resistance [21]. According to data provided by the National Organisation for Drugs, total macrolide consumption in Greece increased from 6.07 defined daily doses ⁄ 1000 inhabitants in 1999 to 9.34 defined daily doses ⁄ 1000 inhabitants in 2003. In 2002, outpatient macrolide consumption in Greece was reported to be the highest among 26 European countries [22]. Various national and international surveillance studies have demonstrated a high global preval-

ence of in-vitro macrolide resistance among pneumococci [1]. In Europe, the European Antimicrobial Resistance Surveillance System (EARSS) has detected a consistent increase in erythromycin non-susceptibility in almost all participating countries between 1999 and 2001, with an overall increase of 5.9% annually during that period [23]. According to the EARSS 2004 annual report, the rates have continued to rise in most participating countries (http://www.earss. rivm.nl). Internationally, results from the Alexander Project have revealed a global increase in the frequency of macrolide resistance, from 16.5% in 1996 [24] to 24.6% by 1998–2000 [25]. The PROTEKT Study demonstrated that erythromycin resistance was higher than penicillin resistance in all regions of the USA, but that it remained stable at c. 30% during the 3-year period of the study [26]. Nevertheless, there was considerable geographical variation in resistance rates [27]. These data emphasise the need to monitor local susceptibility trends on both a national and a regional level. The present study detected an absolute correlation between macrolide resistance phenotype and genotype, although in other large surveillance studies, a minority of macrolide-resistant isolates remain PCR-negative for erm and mef genes, with mutations of ribosomal proteins and ⁄ or 23S rRNA genes being identified as the mechanism of macrolide resistance [4,27]. Another important observation of the present study was the predominance of isolates displaying the M phenotype and harbouring the mef(A) ⁄ (E) gene. This is in contrast with the data from most other European countries, where the spread of macrolide resistance is associated mainly with erm-positive strains [28–33]. However, the M resistance phenotype, mediated by the mef(A) ⁄ (E) gene, is predominant among macrolide-resistant pneumococci in Canada [34] and the USA [27]. Recently, data from surveillance studies have suggested that erythromycin MICs for mef-positive isolates may be increasing (>16 to >256 mg ⁄ L), probably as a result of additional resistance mechanisms [27,30]. The present study also identified an upward shift in erythromycin MICs towards 64 mg ⁄ L in this subgroup of isolates (Tables 2 and 3). A higher prevalence of penicillin resistance was observed among mef(A) ⁄ (E)-positive isolates than among erm(AM)-positive isolates during both study periods (50% vs. 20.6% in 2000,

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Souli et al. Macrolide-non-susceptible S. pneumoniae 75

p <0.02, and 90.8% vs. 50% in 2003, p <0.001; Tables 2 and 3). A higher prevalence of resistance to ampicillin and cefepime was also observed in the year 2000 survey, as well as to cefuroxime and ceftriaxone in the year 2003 survey, among the mef(A) ⁄ (E)-positive subgroups of isolates. A new finding in this study was the identification of a dual resistance mechanism (erm and mef coexistence) in three isolates from 2003. These isolates exhibited high-level resistance to erythromycin and were non-susceptible to penicillin; two were also non-susceptible to cefuroxime and ceftriaxone and belonged to serotype 19F. All three isolates were susceptible to moxifloxacin, linezolid and telithromycin. This is the first report of S. pneumoniae isolates carrying both the mef(A) ⁄ (E) and the erm(AM) genes in Greece, but recent data confirm the worldwide distribution and increasing prevalence of this genotype, with particularly high rates in South Africa and South Korea. Most of these isolates belong to one major clonal group and show limited serotype distribution, with serotypes 19F and 19A being the most common [35]. Furthermore, such isolates often exhibit resistance to multiple classes of antibacterial drugs [35,36]. Their evolutionary advantage has been attributed to the acquisition of two mobile genetic elements, Tn1545 and mega, together with their associated resistance genes [37]. Thus, their introduction and potential spread in Greece is of serious concern. The PFGE results for the isolates from the year 2000 indicated extensive spread of certain macrolide-non-susceptible clones within, but not between, day-care centres. An exception to this observation was the dissemination of the second most common clone (PFGE patterns 2 and 2A) among nine day-care centres with no apparent epidemiological link. This clone was macrolide and penicillin co-resistant and belonged to serotype 19F. The successful spread of this coresistant clone was an alarming finding. Serotype 19F was also the most prevalent serotype among isolates from the year 2003 with dual non-susceptibility. Previous studies [38–40] have shown that the seven-valent conjugate vaccine provides protection against cross-reactive or vaccine-related serotypes. Assuming that serotypes 6A, 19A, 23A and 23B are also covered by, or are cross-reactive with, this vaccine, the theoretical coverage for the macrolide-non-susceptible isolates was c. 85% in

both study periods. There was a theoretical decrease in the coverage against macrolide- and penicillin-non-susceptible isolates, from 96.3% in 2000 to 85.8% in 2003, but this was not statistically significant. Since the majority of macrolide- and penicillin-non-susceptible isolates in this study belonged to vaccine-related serotypes, the prevalence of resistance among pneumococci might be reduced by the use of pneumococcal vaccines. In Greece, the seven-valent conjugate pneumococcal vaccine was introduced during October 2004. The results of future surveillance studies in Greece are much anticipated, since there is evidence that the vaccine could be responsible for a major decline in the incidence of invasive disease and in resistance rates [40–42]. However, there are also concerns regarding possible serotype replacement in pneumococcal clinical isolates [35,39,42,43]. In conclusion, this study detected important trends in the evolution of macrolide resistance among S. pneumoniae isolates colonising the nasopharynx of children in the Athens community. Local surveillance studies remain an important tool for following the evolution of pneumococcal resistance and for guiding empirical treatment of pneumococcal infections. ACKNOWLEDGEMENTS The pooled antisera Pneumotest panel and selected factor sera were purchased with a grant from Abbott Laboratories. These data were presented, in part, at the 15th European Congress of Clinical Microbiology and Infectious Diseases (Copenhagen, 2005).

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