Evolution of resistance patterns and identification of risk factors for Streptococcus pneumoniae colonisation in daycare centre attendees in Athens, Greece

International Journal of Antimicrobial Agents 28 (2006) 297–301

Evolution of resistance patterns and identification of risk factors for Streptococcus pneumoniae colonisation in daycare centre attendees in Athens, Greece Katerina Volonakis, Maria Souli, Anastassios Kapaskelis, Fotini Baziaka, Vassilios Grammelis, Panayiotis D. Ziakas, Helen Giamarellou ∗ Fourth Department of Internal Medicine, Athens University School of Medicine, University General Hospital ‘Attikon’, 1 Rimini Street, 124 62 Chaidari, Greece Received 28 February 2006; accepted 26 May 2006

Abstract Two cross-sectional surveillance studies were conducted during the winters of 2000 and 2003 in Athens, Greece, to obtain nasopharyngeal swabs from healthy pre-school children attending kindergartens. A total of 460 strains were examined in 2000 and 485 strains in 2003, with carriage rates of 31.7% and 34.6%, respectively. Susceptibility patterns were evaluated for penicillin G, erythromycin, ceftriaxone, moxifloxacin, linezolid and telithromycin. Penicillin non-susceptibility increased from 20% to 34.9%, whereas erythromycin non-susceptibility increased from 23% to 30.5%. Resistance to both agents climbed from 7.5% to 22.3% (P < 0.001). No isolates were found to be resistant to any of the other antimicrobial agents. Risk factors for carriage and/or antimicrobial resistance were also assessed. © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Streptococcus pneumoniae; Surveillance; Colonisation; Daycare centres

1. Introduction Streptococcus pneumoniae is the most common respiratory pathogen in the community, causing mainly pneumonia, acute otitis media, bacterial meningitis and bacteraemia. Worldwide concern has been raised owing to the emergence of highly resistant and multidrug-resistant strains in all continents [1–3], prompting several clinical and surveillance studies. Nasopharyngeal carriage of S. pneumoniae is the first step towards clinical infection and is an important source of horizontal spread in the community [4,5]. Daycare centre attendees, in particular, are believed to be the most significant reservoir for dissemination of resistance [4,6,7]. In Greece, epidemiological surveillance resulted in the first identification of resistant pneumococci in the mid 1990s [8,9]. The aim of this study was to follow-up on the suscepti∗

Corresponding author. Tel.: +30 210 583 1990; fax: +30 210 532 6446. E-mail address: [email protected] (H. Giamarellou).

bility patterns of S. pneumoniae in Athens metropolitan area and to identify risk factors for colonisation with resistant isolates. This knowledge is imperative to protect, prevent and effectively treat infections from this very common community pathogen. 2. Materials and methods 2.1. Study population Streptococcus pneumoniae isolates were collected during two cross-sectional surveillance studies conducted in greater Athens metropolitan area, the most densely populated city in Greece, between January and March of 2000 and 2003. Nasopharyngeal samples were obtained from healthy children attending daycare centres. Participating daycare centres were representative of all socioeconomic levels of the population of Athens; 25 of them were common to both studies, permitting a follow-up on the evolution of antimicrobial

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resistance of S. pneumoniae. An informed consent form was signed by the parents. A questionnaire aimed at identifying possible risk factors for colonisation with resistant isolates was distributed in the 2003 survey. The questions intended to reveal demographic data, recent antibiotic consumption, hospitalisations and history of acute otitis media. 2.2. Samples Specimens of nasopharyngeal secretions were obtained per nasally using a cotton-tipped flexible aluminium wire (Medical Wire & Equipment, Corsham, UK) and were appropriately transferred to the laboratory for incubation. 2.3. Bacterial isolation and identification Colony morphology, Gram stain, optochin susceptibility (5 ␮g optochin disks; Becton Dickinson, Sparks, MD), bile solubility and latex agglutination (Slidex pneumo-kit; bioM´erieux, Marcy l’Etoile, France) were used for the identification of S. pneumoniae [10]. 2.4. Antimicrobial agents and susceptibility testing For isolates in the 2000 survey, minimum inhibitory concentrations (MICs) of penicillin, erythromycin and ceftriaxone were determined by Etest (AB Biodisk, Solna, Sweden). For a subgroup of 63 isolates resistant to penicillin and/or erythromycin, MICs of moxifloxacin, linezolid and telithromycin were also determined by Etest. For all isolates in the 2003 survey, MICs of penicillin, erythromycin, ceftriaxone, moxifloxacin and linezolid were determined by Etest [11] and the results were interpreted according to the manufacturer’s instructions, whereas telithromycin susceptibilities were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) [12]. Telithromycin reference standard was provided by SanofiAventis (Paris, France). MICs falling between two values of the Etest were rounded up to the next two-fold value for statistical analysis. Streptococcus pneumoniae ATCC 49619 was used for quality control. 2.5. Statistical analysis In the analysis of risk factors for nasopharyngeal carriage and for carriage of resistant S. pneumoniae, greater Athens metropolitan area was divided into three districts according to geographic criteria, degree of industrialisation, degree of housing density (as determined by the most recent census of 2001) and socioeconomic status of the population. Daycare centres located in districts of the lowest socioeconomic and of the highest population density were designated as level one; daycare centres located in districts of the highest socioeconomic status and the lowest population density were designated as level three; and daycare centres located in

districts of intermediate socioeconomic status and population density were designated as level two. For statistical analysis, children were divided into two age groups: ≤3 years and >3 years of age. As broad-spectrum antibiotics were characterised the following: amoxicillin/ clavulanic acid, clarithromycin, azithromycin, second- and third-generation cephalosporins and sulphamethoxazole/ trimethoprim. Penicillin V, amoxicillin, first-generation cephalosporins, erythromycin and fusidic acid were categorised as narrow-spectrum agents. Multivariate logistic regression was used to assess the risk of pneumococcal carriage among individuals. Separate univariate and multivariate logistic regression models were used to estimate relative risks of colonisation with a penicillinand/or erythromycin-resistant strain. The two-sample proportion test was used for comparison of the rates, and the level of significance was set to 5%. 3. Results 3.1. Demographics A total of 2851 healthy pre-school children aged 1–6 years were included in both studies. In the 2000 survey, 1451 children attending 49 daycare centres were recruited. In 2003, the survey enrolled 1400 children attending 36 daycare centres. Of the children participating in the most recent study, 27 (1.9%) had been hospitalised for various reasons during the last 3 months but were healthy at the time of the study. In the 2003 survey, 495 children (35%) lived in first-level areas, 296 (21%) lived in second-level areas and 609 (43.5%) lived in third-level areas. 3.2. Recent antibiotic use After analysing the data obtained from the questionnaire, it was found that 45.3% of the children who participated in the 2003 survey had received one or more antibiotic prescriptions during the last 3 months. Children up to 4 years old were more likely to have received an antibiotic than older children (52.7% versus 37%; P < 0.001). The most common reasons were acute otitis media (31%), acute bronchitis (18%), unspecified (probably viral) upper respiratory tract infections (17%) and acute tonsillitis (11%). The use of broad-spectrum agents far exceeded that of the narrow-spectrum ones in all types of infections (87% versus 13%; P < 0.001). Paediatricians favoured penicillins, especially broad-spectrum ones, over cephalosporins and macrolides (42%, 33% and 23% of the prescriptions, respectively). 3.3. S. pneumoniae carriage rates and antimicrobial susceptibilities Four hundred and sixty children (31.7%) in the first survey and 485 (34.6%) in the second survey were pneumococci carriers.

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Table 1 Susceptibilities of Streptococcus pneumoniae collected in the 2000 and 2003 surveys Antimicrobial

No.

MIC50

Penicillin Ceftriaxone Erythromycin Moxifloxacin Linezolid Telithromycin

Susceptibility (%)a

MIC (␮g/mL) MIC90

Range

S

I

R

2000

2003

2000

2003

2000

2003

2000

2003

2000

2003

2000

2003

2000

2003

386 386 386 63 63 63

458 458 458 458 458 196

0.015 0.015 0.125 0.125 0.5 0.06

0.03 0.03 0.125 0.125 1 0.03

0.5 0.25 0.5 0.125 1 0.25

2 1 16 0.25 2 0.125

0.004–2 0.004–2 0.06–>256 0.03–0.5 0.25–1 0.015–0.5

0.004–8 0.008–4

80.05 99.5 76.9 100 100 100

65 97.6 69.5 100 100 100

17.9 0.5 2.85

18.8 2.4 0.2

2.07 0 20.2

16.15 0 30.3

0.03–0.5 0.06–2 0.004–0.5

MIC, minimum inhibitory concentration; S, susceptible; I, intermediate; R, resistant. a Defined according to the Etest manufacturer’s breakpoints as follows: penicillin, S ≤ 0.064, I 0.12–1, R ≥2 ␮g/mL; erythromycin, S ≤1, I 2, R ≥ 4 ␮g/mL; ceftriaxone (non-meningeal), S ≤ 1, I 2, R ≥ 4 ␮g/mL; moxifloxacin S ≤ 1, I 2, R ≥ 4 ␮g/mL; linezolid, R > 2 ␮g/mL; telithromycin (according to Clinical and Laboratory Standards Institute), S ≤ 1, I 2, R ≥ 4 ␮g/mL.

In total, 386 and 458 isolates, respectively, were available for further testing. Susceptibility results for both study periods are given in Table 1. A statistically significant increase was noted for penicillin resistance (2.07% versus 16.15%; P < 0.001), erythromycin resistance (20.2% versus 30.3%; P = 0.015) and co-resistance to both of these agents (7.5% versus 22.3%; P < 0.001). Ten of the co-resistant strains from the 2003 survey (2.18% of isolates) also exhibited intermediate susceptibility to ceftriaxone. High-level resistance to penicillin (MIC ≥ 4 ␮g/mL) was only found in three strains from the 2003 survey, two of which had an MIC of 4 ␮g/mL and the third had an MIC of 8 ␮g/mL. Between the two study periods, an increase in the erythromycin MIC50 against penicillin non-susceptible isolates from 0.125 ␮g/mL to 16 ␮g/mL was noted as well as an increase in the ceftriaxone MIC50 and MIC90 from 0.015 ␮g/mL to 1 ␮g/mL and from 1 ␮g/mL to 2 ␮g/mL, respectively. 3.4. Risk factors for pneumococci carriage Streptococcus pneumoniae nasopharyngeal carriage was associated both with age and socioeconomic status. Children aged >3 years were 46% less likely to harbour pneumococci in their nasopharynx (odds ratio (OR) 0.53, 95% confidence interval (CI) 0.39–0.71; P < 0.001) compared with those ≤3 years of age after adjusting for socioeconomic status. Higher socioeconomic level was associated with an increased risk of nasopharyngeal carriage (OR 1.24, 95% CI 1.09–1.43; P = 0.002) for each level increase after adjusting for age. 3.5. Risk factors for carriage of resistant pneumococci Results of the univariate analysis are shown in Table 2. In the multivariate analysis, age, socioeconomic status and prior use of a ␤-lactam agent remained statistically significant risk factors for carriage of a penicillin-resistant isolate after adjusting for other factors. Erythromycin resistance was associated with younger age and prior use of antibiotics, either macrolides or ␤-lactams (Table 3).

Table 2 Univariate analysis of Streptococcus pneumoniae susceptibility to antimicrobial agents in relation to different potential risk factors (2003 survey)

Penicillin susceptibility Socioeconomic statusa Age >3 years Otitis media history Prior use of macrolideb Prior use of ␤-lactamb Prior use of both ␤-lactam and macrolidec Erythromycin susceptibility Socioeconomic statusa Age >3 years Otitis media history Prior use of macrolideb Prior use of ␤-lactamb Prior use of ␤-lactam and macrolidec

OR

95% CI

P-value

0.61 0.42 1.59 1.89 1.76 1.67

0.49–0.77 0.26–0.68 1.01–2.52 0.92–3.89 1.05–2.95 0.36–7.72

<0.001 <0.001 0.045 0.081 0.029 0.509

0.73 0.44 1.49 2.51 1.80 0.49

0.58–0.91 0.27–0.71 0.93–2.39 1.21–5.20 1.05–3.07 0.05–4.20

0.006 0.001 0.096 0.013 0.030 0.517

OR, odds ratio; CI, confidence interval. a Ascending socioeconomic status. b Macrolide and ␤-lactam in the last trimester. c Last 3 months before sampling.

4. Discussion The data presented in this study were derived from two surveillance programmes conducted in 2000 and 2003 in the greater Athens metropolitan area and involved children attending daycare centres. The pneumococcal carriage rates of 31.7% and 34.6% were anticipated and confirmed the results of previous studies in Greece [8,9]. We noticed that children ≤3 years old were twice as likely to be colonised compared with older children, reconfirming the knowledge that young age is an important risk factor for nasopharyngeal pneumococcal colonisation [1,4,13]. Unexpectedly, higher socioeconomic status was associated with a slightly increased likelihood of pneumococcal carriage (OR 1.24; P = 0.002). In our study, daycare centres located in higher socioeconomic status areas (level three) had more attendees of age <3 years. Also, the climate in these areas differed from areas

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Table 3 Multivariate analysis of Streptococcus pneumoniae susceptibility to antimicrobial agents in relation to different potential risk factors (2003 survey) OR

95% CI

P-value

Penicillin susceptibility Socioeconomic statusa Age >3 years Prior use of macrolideb Prior use of ␤-lactamb Prior use of ␤-lactam and macrolidec

0.57 0.48 1.84 2.00 2.49

0.43–0.75 0.29–0.82 0.86–3.95 1.17–3.45 0.51–12.15

<0.001 0.007 0.12 0.012 0.26

Erythromycin susceptibility Socioeconomic statusa Age >3 years Prior use of macrolideb Prior use of ␤-lactamb Prior use of ␤-lactam and macrolidec

0.69 0.46 2.52 1.97 0.61

0.52–0.91 0.27–0.78 1.18–5.39 1.14–3.43 0.07–5.43

0.008 0.001 0.010 0.019 0.516

OR, odds ratio; CI, confidence interval. a Ascending socioeconomic status. b Macrolide and ␤-lactam in the last trimester. c Last 3 months before sampling.

of lower socioeconomic status, as they were located in the northern part of the city at a higher level above the sea and were colder and more humid. These factors could account for the discordance with similar studies [1,4,13]. None the less, age >3 years and higher socioeconomic status were protective factors against carriage of resistant S. pneumoniae. This probably reflects fewer infections, fewer doctor visits and fewer antibiotic prescriptions, as children’s immune systems mature by aging. A history of acute otitis media in the past year was a risk factor for penicillin resistance but not for nasopharyngeal carriage in our study. Non-susceptibility to penicillin reached 34.9% in 2003, suggesting that empirical use of this antibiotic in invasive pneumococcal infections such as meningitis or bacteraemia is not safe in our community. However, only three of the isolates demonstrated a high level of resistance to penicillin with MICs ≥4 ␮g/mL, suggesting that ␤-lactams remain the drugs of choice for pneumococcal respiratory tract infections in the community. Ceftriaxone remains a safe therapeutic choice for invasive pneumococcal infections in hospitalised patients. On the other hand, erythromycin resistance has increased during the last 3 years, compromising the role of macrolide monotherapy for the empirical treatment of respiratory tract infections. Based on these findings, the Hellenic Society for Infectious Diseases and the Hellenic Society for Chemotherapy issued the Greek guidelines for the treatment of community-acquired pneumonia in 2005, which recommend that monotherapy with macrolides should be restricted to a specific patient subgroup (i.e. those without comorbidities, without risk factors for drug-resistant S. pneumoniae and without severe illness or those with Mycoplasma pneumoniae or Chlamydia pneumoniae pneumonia). Antimicrobial agents released more recently, i.e. telithromycin, moxifloxacin and linezolid, retained efficacy against all isolates in both studies, urging for careful prescribing to preserve their potency [2,3].

Antibiotic overuse and misuse are considered the major causes of emergence of resistance. Indeed, in Greece, consumption of ␤-lactams and macrolides increased from 22.7 daily defined doses (DDDs)/1000 inhabitants/day in 1999 to 25.4 DDDs in 2002. The European Surveillance of Antimicrobial Consumption (ESAC) project ranked Greece second in total outpatient antibiotic use between 1997 and 2002 [14]. A conclusion can easily be drawn if these facts are combined with the high resistance rates in Greece. In our study, prior use of ␤-lactams was identified as a risk factor for carriage of ␤-lactam- and/or macrolide-resistant isolates, whereas prior macrolide use was only associated with macrolide resistance. Surveys have produced contradictory results, as different antibiotics appear to exhibit different selection powers [5,15]. Apart from antibiotic consumption, other factors such as the high turnover of respiratory pathogens in daycare centres, clonal spread of resistant strains, natural fluctuations and the high population density have all been held responsible for resistance [16]. Further evaluation of the S. pneumoniae isolates will provide valuable information on the possible clonal spread of multidrug-resistant strains and the applicability of the new 7-valent conjugated pneumococcal vaccine in Greece.

Acknowledgments We thank all the participating children and the Demotiko Vrefokomio of Athens for their support. We also thank the laboratory technicians Dimitra Katsala and Konstantina Orlandou for valuable assistance. Etests were purchased with grants from GlaxoSmithKline, Sanofi-Aventis and Bayer.

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