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The susceptibilities of pneumococci recently collected (up to June 2009) in Spain (500 isolates nonsusceptible to oral penicillin and 150 susceptible isolates) from serotypes not included in the conjugate vaccine were determined. Most nonsusceptible isolates (53.6%) belonged to serotype 19A. Susceptibility rates in serotype 19A penicillin-intermediate (n = 201)/penicillin-resistant (n = 67) isolates were <33%/≤6.0% (erythromycin and oral cephalosporins with defined breakpoints), 85.1%/11.9% (amoxicillin), and 96.0%/52.2% (cefotaxime), respectively. Low susceptibility to common oral β-lactams was also found in serotypes 11A (95.5% susceptibility to cefotaxime and erythromycin) and 35B.
From 1979 to 2009 the introduction of new antibiotics and of the 7-valent pneumococcal conjugate vaccine (PCV-7) has altered distributions of serotypes and/or antibiotic nonsusceptibility phenotypes. In Spain the increase in antibiotic nonsusceptibility in the 1980s and 1990s was related to antibiotic consumption (6); the selection of PCV-7 serotypes in the 1990s (after the launch of long-half-life macrolides/cephalosporins) was attributed to penicillin and erythromycin co-nonsuscepti-bility (8). This situation reversed in the 2000s when PCV-7 distribution increased among children, producing a herd effect in adults (5). However, a vaccine-induced replacement has occurred (6) with an increase in the incidence of specific non-PCV-7 serotypes such as 19A and an increase in nonsusceptibility (1) in certain non-PCV-7 serotypes that can complicate selection of therapeutic empirical treatments, especially in the case of oral compounds.
The aim of this study was to explore the susceptibilities of penicillin-nonsusceptible Streptococcus pneumoniae isolates (with penicillin-susceptible isolates as a control group) recently collected (up to June 2009) in Spain, belonging to serotypes not included in PCV-7.
From January to June 2009 the Spanish Reference Laboratory for Pneumococci (SRLP; Instituto de Salud Carlos III) received 2,778 pneumococcal isolates (71.7% invasive and 28.3% noninvasive) from all 17 autonomous communities in Spain through its passive, laboratory-based surveillance system. A total of 2,369 (85.3%) isolates belonged to non-PCV-7 serotypes. Of them, 479 (20.2%) were nonsusceptible to penicillin (MIC, ≥0.12 μg/ml). All these isolates and (in order to obtain 500 isolates) the last 21 isolates from 2008 fulfilling these requirements (non-PCV-7 serotypes and nonsusceptibility to penicillin) were tested in the present study. As controls, the first 150 isolates from 2009, not belonging to PCV-7 serotypes and being susceptible to penicillin (MIC, ≤0.06 μg/ml), were also included in the study collection.
MIC determination was performed by agar dilution using Mueller-Hinton agar (Difco Laboratories, Detroit, MI) supplemented with 5% sheep blood (Biomedics, Madrid, Spain) as the culture medium and incubation under a 5% CO2 atmosphere, as suggested. S. pneumoniae ATCC 6303 and S. pneumoniae ATCC 49619 plus five clinical strains were used as quality controls, as in all determinations in the SRLP. Concentrations tested ranged from 0.015 to 16 μg/ml for penicillin and cefotaxime; 0.06 to 16 μg/ml for amoxicillin; 0.12 to 128 μg/ml for cefaclor; 0.015 to 32 μg/ml for cefuroxime, cefpodoxime, and cefdinir; 0.007 to 8 μg/ml for cefditoren; 0.12 to 128 μg/ml for erythromycin; and 1 to 64 μg/ml for levofloxacin. CLSI breakpoints (4) were used for interpretation (susceptibility/resistance [μg/ml]): penicillin (oral), ≤0.06/≥2; amoxicillin, ≤2/≥8; cefaclor and cefuroxime, ≤1/≥4; cefpodoxime and cefdinir, ≤0.5/≥2; cefotaxime (nonmeningitis), ≤1/≥4; erythromycin, ≤0.25/≥1; and levofloxacin, ≤2/≥8. There are no defined CLSI breakpoints for cefditoren.
Serotyping was performed by the quellung reaction and/or dot blot assay (7).
Among the last non-PCV-7 serotype isolates nonsusceptible to penicillin received in the SRLP, the most prevalent serotype was 19A (53.6%), followed by 24F (9.8%); 35B (7.0%); 6A (5.4%); 11A, 15A, and 23B (4.4% each); and others (11.0%). In contrast serotype prevalences among the 150 penicillin-susceptible isolates from non-PCV-7 serotypes (controls) were 17.3% for serotype 3; 14.0% for serotypes 1 and 7F; 4.7% for 23A; 4.0% for 11A, 22F, and 9N; and 38.0% for other serotypes. Penicillin-susceptible isolates were 100% susceptible to all β-lactams, with MIC90 values of ≤0.06 μg/ml except for cefaclor, to which isolates exhibited 2.7% nonsusceptibility and a MIC90 of 1 μg/ml. Among the 500 non-PCV-7 penicillin-nonsusceptible isolates, 95 (19%) were fully resistant to penicillin (MIC, ≥2 μg/ml). Susceptibility to levofloxacin was ≥99% regardless of the penicillin susceptibility category, while susceptibilities to erythromycin were 89.3% for penicillin-susceptible, 36.0% for penicillin-intermediate, and 23.2% for penicillin-resistant strains.
A total of 53.6% of penicillin-nonsusceptible isolates belonged to serotype 19A. Only two serotype 19A isolates were found among the 150 penicillin-susceptible control strains. Multiresistant serotype 19A has been reported both in countries where PCV-7 has been introduced and in those where it has not (2, 9-11) and is becoming increasingly more drug resistant (3, 12). In Spain, until 2002 serotype 19A accounted for <5% of invasive isolates, but between 2003 and 2007 its incidence rose progressively up to 10% (6). In the present study serotype 19A accounted for 9.7% of the 2,778 strains received from January to June 2009. A previous study in Spain has related the rise in penicillin-resistant serotype 19A to the emergence and clonal spread of two worldwide-disseminated multiresistant clones (ST276 and ST320) (1). Table Table11 shows the susceptibilities of 268 penicillin-nonsusceptible serotype 19A isolates distributed by penicillin-intermediate (201 out of 268 isolates; 75%) and penicillin-resistant (67 out of 268; 25%) categories. In penicillin-intermediate isolates, susceptibility was 1.5% for cefaclor, 27.4% for erythromycin, 32.8% for cefdinir, 33.8% for cefuroxime and cefpodoxime, 85.1% for amoxicillin, 96.0% for cefotaxime, and 99.5% for levofloxacin. This situation worsened in penicillin-resistant strains, where although susceptibility was 100% for levofloxacin, it was only 52.2% for cefotaxime, 11.9% for amoxicillin, 6.0% for erythromycin, and 0% for all oral cephalosporins with defined CLSI breakpoints (Table (Table11).
Table Table22 shows the susceptibility profiles of those penicillin-nonsusceptible (MIC, ≥0.12 μg/ml) isolates belonging to serotypes (other than 19A) with ≥20 isolates. Rates of susceptibility to cefotaxime were >95% for all these serotypes. While susceptibility rates were 100% for levofloxacin for all serotypes shown in Table Table2,2, rates of susceptibility to macrolides (using erythromycin as a resistance marker) were low for serotypes 6A (33.3%), 24F (4.1%), and 15A (0%). Serotype 11A was the most troublesome with 0% susceptibility to oral β-lactams but with 95.5% susceptibility to cefotaxime and erythromycin. Low rates of susceptibility (from 5.7% to 51.4%) to oral β-lactams were also found in serotype 35B, with 91.4% susceptibility to erythromycin and 100% to cefotaxime.
In contrast to previous studies analyzing susceptibility and serotype distribution in S. pneumoniae strains, the current study was exclusively focused on penicillin-nonsusceptible isolates belonging to non-PCV-7 serotypes. According to data from pneumococci received in the SRLP from January to June 2009, non-PCV-7 serotypes are now prevalent among pneumococcal isolates (85.3%) and 20.2% of them are penicillin nonsusceptible. This makes it worthwhile to maintain active surveillance systems exploring the intrinsic activity of oral compounds. The results of this study show that penicillin resistance in specific non-PCV-7 serotypes such as serotypes 19A (the most prevalent), 11A, and 35B is associated with low rates of susceptibility to oral β-lactams commonly used in the community.
This study was supported by an unrestricted grant from Tedec-Meiji Farma S.A., Alcalá de Henares, Madrid, Spain.
O.R. belongs to the Spanish Network for Research in Infectious Diseases (REIPI).
Published ahead of print on 22 March 2010.