Long-Term Epidemiology of Streptococcus pneumoniae Serogroup 6 in a Region of Southern Europe with Special Reference to Serotype 6E

Streptococcus pneumoniae serotype 6E has recently been described, but its long-term epidemiology is not well known. From 1981–2013, 704 serogroup 6 clinical isolates were obtained in Gipuzkoa, Basque Country, Spain. All invasive and one in four non-invasive isolates were included. Overall, 75, 97, 51 and 45 serotypes 6A, 6B, 6C and 6E isolates, respectively, were detected. No serotype 6D isolates were identified. The prevalence of serotypes 6E and 6B, but not that of serotypes 6A and 6C, declined after the introduction of pneumococcal conjugate vaccines. Serotype 6E isolates showed the highest resistance rate. Most serotype 6E isolates were ST90.


Introduction
Among the nearly 100 different pneumococcal serotypes currently described, Streptococcus pneumoniae serogroup 6 serotypes have been extensively studied, especially serotype 6B [1]. When pneumococcal capsular genes were described in 2006 [2], new serotypes (6C, 6D [3,4] and 6E [5]) were added to the classical serotypes (6A and 6B) in serogroup 6. Because serotype 6E has been described so recently, there is scarce information on its clinical burden and epidemiological trends over long periods.
The main objective of this work was to describe the clinical and microbiological characteristics of serotype 6E pneumococci in our region during a 33-year period.

Materials and Methods
This retrospective study was conducted in San Sebastian, Basque Country, northern Spain. Serotyping was performed with the Quellung reaction using polyclonal antisera (Statens Serum Institut, Copenhagen, Denmark) and multiplex PCR [6].
Serotype 6E was investigated in all serogroup 6 isolates from invasive infections and in a random selection of one in four non-invasive serogroup 6 isolates to accomplish a similar number of invasive and non-invasive isolates. Serotype 6E isolates were detected by PCR amplification and sequencing of the four open reading frames (orf1 to orf4) present between the dexB and wzy genes [5]. Genotyping was performed on all serotype 6E isolates by PFGE and MLST [7,8]. Antibiotic susceptibility was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) [9].
The epidemiological and clinical data of patients with serotype 6E infection, including the 30 days mortality in invasive infections, were recorded. For statistical analysis, categorical variables were compared using the chi-square test or the Fisher exact test when appropriate.
This study was approved by the Ethical Committee for Clinical Research of the Health Area of Gipuzkoa with a waiver of informed consent documentation since this was a retrospective study and patients' identities were safeguarded.

Results
Between 1981 and 2013, 2,082 invasive and 7,595 non-invasive S. pneumoniae isolates were serotyped. The present investigation included all 124 invasive and 144 (one in four of the 577) non-invasive serogroup 6 isolates. The serotypes found among these 268 isolates were as follows: 75 (28.0%) 6A; 97 (36.2%) 6B, 51(19.0%) 6C and 45 (16.8%) 6E (Table 1). There were no serotype 6D isolates. Serotype 6E, consisting of 10 invasive and 35 non-invasive isolates, caused mainly respiratory infections (10 cases of pneumonia, 10 COPD exacerbations, and 6 lower respiratory infections). Of the patients with serotype 6E invasive infections, three had cancer (stomach, leukemia and lymphoma); two patients had chronic bronchitis (one of them also with non-alcoholic cirrhosis and diabetes); two were HIV coinfected patients, one had senile dementia and mild respiratory insufficiency and myocardial sclerosis; in the other two patients no underlying diseases were recorded. The first serotype 6E isolate was collected in 1981 from the pleural fluid of a 45-year-old man with pneumonia.
Serotype 6E caused fewer invasive infections than serotypes 6A, 6B or 6C (p<0.01) but had a similar prevalence to the other three serotypes in non-invasive infections (Table 1).
The mean and median age of patients infected with serotype 6E was 46 years and 57 years, respectively (range 25 days to 95 years) and 34% were women. The mean/median age of patients infected with serotypes 6A, 6B and 6C were 39.4/50 years, 44.5/56 years, and 44.7/55 years, respectively.
Among serogroup 6 isolates, serotype 6E isolates showed the higher resistance rates. The highest percentage of resistance, with significant differences, was obtained between serotype 6E and serotypes 6A and 6C against beta-lactams: penicillin, amoxicillin and cefotaxime; macrolides and lincosamides; sulfamethoxazole trimethoprim (SXT); tetracycline; and chloramphenicol (Table 2). No differences in resistance were found between serotype 6E and 6B isolates except for SXT and chloramphenicol, against which 6E isolates also showed the highest resistance rate (p< 0.003). Resistance to fluoroquinolones and rifampin was infrequent and was similar among isolates of the four serogroup 6 serotypes. Overall, 86.7% (39/45) serotype 6E isolates were multiresistant (non-susceptible to three or more antimicrobial classes).

Discussion
S. pneumoniae serotype 6E has a worldwide distribution [5,10]. However, the current prevalence rates of serotype 6E among serogroup 6 isolates vary widely between different countries, with the highest rates being found in Asian regions [10,11]. Due to the high rates of antibiotic resistance among serotype 6E isolates, if prevalence studies of serotype 6E are done in selected multiresistant isolates, a higher rate of 6E isolates will be expected. The present series has the advantage of being obtained from unselected invasive and non-invasive serogroup 6 isolates.
Although most initial studies considered that the introduction of PCV7 had no influence on the prevalence of serotype 6E [10,12], a recent work demonstrated that serotype 6B antibodies induced by PCV7 had a protective effect against serotype 6E pneumococci [13]. Our population-based data study also supports this latter hypothesis, as serotype 6E was isolated more frequently before the introduction of PCV7 in late 2001. Genotyping showed a clonal distribution of serotype 6E isolates in our region, with CC156 (ST90) being the most common. This clonal distribution is very similar to that of other countries and regions [10,[12][13][14] with the exception of China, where ST982 was the most common ST, followed by ST90 [14].

Conclusions
In Gipuzkoa, northern Spain, serotype 6E pneumococci were isolated in all age groups causing a wide variety of diseases, except meningitis. Most serotype 6E isolates were multiresistant and the decrease in all serotype 6E infections after the introduction of PCV7 in our country supports the hypothesis that the serotype 6B included in PCV formulations confers cross-protection against this long-standing but recently described serotype.  * Susceptibility to all antibiotics was not available in two 6A and 6B isolates