Characteristics of serogroup 20 S.pneumoniae isolates from Brazil

From 2007 to 2012, 16 out of 336 pneumococci recovered from patients with IPD belonged to serogroup 20. Yildirim and co-workers (2012) [17] evaluated prevalence of serotypes causing invasive diseases in two periods: directly after PCV7 implementation in USA (2000-2002) and a few years later (2009-10) and also observed an increase in the proportion of invasive disease caused by serogroup 20. However, other studies have reported a stable participation of this serogroup in invasive disease over the years [15].

Yearly, the proportion of serogroup 20 increased from 2007 (1/43, 2 % were serotype 20) to 2011 (with 11/125, 9 % of all pneumococci serotyped as 20) and decreased after that (1/76, 1 % in 2012). Indeed, most of the cases of invasive disease caused by serogroup 20 were detected after the implementation of PCV10 (69 %; 11/16), mainly in the year 2011. However, such effect of the vaccine would be unlikely only one year after the beginning of the vaccination program, especially among adults, and in addition only one serogroup 20 case was observed in 2012. It is well known that temporal variations in the distribution of pneumococcal serotypes are expected, independent of selective pressure due to antibiotic use or vaccination and is a more likely explanation for the changes in prevalence seen in this study than is vaccine-related serotype replacement [32].

Most patients were male (56 %; 9/16). For two patients, no gender data were available. The age of patients varied from 37 to 85 years old and the average and median ages were 61.6 and 62 years old, respectively. Eight (50 %) patients were ≥65 years old. Most isolates were recovered from patients admitted at GHC (81 %; 13/16). Isolates from HCPA and HMD represented 12 % (2/16) and 6 % (1/16), respectively.

The majority of isolates (87 %; 14/16) were from blood and the remainder from cerebrospinal fluid (CSF). Although some authors have reported both invasive [13, 33] and non-invasive [20] infections caused by pneumococci from serogroup 20 in adults, studies consistently describe this serogroup in invasive disease among children [15, 17, 19, 36]. Most studies report serogroup 20 associated with bacteremia and/or meningitis [21, 34]. Also, serogroup 20 appears to be found in a very small proportion of pneumococci in the nasopharynx of children [7‐12].

Medical records were available for 12 patients (Table 2). All of them presented at least one underlying condition. The most common underlying conditions were chronic obstructive pulmonary disease (COPD) (4/12; 33 %), alcoholism (4/12; 33 %), systemic arterial hypertension (5/12; 42 %) and smoking (5/12; 42 %). Indeed, it is well-established that pneumococcal diseases (considering all serotypes) are facilitated by abnormal conditions of the respiratory tract or underlying conditions [6]. Three patients (25 %) needed admission to ICU.

Five of the 12 (42 %) patients with known outcome died. However, it is difficult to established mortality attributable to pneumococcal infection as underlying conditions were a common feature. Despite these confounders, some studies have demonstrated an increased risk of invasive disease and/or poor outcome related to serogroup 20. In this context, Grabenstein and co-workers (2014) [33] performed a systematic review to characterize differences in serious outcome between pneumococcal serotypes; among seven adult studies evaluating meningitis, serogroup 20 was among the group with elevated risk. Also, Jansen and co-workers (2009) [13] demonstrated that serogroup 20 was among the serotypes that caused meningitis and bacteremia without focus in a relatively high proportion compared to other serotypes. Other studies focusing on analysis of invasiveness demonstrate that this serogroup, along with others, was found to have an enhanced propensity to cause invasive disease [21]. Despite this important characteristic of invasiveness, serogroup 20 was associated with low rates of case-fatality [13]. In contrast, eight deaths among 569 IPD cases reported by Hsu and co-workers (2010) [15] were associated with a group of non-PCV7 isolates, including serogroup 20.

All pneumococci were resistant to tetracycline and 31 % (5/16) were non-susceptible to trimethoprim/sulfamethoxazole (2 with intermediate resistance and 3 fully resistant). Interestingly, we had previously characterized the antimicrobial susceptibility profile of 159 pneumococci recovered from invasive disease in our region [35] and resistance to tetracycline was observed in 22 % of the isolates. Penicillin MICs ranged from 0.004 to 1 μg/mL and resistance (MIC ≥ 0.12 μg/mL) was observed in 31 % (5/16) MIC₅₀ for penicillin was very low (<0.03 μg/mL); MIC₉₀ was 0.5 μg/mL and one isolate had an MIC of 1.0 μg/mL. Among the limited number of available studies describing antimicrobial susceptibility profiles of serogroup 20 isolates, non-susceptibility to penicillin was not observed by Dunais et al (2011) [7], while one paper reported MICs higher than 0.12 μg/mL [36]. Our isolates were susceptible to all other antimicrobials tested. Indeed, as described in literature, resistance to other antimicrobials appears to be low. Rudolph and co-workers (2013) [37] found a very small proportion of isolates of serogroup 20 non-susceptible to erythromycin among invasive pneumococci recovered from Alaska (1986–2010). Recently, Guo and co-workers (2014) [22] reported two pneumococci belonging to serotype 20B presenting resistance to quinolones in China.

All but one isolate (121–11) were previously submitted to the MLST website; all isolates represented a unique and newly described sequence type: ST8889 [23]. Rudolph and co-workers (2013) [37] typed an erythromycin non-susceptible serogroup 20 isolate and found it to be ST1030, which is one of the 95 serogroup 20 sequence types listed at the MLST website. These STs are distributed in 12 groups (with 59 STs) and 35 singletons. ST 8889 is part of the major clonal complex (CC235) and is a single locus variant of the ancestor, ST235 (Fig. 1).

PFGE grouped all the 16 invasive isolates in a single clone (similarity of 80 % or more in the band pattern). Most isolates (87 %; 14/16) had an identical band profile, and the remaining two pneumococci (044–99 and 045–99) presented a very similar band pattern (Fig. 2). The two isolates recovered from CSF were included in the major PFGE band profile and the ones recovered from patients attending different hospitals (HMD and HCPA) also presented the major band profile. Interestingly, the non-invasive isolate (079–12) included for comparison purposes presented the most distinct band pattern, as shown in Fig. 1, and it was not included in the same clone.

According to Calix and co-workers (2012) [24], serotype 20B (GenBank™ accession number JQ653093.1) is identical to the reference strain of serogroup 20 (GenBank™ accession number CR931679.1), except for the occurrence of a silent mutation in wcjE (936 c::t). On the other hand, serotype 20A (GenBank™ accession number JQ653094.1) presents three alterations in genes of the cps locus: mutation in wchA (659 t::g), mutation in whaF (898 a::c) and an adenosine insertion within a polyadenosine tract in whaF (position 881). None of the 16 pneumococci had these alterations. The wchaA gene of our isolates was identical to the strains CR931679.1 and JQ653093.1 (20B), as well as for whaF. The wcjE gene of isolates included in this study did not have the 936 c::t mutation observed in 20B (JQ653093.1). However, as this is a silent mutation, we can deduce that the protein constitution of our isolates is the same as the previously described 20B. Serotype 20B seems to be the more common subtype identified amongst serogroup 20 isolates, at least among the restricted number of serogroup 20 isolates subtyped so far. Our subtyping results correlated with Calix et al (2012) [24] who identified serotype 20B amongst their isolates, which were also all from IPD.PPV23 includes serotype 20A and although Calix and co-workers [24] infer there might be effective cross-protection against 20B, they suggest epidemiological analyses are warranted to confirm these data.