Characteristics of serogroup 20 S.pneumoniae isolates from Brazil

Background Although serogroup 20 is not part of any conjugate pneumococcal vaccine, its serotype 20A, but not 20B, belongs to the polysaccharide 23-valent formula. Little is known about its clinical, laboratorial and epidemiological characteristics. Methods The purpose of this study was to evaluate the bacterial genotypes (by PFGE and MLST), clinical characteristics of patients (from review of medical records) and antimicrobial susceptibility of serogroup 20 isolates which were recovered from patients with invasive pneumococcal disease (IPD) from 2007 to 2012. Subtyping to determine 20A and 20B types was also performed by sequencing the genes of the cps locus. Results Sixteen isolates were genotyped and were highly related. All pneumococci were resistant to tetracycline and 31 % were non-susceptible to trimethoprim/sulfamethoxazole. Penicillin MIC ranged from 0.004 to 1 μg/mL and non-susceptibility (MIC ≥ 0.12 μg/mL) was observed in 5/16 isolates (31 %). All isolates belonged to subtype 20B. Most patients were male with a median age of 62 years and presented at least one underlying disease (mostly respiratory conditions). All isolates belonged to ST8889 and to a unique PFGE clone. Conclusions A high clonal occurrence of serotype 20B pneumococci recovered from patients with IPD in Brazil was observed. As a non-PCV10 serotype, selective pressure may be responsible for this unusual occurrence of serogroup 20. However, temporal variation effect should not be underestimated; therefore it is an issue that warrants continued monitoring.

Serogroup 20 is part of the 23-valent polysaccharide pneumococcal vaccine (PPV23), but it is not included in any of the available conjugate formulations. Albeit in a low proportion, this serotype has been found in the nasopharynx of children [7][8][9][10][11][12] and has also been reported as a cause of invasive disease [13][14][15][16][17][18][19][20]. Although little is known about specific characteristics related to its virulence, this serogroup has been associated with increased disease severity, invasiveness and mortality [15,21]. In addition, serogroup 20 has also been linked with some clinically relevant resistance phenotypes, such as levofloxacin resistance [22].
In Brazil, serogroup 20 has recently been recognized among the more prevalent serotypes in the post-vaccine period [23]. Analysis of these isolates by multilocus sequence typing (MLST) identified them as belonging to the same (and newly described) sequence type (ST) 8889. Cálix and co-workers (2012) [24] subtyped isolates belonging to serogroup 20 based on differences in the cps locus and designated these variants as 20A and 20B. They showed distinguishable antigenicity when human sera from patients who were vaccinated with the PPV23 were used. The authors concluded that this vaccine contains serotype 20A polysaccharide, but not 20B.
Compared with other serotypes, there is a restricted number of strains of serogroup 20 described in the MLST database (http://pubmlst.org/spneumoniae/) and therefore additional molecular epidemiology analyses remain to be explored. The aim of this work was to characterize isolates of serogroup 20 recovered from patients with IPD in Porto Alegre, South Brazil.

Study setting and bacterial isolates
S. pneumoniae isolates from patients with IPD have been systematically collected as part of our surveillance studies. From January 2007 to December 2012, 358 pneumococci were recovered from patients attending three hospitals in Porto Alegre, Brazil: Hospital Mãe de Deus (HMD), Grupo Hospitalar Conceição (GHC) and Hospital de Clínicas de Porto Alegre (HCPA). One isolate per patient was considered. Identification was confirmed by optochin susceptibility and bile solubility tests [25]. Serotyping was performed for 336 isolates by multiplex PCR [26] and/or Quellung reaction. Among all S. pneumoniae, 4.8 % (16/336) were identified as serogroup 20 and included in the present study. Isolates were stored in Skim Milk® (Difco) with 5 % glycerol at -80°C.

Antimicrobial susceptibility tests
Minimal inhibitory concentration (MIC) to the following antimicrobials was determined by broth microdilution, as recommended by CLSI, 2013 [27]: penicillin, ceftriaxone, erythromycin, tetracycline, trimethoprim/sulfamethoxazole, levofloxacin, chloramphenicol and vancomycin. The reference strain S. pneumoniae ATCC 49619 was used for quality control.

Genetic characterization of serogroup 20 cps locus
Regions of the capsular genes that previously demonstrated genetic variability between serotypes 20A and 20B [28] were sequenced: wcjE, wchA and whaF. DNA extraction was performed using a 5 % suspension of Chelex® 100 resin (Biorad). The oligonucleotide sequences used were designed based on the nucleotide sequence of the published serogroup 20 cps locus (GenBank™ accession number CR931679.1) and are shown in Table 1. PCR reaction included 1.2 μM of each primer, 2.5U of Taq DNA polymerase and 3.5 mM of MgCl 2 . Reaction conditions included an initial denaturation at 94°C for 4 min, 30 cycles of denaturation (94°C for 45 s), annealing (56°C for 45 s) and extension (72°C for 2.5 min) and a final extension at 72°C for 5 min. PCR products were purified with ExoSAP-IT (Affymetrix USB, Santa Clara, CA) and cycle sequenced using the BigDye Terminator V3.1 chemistry (Life Technologies, Carlsbad, CA). Sequencing reactions were analyzed on an ABI 3130xl genetic analyzer (Applied Biosystems, Carlsbad, California, USA).

Molecular typing
Pulsed-field gel electrophoresis (PFGE) and MLST techniques were used for molecular typing. PFGE was performed according to McEllistrem et al. (2000) [28] and Pinto et al. (2013) [29]. PFGE patterns were clustered by UPGMA. A dendrogram was generated from a similarity matrix calculated using the Dice similarity coefficient with an optimization of 0.5 % and a tolerance of 1.5 %. A non-invasive serogroup 20 isolate (079-12), recovered during the same period from a sputum sample, was included for comparative purposes. Clonal relationship among isolates was defined according to parameters published by Tenover and co-workers (1995) [30].

Analysis of medical records
Patient's records were evaluated to obtain information such as age, gender, intensive care unit (ICU) admission, outcome and occurrence of the following underlying conditions: diabetes, hypertension, HIV infection,

Ethical considerations
This retrospective study was approved by the Research Ethical Committee of Grupo Hospitalar Conceição (Project number . Patient records and information were anonymized and de-identified prior to analysis. 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].
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 wellestablished 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 casefatality [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) MIC 50 for penicillin was very low (<0.03 μg/mL); MIC 90 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 nonsusceptible 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 (Gen-Bank™ 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  Each ST encountered is indicated by a circle, with their diameters being proportional to the numbers of isolates. ST described by our group [23] is highlighted in red 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.

Conclusions
Serogroup 20 is an infrequent non-PCV serotype and little is known about its molecular epidemiology and clinical disease presentation. As changes in the pneumococcal population are expected due to temporal variation and/or selective pressure of vaccination/antimicrobial use, it is important to generate data to better understand the evolution of serotypes not included in available conjugate vaccine formulations. As far as we know, this is the first study devoted to this serotype in Latin America. We focused on the occurrence of a specific ST of serotype 20B, a serotype not included neither in the conjugate nor polysaccharide vaccines. This observation leads to the need of further surveillance for this non-vaccine serotype.