Features of Streptococcus agalactiae strains recovered from pregnant women and newborns attending different hospitals in Ethiopia

Group B Streptococcus (GBS) is a recognized cause of infant sepsis and meningitis globally and is a leading cause of morbidity and mortality in Africa [1]. GBS is also a common commensal colonizer of the gastrointestinal and urogenital tracts of women and maternal colonization is a major factor in mother-to-child GBS transmission. Early onset (< 7 days) GBS disease has been well characterized, whereas the epidemiology of late onset disease (LOD, 7–89 days) is less well understood [2].

Penicillin G is the drug of choice for intrapartum prophylaxis [3] and GBS isolates remain mostly susceptible to penicillin. Its prophylactic use has been instrumental in significantly reducing the incidence of early-onset diseases in neonates [4]. However, GBS isolates with reduced susceptibility to penicillin have been reported for more than a decade [5]. Erythromycin and clindamycin have been used as alternatives to prevent vertical transmission of GBS among pregnant women who are allergic to penicillin, but resistance to these antibiotics has emerged in several countries, including reports from Africa [6, 7].

GBS strains are subdivided according to type-specific capsular polysaccharides into 10 unique serotypes which are also a major focus of vaccine development [8]. Serotypes I to V account for about 98% of colonizing GBS isolates worldwide with serotype III usually associated with invasive disease and less common among colonization isolates. GBS serotype distribution is not uniform across different geographic regions and temporal variations have also been described [9]. As information on GBS from their genomes has accumulated, molecular methods have also proved very useful for investigating the population structure of GBS and discriminating differences between strains isolated from different sources [5, 10‐12]. For example, the ST17 lineage has been associated with neonatal infections, particularly with late-onset disease [11]. There are previous reports showing diversification and shifts in serotypes [5, 12, 13] and abundant evidence of past capsular switching within several MLST-based lineages [12, 14] which also pose potential challenges for the development of a vaccine. Global data on circulating GBS strains is important for disease control and for informing the development of effective vaccines. However relatively few studies are available from Sub-Saharan Africa [1] and there are limited data on serotypes and strain characterization [7].

Ethiopia is an important country with a substantial birth cohort in Africa, and while there are previous data published from Ethiopia on antibiotic susceptibility patterns for GBS, there are limited available detailed descriptive data on circulating strains [7, 15]. Here we used whole genome sequencing to investigate serotype distribution, clonal relationships, lineage distributions, virulence factor determinants, and antimicrobial susceptibility patterns of S. agalactiae strains recovered from pregnant mothers and their newborns attending three hospitals in Ethiopia.

Vaccination of pregnant women against GBS is a promising strategy to prevent invasive GBS disease in their infants [8]. Vaccine candidates include protein-based formulations and serotype-specific polysaccharide-protein conjugates [20] and thus an understanding of serotype and surface-protein antigen distribution in maternal colonization and infant disease worldwide is important. While there have been several reports published from Ethiopia on maternal and infant GBS colonization and disease over the past decade, little information on GBS strain characteristics has been described. In this study, serotype II was the most common predicted serotype, with five types (Ia, Ib, II, III and V) accounting for the vast majority of isolates. This is consistent with maternal colonization serotypes described from other studies in Africa and globally [7, 9], however, serotype II is not usually the predominant serotype but rather serotypes III and V [9]. Globally, the vast majority of invasive and colonizing GBS isolates are grouped into five CCs (CC1, CC10, CC17, CC19, and CC23) [21, 22]. In this study, 88% of isolates were grouped into one of these CCs, although no isolates belonging to CC1 and CC17 were identified, emphasizing the diversity of S. agalactiae in human isolates and highlighting the potential for local geographic differences. A recent study from Northwest Ethiopia grouped GBS in four CCs (CC1, CC10, CC19 and CC23) [23]. Similar to findings by others [21, 22] we also saw evidence of past capsular locus replacement events within 2 genetic lineages (ST19 and ST196) which may have implications for vaccine development strategies.

Surface protein antigens play an important role in the pathogenesis of GBS infection, and several of these antigens have been documented as promising vaccine targets [24]. Our data are consistent with past work indicating that a vaccine containing the 3 pilus protein components could be effective in preventing disease caused by GBS as all strains carried at least one of the pilus proteins [25]. Similarly, in all of the isolates one of four highly related Alpha family proteins (Alpha, Rib, Alp2/3, Alp1) were detected suggesting possible broad coverage of the fused N terminal domain Rib-Alpha (GBS-NN) vaccine tested in phase I clinical trials in pregnant women [26]. The HvgA surface-anchored protein has been found to be critical for GBS intestinal colonization and translocation across the blood brain barrier during the onset of meningitis [27]. There were no isolates from meningitis cases in this study but a small proportion of GBS colonization isolates (2.4%) contained the hvgA gene. The hvgA gene has been detected in previous studies, primarily among highly virulent S. agalactiae belonging to serotype III/ST17 [27] and rarely found among non-serotype III isolates [14, 28]. Two hvgA positive isolates in our sampling were of serotype Ia/ST934, which is genetically divergent from other characterized GBS. The closest MLST match within the global database was the triple locus variant ST1232 from an invasive, hvgA-negative serotype II strain recovered during 2017 [12http://​pubmlst.​net].

S. agalactiae have generally been considered universally susceptible to penicillin although there have been several reports of isolates with mutations associated with decreased susceptibility over the last decade [12, 29, 30]. Here, PBP 2X types were restricted to types, 1, 4 and 5, commonly seen in penicillin-susceptible US isolates [5] and all isolates were sensitive to penicillin and cefotaxime. This supports data published from Jimma, Ethiopia [31], however, several previous colonization studies in Ethiopia have documented large numbers of GBS strains resistant to penicillin [15, 32]. Differences in these reported rates warrants further investigation to determine if these rates are indeed real or due to challenges with appropriate and accurate laboratory testing for GBS species identification and antibiotic resistance. The proportion of isolates with in vitro resistance to both erythromycin and clindamycin was similar to rates described by Mengist et al. [31] but lower than that reported from other regions of Ethiopia [7, 32] and other countries [7, 12, 33]. Combined resistance to erythromycin and clindamycin in GBS is most commonly due to 23S rRNA methylases encoded by different erm genes which supports our findings with ermTR and ermB determinants predominantly associated with macrolide and lincosamide resistance [5]. The high level of tetracycline resistance, associated with tetM resistance determinant, may strengthen the hypothesis that current globally circulating S. agalactiae strains in humans were selected by tetracycline usage in 1940s [34]. The proportion of isolates resistant to levofloxacin were similar to that from United States [5, 12], Taiwan [35], Italy [36] and Brazil [37] and lower compared to data reported from China [38] and Canada [39].

A key strength of our study was the molecular characterization of GBS isolates from a region of the world with limited data on this subject. A major challenge and limitation were the reduced recovery rate of GBS strains at CDC allowing only half of the isolates available for additional testing. The inability to recover GBS from the initial frozen stocks could partly be due to loss of viability during storage and transport, and a number of samples were also heavily contaminated which may have contributed also to reduced recovery.

In summary, the most prevalent GBS serotype was serotype II followed Ia, V, Ib, III and IV. This study suggests that circulating GBS in mothers and infants is primarily restricted to five major genetic lineages. All isolates were susceptible to penicillin and resistance to macrolides was relatively low. This study highlights the importance of additional studies to assess GBS epidemiology and develop accurate GBS prevention strategies in Ethiopia. Further epidemiological studies are needed for a more detailed understanding of GBS strain distributions and for subsequent development of vaccination strategies.