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Indian Journal of Medical Microbiology, Vol. 28, No. 4, October-December, 2010, pp. 313-319 Original Article Serotype markers in a Streptococcus agalactiae strain collection from Zimbabwe RT Mavenyengwa1, JA Maeland2, SR Moyo3 1 Department of Laboratory Medicine, Children's and Women's Health, Norwegian University of Science and Technology (NTNU), Trondheim, Postcode: N-7006, Norway; Department of Medical Microbiology, College of Health Sciences, University of Zimbabwe, P. O. Box Postcode: A178, Avondale, Harare, Zimbabwe, Date of Submission: 11-Mar-2010 Code Number: mb10097 PMID: 20966561 Abstract Objective: Group B streptococci (GBS) from Southern African areas have been less well characterized. Our objective was to study serotype and serovariant distribution of carrier GBS strains as part of a study of the epidemiology of GBS carriage in pregnant women from Zimbabwe. Keywords: Serotype markers, Zimbabwean Streptococcus agalactiae Introduction Research during the last decades has revealed that group B streptococci (GBS) can be classified into a large number of types or variants, by the use of gene-based molecular methods, detection of phenotypic traits such as identification of serotypes or by using combinations of methods. Typing is important in epidemiological settings, including in identification of highly pathogenic GBS clones. In the context of vaccine developments, identification of antigenic markers, which are able to induce protective immunity, is of particular importance, notably in highly pathogenic GBS variants. Ten different CPS types are known in GBS, Ia, Ib and II through IX. The CPSs have been of substantial importance as serotype markers, in addition to surface-anchored protein antigens, which include the proteins Cί, Cα, the alpha-like proteins (Alps) Alp1 (epsilon), Alp2, Alp3, Alp4 and R4(Rib). The Alps are encoded by allelic genes [1],[2] and are chimeras with structural arrangements, which result in immunological cross reactivities. A prominent feature of these proteins is a major stretch composed of a variable number of large, identical, and tandem repeat units. [2] Less well studied are the R3 protein of GBS, [3] the R5 protein that has been sequenced, [4] and a most recently described high-molecular mass antigen of protein nature called protein Z. [5] Both R3 and the Z antigen were expressed by approximately 30% of carrier GBS strains from Zimbabwe. [5] Both Cί, [6] the Alps [2] and the R5 protein [4] have the capacity to induce increased resistance to GBS infection in animal models, and both these and the CPSs have been considered potential vaccine candidates, for instance as CPS-protein conjugates. [6] Less is known of the R3 and Z proteins with regards to capacity to induce protective immunity. In this report, we present an overview of the CPS markers and of nine different serosubtype protein markers detected in a collection of carrier GBS strains from Zimbabwe. Data already described on markers occurring in the Zimbabwean strain collection [5],[7] have been supplemented with the results of testing of the isolates for possession of the gene SAR5, which encodes the strain-variable protein R5, [4] data on CPS association with the various protein markers, and the association between the markers R3, Z and Sar5(R5). To our knowledge, the Sar5(R5) PCR testing is presented for the first time. The results emphasize the predominant role of the R3 and Z antigens and Sar5 as serosubtype markers in GBS strains from Zimbabwe and have provided evidence that the Zimbabwean CPS type V strains differed markedly from type V strains from the Western World with respect to serosubtype markers. Materials and Methods Bacterial strains The prototype and reference strains used in this study and the carrier GBS strains, which have been analysed, have been described in previous reports. [5],[7] Briefly, the reference strains included isolates of nine CPS types of GBS and strains which produced one or more of the well defined and surface-anchored protein serotype markers. The strains under study were 121 GBS carrier isolates from Zimbabwe, 109 vaginal isolates from healthy pregnant women, and 12 isolates from colonized children, collected during the period 2003−2005. [7] Specimen collection, transport, culture, and species identification have been described. [7] The isolates were brought from Harare, Zimbabwe, to Trondheim, Norway, in Stuart's transport medium and immediately cultured on blood agar plates. SAR5(R5) PCR Selection of primers for the SAR5(R5) PCR was based on the published SAR5 sequence [4] with accession number AJ133114. The oligonucleotides 287 5΄-CGTAAATTTTCGGTTGGAATAGC-3΄ 309 (forward) and 704 5΄-GACGAACCACCGTTGTTTCAG-3΄ 683(reverse) were the primers used, synthesized by Eurogentech, S.A., Liege, Belgium. Expected amplicon size was 416 bp. The SAR5 PCR was performed essentially as described previously. [7] Briefly, initial denaturation was performed at 96ºC (3 minutes), denaturation at 95C (60 seconds), annealing at 58ºC (45 seconds.), extension at 72ºC, with 36 cycles, and finally at 72ºC (10 minutes). PCR products were detected using bioanalyser 2100, performed as recommended by the manufacturer (Agilent Technologies). Serotyping Molecular serotyping (PCR) was used to identify CPS types instead of antibody-based typing methods. Primer sets designed to target genes involved in CPS synthesis were used in CPS-specific PCRs as described previously. [7],[8],[9] Multiplex PCR was used to detect the genes encoding the protein markers Cα, Alp1 (epsilon), Alp2, Alp3, Alp4 and R4(Rib), with performance of the tests as described previously. [10] The gene BAC encoding the Cί protein was detected by a separate Cί-specific PCR. [11] The R3 protein and the Z protein were searched for by antibody-based methods, mainly by probing with polyclonal anti-R3 and anti-Z antibodies, both prepared by appropriate cross-absorption of rabbit antiserum raised against whole cells of the R3 reference strain ATCC 49447 (strain 10 out of 84; serotype V/R3, Z and SAR5 PCR positive), diluted in the ratio of 1:1000. [5] The probing was done with ELISA, prepared with whole cells of GBS. [7] Strains that showed inconclusive results were further examined by an exhaustive absorption test. [5] ELISA inhibition test ELISA inhibition was used to compare strains with respect to the capacity to neutralize anti-R3 and anti-Z protein antibodies. [5] Briefly, dilutions of bacteria in a density of 1010 mL-1 were incubated with appropriately diluted antiserum, incubated, centrifuged, and the supernatant tested in indirect ELISA against HCl-extracted antigens. Percentage reduction of optical density recordings was calculated. Results The SAR5(R5) PCR Several primer sets for SAR5 PCR were constructed and evaluated. The set described above showed adequate function and was used throughout this study. PCR results obtained with SAR5 positive and negative isolates, when using the primers selected are shown in [Figure - 1], including the amplicon generated by the GBS strain ATCC 9828 (NT/Alp4, R3, SAR5), the strain from which SAR5 was sequenced. [4] All SAR5 PCR positive strains generated an amplicon of the expected 416 bp size. A variety of reference and prototype strains were tested by the SAR5 PCR, including isolates that produced one or more of the strain-variable proteins Cί, Cα, Alp1 , Alp2, Alp3 or R4(Rib). Only the strains ATCC 9828 and ATCC 49447 (10/84; V/R3, Z and SAR5) showed positive SAR5 PCR. These results supported the notion that the PCR was specific for SAR5. Antigen expression variation Previously we had found that 26 out of 121 (21.5%) of the carrier GBS strains from Zimbabwe showed unambiguous results for R3 protein expression in whole cell-based ELISA. [7] When the isolates, which had shown equivocal results, were examined by means of an exhaustive absorption test, R3 antigen expression was confirmed in seven additional isolates, that is 33 (27.3%) of the 121 isolates examined expressed R3 at detectable levels. We suspected that the ambiguous ELISA results were due to low-level expression of R3 by some of the isolates examined and tested this possibility by performing an absorption ELISA, illustrated by the examples shown in [Figure - 2]. For instance, 10 out of 84 strains (c in [Figure - 2]) produced at least 30 times more of the R3 antigen per bacterial cell unit than strain HMSV391T(d), which previously had shown uncertain results in the whole cell-based ELISA. The isolates d and e are examples of strains which showed equivocal results for R3 protein expression in whole cell-based ELISA. These results underscore a pitfall, which hampers serotyping when performed by means of gene product detection. The final data for the R3 antigen and/or Z antigen expression are included in [Table - 1]. Isolates which expressed the Z antigen at levels not confirmed by the whole-cell ELISA were not detected. Serotype distribution of the Zimbabwean GBS strains [Table - 1] shows the serotype distribution of 121 carrier GBS strains from Zimbabwe. These data are similar to those shown in a previous report, [7] except that [Table - 1] includes: the final results of testing of R3 protein expression; results of testing of all isolates for Z antigen expression and for SAR5(R5) possession; and results which could not be revealed in the former study. [7] By adding the markers Z and SAR5(R5), and the final data for R3 expression, the number of serovariants included in the strain collection increased from 25 to 32. Only 7 (5.8%) of the isolates did not possess any of the nine protein markers searched for in this study while 13 (10.7%) of the isolates possessed as many as four of the protein markers, the maximum number of protein markers associated with single isolates. A total of 111 out of 121 (91.7%) isolates possessed alpha-like protein gene, BCA, Alp1, Alp3 or Rib, but only two of the isolates possessed more than one of the Alp-encoding genes. BAC encoding Cί, a non-alpha-like protein, was possessed by 19 out of 121 (15.7%) of the isolates. Protein marker distribution [Table - 2] details data on the nine protein markers, deducted from the data presented in [Table - 1]. Although each of the protein markers occurred in association with GBS of different CPS types, from three to five CPS types, each marker favoured association with GBS of one or two distinct CPS types: Alp10 with the CPS type Ia strains; the Cα/Cί combination and Z antigen expression with the CPS Ib strains; the R4(Rib) protein with the CPS III strains; the R3 antigen, the Z antigen and sar5(R5) with the CPS type V strains. For all of these markers/CPS type relationships, the association was significantly stronger than the marker association with the rest of the isolates (P ≤ 0.001; Chi-squared test). Among the protein markers, R4 (Rib) predominated and was harboured by 56 (46%) of the isolates, followed by SAR5(R5) and the R3 antigen, both detected in 33 (27%) of the isolates, and then Alp1 detected in 27 (22%) of the strains. Strains of the various CPS types varied with respect to the number of protein markers harboured per strain, from an average of 1.0 marker per CPS type III strain, mostly only R4(Rib), to an average of 3.5 markers per CPS type V strain. These data show that there was a great variation in the favouring of protein markers by GBS of different CPS types and in the number of markers possessed by single isolates of different CPS types. A striking feature was that while the alpha-like proteins Cα, Alp1 and R4 (Rib) occurred with a high frequency, Alp2 was not detected and that only 6 (5%) isolates possessed Alp3 encoding the Alp3 protein. None of the six Alp3-positive isolates possessed any of the markers R3, Z or SAR5(R5), while, otherwise, these markers occurred in combination with Cα, Alp1, and R4 (Rib). These data are consistent with great variability in the preference of GBS of different CPS types for combination with different surface-exposed protein markers, or, vice versa, differences between the protein markers in their favouring of CPS type. R3 antigen, Z antigen, and SAR5(R5) associations The R3, Z antigens and SAR5(R5) most often were found in CPS type V strains but not exclusively [Table - 1] and [Table - 2]. These three markers most often occurred in combination with details shown in [Table - 3]. Nearly all strains (97%), which expressed the R3 antigen, also harboured SAR5(R5) and the majority of R3 positive strains (73%) also expressed the Z antigen. Of the three markers, the Z antigen most often occurred without combination with any of the markers R3 and SAR5. This was the case with 8 out of 14 CPS type Ib isolates [Table - 1] and [Table - 2]. Interestingly, Z was not expressed by any of the 47 CPS type III strains, which also rarely expressed the R3 antigen or possessed SAR5(R5). Only 4 out of 47 (8.5%) of the type III strains possessed one or two of the markers R3, Z and SAR5. Above all, this was distinct from the CPS type V strains of which 27 out of 29 (93.1%) strains harboured at least two of the three protein markers. These data further emphasize the CPS-to-CPS type variability in protein marker association and the inclination of the markers R3, Z and SAR5(R5) to occur together, which may be related to genome localization of the encoding genes. Discussion During recent years, molecular methods have become increasingly important in the subclassification of GBS, in particular to identify highly pathogenic GBS clones. [12] Nevertheless, identification of antigens such as serotype markers and characterization of markers should be important, notably in relation to GBS vaccine developments, as such markers often possess protective epitopes. [2] In our studies of GBS from Zimbabwe, we have combined PCR for identification of surface-exposed CPS serotype and serosubtype protein markers, when technology for this has been available, and antibody-based marker identification when only such methods have been available. It is well known that GBS genes may not always be expressed, or expressed in quantities insufficient for detection of the gene product. [13] We observed this with the R3 antigen, without knowing the reason for some of the isolates produced antigen at low levels. This is a drawback of phenotype-based testing methods and substantiates the advantage of gene-based identification of phenotypic markers such as serotype markers or preferably, combinational testing. As we did not detect isolates with low-level expression of the Z antigen, this substantiates GBS gene differences with respect to efficiency of the process resulting in gene product expression. Both the R3 and Z antigens were detected in 27% of the Zimbabwean GBS strain collection, SAR5 in 29%. These markers were among the most frequent protein antigens detected in the Zimbabwean strain collection, only surpassed by the R4 (Rib) protein, which was found in 46% of the isolates. For the R3 antigen, this finding is in agreement with results obtained when testing isolates of another GBS strain collection from Zimbabwe. [14] Z antigen and SAR5 testing were not available at that time. High prevalence of these markers may be the case for GBS from larger areas of Southern Africa but this remains to be verified. Knowledge of the prevalence of these markers among GBS from Western countries is sparse, but ten years ago, R3 expression was detected in 6.5% of 153 clinical isolates of a Norwegian strain collection. [15] The R3 antigen was not detected in 131 clinical USA isolates reported in 1989, [16] but the R5 antigen was found in 4.4% of 1627 USA strains reported in 1999. [17] Recently, both R3 and the Z antigen were detected when 173 Norwegian clinical strains were examined, R3 in 12, and Z in 14 of the isolates, both antigens in 9 of the strains [Radtke A, personal communication]. As the Z antigen was described quite recently, [5] other data concerning this antigen are not yet available. However, the available data support the probability that the markers R3, Z, and SAR5 occur worldwide but that they play a much more dominating role in Southern African areas than in the Western world, thus illustrating regional differences in the prevalence of serotype markers, important in the context of vaccine developments. Out of 44 isolates which possessed at least one of the markers R3, Z and SAR5(R5), 34 strains signalled positive test with two or all three of the markers. Thus, co-expression of these antigens predominated. As the encoding genes have not been sequenced except for SAR5, and localization in the genome has not been determined, the genetic basis for the marker associations is unknown. Since S. agalactiae strains possess putative mobile genetic elements, [18],[19] anchoring of one or more of the genes in a certain mobile element is a possibility. The gene Alp3 encoding the GBS protein Alp3 is frequently found as the well-characterized R28 antigen in S. pyogenes strains, where the R28 gene is anchored to a 37.4-kb mobile gene element. [20] Preference of the GBS of each particular CPS type for some of the protein markers has been recognized for a long time. This was also the case with the Zimbabwean GBS and with essentially the same preferences as found with GBS from non-African areas [2] with one exception that, the CPS type V strains which, when originating in the Western World, more than 50% of the strains express Alp3 or harbour Alp3. [11],[21],[22] Only 2 out of 29 (7%) of the Zimbabwean type V strains possessed Alp3, while 27 out of 29 (93%) of these isolates possessed two or three of the markers R3, Z and SAR5(R5), 21 out of 29 (72.4%) of the isolates possessed all three markers. These findings are consistent with major differences between type V isolates from these distant areas of the world. It seems likely that whole genome sequencing of type V strains from Zimbabwe may reveal additional major differences between type V GBS from these distant areas. Two sequenced type V strains from USA, strain CJB111 (V/Alp3) and strain 2603(V/Rib), were negative for the markers R3, Z, and SAR5(R5) in our test systems (not described in detail). Our findings argue in the direction of distinct evolutionary lineages of CPS type V GBS from Zimbabwe and those originating from Western areas. CPS type V strains which usually express Alp3 are important pathogens. The importance in this context of the Zimbabwean version of type V strains will remain unknown until isolates from invasive disease cases have been collected and analysed. The finding that GBS, which expressed the R3 antigen, nearly always possessed SAR5 raises the question if R3 and R5 actually are distinct antigens, although one R3-positive isolate in our collection was SAR5 PCR negative and three SAR5 positive strains were R3 negative. Originally, R5 was identified by immunoprecipitation in agarose gels. [4] We have performed similarly designed experiments without being able to identify a precipitinogen that possibly could be the R5 protein. Sequencing of the gene which encodes the protein targeted by R3-specific antibodies, notably the R3-specific monoclonal antibody [15] could provide an answer to the question. Our studies have substantiated that a great diversity of surface-exposed markers occurred among the Zimbabwean GBS strains and have also demonstrated which of the markers predominated, the CPSs Ia, III and V, and the protein markers R4(Rib), R3, Z, R5(SAR5) and Alp1. However, since all of the isolates tested were carrier strains, our studies do not permit conclusion with regard to the role of distinct serotypes and serosubtypes in invasive disease and potential as vaccine candidates in Southern African areas. In particular, the role of R3 and Z as virulence factors and/or inducers of protective immunity is largely unknown while for the other proteins tested and the CPSs, several studies have substantiated important functions in these contexts. [2] Since the proteins R3 and Z were prevalent among the Zimbabwean GBS, studies of the immunobiological role of these proteins deserves priority. When local circumstances make it feasible to collect in larger scale GBS from clinical cases, the data presented here may make a useful background for characterization of the isolates, in particular in relation to vaccine development. References
Copyright 2010 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb10097f1.jpg] [mb10097f2.jpg] [mb10097t2.jpg] [mb10097t1.jpg] [mb10097t3.jpg] |
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