|Year : 2016 | Volume
| Issue : 4 | Page : 421-426
Understanding the viridians group streptococci: Are we there yet?
Department of Microbiology, Dr AL Mudaliar, Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, Tamil Nadu, India
|Date of Submission||06-Jul-2016|
|Date of Acceptance||30-Aug-2016|
|Date of Web Publication||8-Dec-2016|
Department of Microbiology, Dr AL Mudaliar, Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
The viridans group streptococci are a heterogeneous group of organisms which exist as commensals in the oropharynx and the gut. They cause serious infections when they gain entry into sterile sites particularly in patients with predisposing conditions. Classification and species differentiation of these organisms has always been a challenge because of phenotypic differences between strains of the same species. Facklam's typing scheme based on six metabolic properties has been the most widely used and many commercial identification systems are based on it. Due to the ambiguity in species differentiation based on phenotypic tests, nucleic acid-based methods have been developed to improve the identification of these organisms. Results using genotypic methods such as 16S rRNA and sodA gene sequencing have been promising. Multilocus sequence analysis of seven house-keeping genes map, pfl, pyk, ppaC, rpoB, soda and tuf amplified by polymerase chain reaction was found to be an accurate alternative to other methods and could be useful in the characterisation of larger collections of isolates.
Keywords: Differentiation, polymerase chain reaction, phenotypic, viridians streptococci
|How to cite this article:|
Menon T. Understanding the viridians group streptococci: Are we there yet?. Indian J Med Microbiol 2016;34:421-6
| ~ Introduction|| |
The viridans group streptococci (VGS) are a heterogeneous group of organisms which form the predominant commensal flora in the oropharynx. Although these bacteria exist in the mouth and gut as harmless commensals, they are capable of causing serious diseases when they gain entrance to sites that are usually sterile. The viridians streptococci cause a significant percentage of all cases of infective endocarditis (IE), Streptococcus mutans is responsible for dental caries and Streptococcus anginosus causes deep seated abscesses in the brain, liver, etc., and has also been reported to cause neonatal infections. The VGS are generally thought not to have high pathogenic potential, yet they can cause infections such as cellulitis and septicaemia in patients with underlying conditions, particularly immunocompromised states and IE in those with cardiac abnormalities.,,
Several typing schemes for this group of organisms have been proposed, yet they remain poorly classified. There are now at least thirty recognised species of VGS which are classified into six major groups: The S. mutans group, Streptococcus salivarius group, S. anginosus group, Streptococcus mitis group, S. sanguinis group and Streptococcus bovis group. Among them, the S. anginosus group has been the one which is most difficult to classify since all isolates are not uniformly alpha haemolytic; some are non-haemolytic or beta haemolytic. Further, haemolysis may also depend on external factors such as temperature of incubation and medium constituents.
They usually do not react with Lancefield grouping sera, exceptions being some types of S. anginosus which may react with Lancefield A, C, F or G antiserum; S. bovis has some characteristics of the enterococci and reacts with Lancefield D antiserum. Hence for practical purposes, this heterogeneous group of organisms is often defined by the exclusion of other streptococci.
All VGS are catalase-negative, Gram-positive cocci in chains which are leucine aminopeptidase positive, pyrrolidonylarylamidase negative and do not grow in bile esculin agar or 6.5% NaCl. The differentiation of Streptococcus pneumoniae from the VGS using optochin susceptibility and bile solubility can be difficult in certain instances particularly in case of S. mitis and Streptococcus oralis which are known to be genotypically similar (>99% sequence homology) to S. pneumoniae.
| ~ History and Taxonomy|| |
One of the first classifications of VGS was done in 1906 by Andrewes and Horder, who described the first three species. One was a rarely pathogenic resident of the mouth and intestine, which they called S. mitis and was poorly defined for many years because of the small number of substrates hydrolysed by the group. The second one fermented sucrose, lactose and usually raffinose and was so common in saliva that they named it S. salivarius.
The third was a disease causing haemolytic species that fermented sucrose and lactose and often fermented raffinose which they named as S. anginosus. Today, we now know that there are many non-haemolytic variants of S. anginosus, which have been described as Streptococcus milleri, Streptococcus constellatus, S. intermedius and S. milleri group.
In 1919, Orla-Jensen  described S. bovis, an organism common in the bovine gut. It fermented starch, inulin, raffinose and arabinose, but not mannitol. This species is common in the gut of humans with colonic disease and frequently causes IE.
We now know that the most common species isolated from human sources particularly the oral cavity are S. mutans, the aetiological agent of dental caries  and Streptococcus sobrinus. Strains of S. mutans ferment melibiose, hence this test is used to differentiate it from S. sobrinus. In the 1930s and 1940s, several reports were published on the differential characteristics of the viridians streptococci. Members of the group other than the common S.mitis were recognized. Such organisms which were isolated from the blood of patients with endocarditis were found to be resistant to penicillin. This new species was named S. sanguis and in addition to the characteristics noted above, it produced strong green discolouration on blood as well as an extracellular polysaccharide glucan from sucrose. It was later renamed as S. sanguinis.
S. sanguinis binds directly to saliva-coated teeth by a variety of mechanisms. Some of the salivary components to which S. sanguinis binds have been identified, including salivary immunoglobulin A and α-amylase. Once bound, S. sanguinis serves as a tether for the attachment of other oral microorganisms that colonise the tooth surface, form dental plaque and contribute to the development of caries and periodontal disease.S. sanguinis may also interfere with colonisation of the tooth by S. mutans, the primary species associated with dental caries and its presence therefore may also be beneficial for oral health.
| ~ Biotyping of Viridans Group streptococci|| |
The phenotypic characterisation of VGS described by many investigators in the past was mainly based on the conventional typing of biochemical characteristics, such as sugar fermentation and amino acid hydrolysis.
Carlsson in 1967 characterised the strains of VGS by employing seventy different common physiological characteristics which included fermentation of mannitol, sorbitol, lactose, inulin, raffinose and trehalose; hydrolysis of esculin, arginine and hippurate; haemolytic reaction on blood agar, glucan formation on 5% sucrose agar; tolerance to various pH limits of growth; tolerance to salt and bile concentrations in culture media. He described seven groups including S. sanguis I and S. sanguis II.
In the 1970's, two biochemical schemes for the identification of viridans streptococci were published and subsequently widely adopted.
The scheme of Colman and Williams  recognised five species of viridans streptococci isolated mainly from the mouth (S. mutans, S. milleri, S. sanguis, S. salivarius and S. mitior) and became established in European laboratories.
Their identification scheme includes haemolysis on blood agar; formation of glucans; hydrolysis of arginine, esculin and starch; fermentation of inulin, trehalose, salicin, mannitol and sorbitol; tolerance to growth at 45° and 10°C and on 10% and 40% bile; susceptibility to bacitracin and nitrofurazone; production of acetoin from glucose; the presence of rhamnose and ribitol in the cell walls.
The second scheme for the identification of VGS was devised by Facklam and was widely used in the United States. It was based on enzymatic reactions rather than on results of tolerance tests. More than 1200 strains were included in the study and 97% of them could be speciated by using around 25 different biochemical tests for 11 type strains of VGS (S. mutans, S. salivarius, Streptococcus morbillorum, Streptococcus acidominimus, S. sanguis biotype I, S. sanguis biotype II, S. mitis, S. anginosus, S. uberis, S. anginosus–S. constellatus and S. milleri group–S. intermedius). Tests such as acid formation in mannitol, lactose, inulin and raffinose broths; hydrolysis of hippurate, arginine and esculin; growth in litmus milk; tolerance to 40% bile; glucan production on 5% sucrose agar and broth; and haemolysis were used to differentiate the species in the viridans group. Other acid reactions in carbohydrate broths, such as trehalose, salicin and melibiose, were included but could not differentiate the species.
This scheme, devised by Facklam, was more widely used in the USA and differed in assigning strains classified by Colman and Williams as S. sanguis and S. mitior to S. sanguis I and S. sanguis II or S. mitis. It also classified strains of S. milleri into S. anginosus–S. constellatus and S. milleri group–S. intermedius. Facklam's scheme, has formed the basis of several commercial identification kits.
Setterstrom et al., in 1979, introduced the commercial Minitek system (paper discs impregnated with biochemical substrate) for the identification of VGS strains and demonstrated an overall agreement of 98.9% when compared with conventional methods.
Beighton et al., in 1991, devised an improved scheme for biotyping of VGS based on the production of a range of glycosidase activities with 4-methylumbelliferyllinked fluorogenic substrates. His scheme also included 14 conventional fermentation and hydrolytic tests, which enabled the differentiation of all species and distinguished three biotypes within S. sanguis.
Facklam in 2002 introduced a short, reliable biotyping scheme with six tests for the classification and identification of VGS which includes the production of acetoin, fermentation of mannitol and sorbitol, hydrolysis of arginine, aesculin and urea. On the basis of his biotyping method, he classified the VGS into five major groups, such as mutans, salivarius, sanguinis, anginosus and mitis groups with several species under each group. A total of 26 streptococcal species that have the phenotypic characteristics of typical viridans streptococci could be arranged according to the six phenotypic characteristics. With this system, most individual species cannot be identified but are placed in one of the six groupings. This scheme is widely accepted for the speciation of VGS and easy to perform in a routine busy laboratory. The phenotypic characteristics of some of the common species of VGS are shown in [Table 1].
|Table 1: Biochemical characteristics of representative species of viridans group streptococci|
Click here to view
With the exceptions of the group D streptococci, serological differentiation of the viridans streptococci has not been successful. Lancefield attempted to use nucleoprotein and carbohydrate antigens in several serological tests and found cross reaction among the carbohydrate antigens. She concluded that viridans streptococci could not be serologically differentiated.,
Despite introduction of many shortened biotyping scheme by different investigators, classification and identification of the VGS still remains problematic, since the phenotypic-based identification of VGS does not allow the unequivocal identification of some of the species due to the variability in common phenotypic trait among the strains of the same species.
| ~ Nucleic Acid-Based Methods of Species Differentiation|| |
Since species determination of VGS based on phenotypic tests alone remains inaccurate  nucleic acid-based analyses have been developed to improve the identification.
Maeda et al. have compared the ability of five gene loci, namely rnpB, 16S rRNA, 16S-23S rRNA, sodA and dnaJ, to speciate VGS through a sequence typing-based approach. Reference organisms consisting of six VGS species were compared based on sequence typing, followed by comparison of wild-type respiratory isolates, which showed that the employment of sequence typing using the rnpB gene locus was the most specific and reliable and could be used for the identification of VGS to species level.
We compared phenotypic identification of VGS strains with 16S rRNA sequencing. A polymerase chain reaction (PCR) targeting 16S rRNA gene was performed for 48 VGS strains by using broad- range eubacterial 16S rRNA primers described by Weisburg et al. 1991. PCR amplicons were resolved in 0.8% agarose with ethidium bromide by gel electrophoresis and analysed by Bio- Rad Gel documentation system (Bio- Rad, USA).
The 16S rRNA gene sequencing reactions were performed in a MJ Research PTC-225 Peltier Thermal Cycler using an ABI PRISM ® BigDye™ Terminator Cycle Sequencing Kits and ABI 3730 × l sequencer (Applied Biosystem, USA). Single- pass (Unidirectional) sequencing was performed on each template using the aforementioned forward primer (fD1), 5′-AGAGT TTGATCCTGGCTCAG-3′. Sequence chromatogram files were examined for quality by using Bio- Edit version 7.0.9 (Isis Pharmaceuticals) and the low quality ends of 16S rRNA sequences were trimmed by using Codon Code Aligner version 4.0 software (CodonCode Corporation, MA, USA). Species identification of the each strain was achieved by comparing the nucleotide sequence of 16S rRNA gene against known sequences available in the GenBank microbial genomes database using the Basic Local Alignment Search Tool (BLAST) available online (http://www.ncbi.nlm.nih.gov/BLAST/).
Twenty-seven of the 48 strains belonged to the mitis group by 16S rRNA gene sequencing; however, only 20/27 were characterised as belonging to mitis group by biotyping and the remaining seven were identified as salivarius group. In case of the sanguinis group, only one of the nineteen isolates could be identified by biotyping. The rest were identified as mitis group (16), salivarius group (1) and one was unidentified by biotyping. One strain each of S. anginosus and S. mutans gave similar identification by both methods [Table 2].
|Table 2: Comparison of 16s rRNA sequencing and phenotypic identification|
Click here to view
This study showed that the speciation of VGS based on the shortened scheme of Facklam (2002) does not allow unequivocal identification of species of some groups such as the sanguinis group. However, in case of the other members of VGS, such as S. mitis, S. mutans and S. anginosus, phenotypic identification correlated better with genotypic methods.
Sequencing of the manganese-dependent superoxide dismutase (sodA) gene and rpoB gene, encoding the beta-subunit of RNA polymerase have been used in the phylogenetic analyses and identification of bacteria. We compared 16S rRNA PCR with sodA and rpoB gene sequencing and found that the 16S rRNA sequencing was found to be the less discriminative than sodA and rpoB sequencing for very closely related VGS species, however 16S rRNA aided in identification of new species of VGS.
Species such as S. sanguinus, S. gordoni, S. parasanguinis, S. mutans and S. anginosus were identified by both 16S rRNA, sodA gene sequencing, whereas 16S rRNA sequence analysis was found to be less reliable than sodA sequencing for the identification of closely related species such as S. mitis and S. oralis.
Teles et al. in 2011 assessed two phenotypic and three molecular methods to identify VGS and reported that sequence analysis of the sodA gene provided a correct identification for 95% of the strains; however, they concluded that no single method could be considered accurate for identifying strains of VGS.
In situations where it is difficult to assign strains to bacterial species, analysis of sequences of multiple house-keeping genes have been shown to be able to define bacterial species as sequence clusters. This approach, multilocus sequence analysis (MLSA) is done using a strain database, which can be produced and combined with software that allows query strains to be assigned to species via the internet. It is especially useful for groups of organisms such as, the viridans streptococci, which are very difficult to assign to species using standard taxonomic procedures.
Using MLSA, we have examined viridians group streptococci and bovine group streptococci (BGS) isolates that caused IE in different geographic regions. Seven house-keeping genes map, pfl, pyk, ppaC, rpoB, sodA and tuf were amplified by PCR and amplicons were sequenced in both directions. Sequences of type strains were retrieved from the GenBank database. Sequences were aligned, trimmed, edited and concatenated using the software BioEdit 7.0.1 (Isis Pharmaceuticals, CA, USA). Cluster analysis of the concatenated nucleotide sequences (3063 bp) of aforementioned seven house-keeping genes was conducted with MEGA 5.1 using the neighbour-joining method. Robustness of the nodes was tested by bootstrapping with 500 replicates. Species of the isolates were assigned based on the MLSA.
Based on the BLAST analysis of seven housekeeping genes, sodA and rpoB genes sequencing were found to be better for the species identification of VGS by using MLSA typing as gold standard. The other housekeeping genes have low discriminative efficiency for the species identification of VGS [Table 3].
|Table 3: Comparison of multilocus sequence analysis with sequencing results of individual housekeeping genes|
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Comparing aetiology of IE in India and Germany, BGS endocarditis was more frequent in Germany. Only a single Indian cases of IE was caused by S. gallolyticus subspecies gallolyticus, while 25% of the German cases were caused by this subspecies or other members of the BGS. Three out of the 15 cases were caused by S. gallolyticus subsp. pasteurianus and two by S. infantarius. All BGS isolates from IE formed distinct clades in the neighbour-joining tree together with the respective type strains that were used for species assignment. In both regions, the majority of cases were caused by mitis group streptococci; however, there was considerable difference in the spectrum of causative species.
MLSA studies are important in determining phylogenetic relationships of species and are currently widely used to deduce phylogenies by studying partial sequences of genes coding for proteins with conserved functions. However, it is performed in different ways and there are no common generally accepted recommendations on date for applications of individual MLSA schemes. Though it is an accurate alternative to other methods and can be applied in the characterisation of larger collections of isolates, MLSA still needs distinct recommendations and improvements in order to make the application more feasible and generally applicable. Meanwhile, single gene-based identification, particularly the widely used 16S rRNA gene sequence-based phylogenies will remain as the basic approach for genus assignment for VGS. In addition, as our knowledge of disease manifestations and antibiotic profiles of individual species of VGS continues to grow, accurate identification of VGS assumes a greater importance.
Some of the data reported in this paper formed part of the PhD thesis of V. Naveenkumar. Studies on MLSA were done in collaboration with Dr. Patric Nitsche-Schmitz, Helmholtz Centre for Infection Research, Braunschweig, Germany. The financial assistance provided by ICMR, New Delhi and BMBF, Germany, is gratefully acknowledged.
Financial support and sponsorship
Financial assistance was provided by ICMR, New Delhi and BMBF, Germany.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]