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The viridans group streptococci

Maeda, Yasunoria,b; Goldsmith, Colin Ea; Coulter, Wilson Ac; Mason, Charlenec; Dooley, James SGb; Lowery, Colm Jb; Moore, John Ea,b

Reviews in Medical Microbiology: October 2010 - Volume 21 - Issue 4 - p 69–79
doi: 10.1097/MRM.0b013e32833c68fa

The viridans group streptococci (VGS) are an extremely diverse range of organisms within the genus Streptococcus, which are characterized by a greening of the blood in blood agar, on which they are normally grown. Oral streptococci largely comprise members of the VGS, which currently include 20 species, which are commensal inhabitants of the oropharyngeal cavity, and the gastrointestinal and genital tracts of mammals. On the basis of 16S rRNA sequence homology, these bacteria are categorised into four groups: the salivarius rRNA homology group, including Streptococcus thermophilus, Streptococcus vestibularis and Streptococcus salivarius; the mitis group, including Streptococcus cristatus, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus sanguinis and Streptococcus parasanguinis; the anginosus group, including Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius; and the mutans group, including Streptococcus mutans, Streptococcus criceti, Streptococcus downei, Streptococcus ferus, Streptococcus macacae, Streptococcus ratti and Streptococcus sobrinus. This review aims at comparing conventional and molecular detection methods and the emergence of antibiotic resistance within the VGS.

aNorthern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast, UK

bSchool of Biomedical Sciences, University of Ulster, Coleraine, UK

cSchool of Dentistry, Queen's University of Belfast, Royal Group of Hospitals, Belfast, Northern Ireland, UK.

Received 26 April, 2010

Accepted 24 May, 2010

Correspondence to Professor John E. Moore, Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK. Tel: +44 28 9026 3554; fax: +44 28 9026 3991; e-mail:

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The viridans group streptococci (VGS) include a variety of commensal bacteria, which used to be considered as one species, namely Streptococcus viridans, characterized by the production of a greenish halo around the colony when they were cultured on blood agar. The term viridans comes from the Latin word ‘viridis’, meaning green. VGS have several common phenotypic features, including being facultatively anaerobic, Gram-positive cocci, catalase-negative, where the majority of strains show alpha haemolysis on blood agar. They are mainly commensal bacteria and inhabit the oral cavity, the upper respiratory tract and the gastrointestinal tract. They are often called ‘oral streptococci’ because of their association with their habitat in the human mouth. Oral streptococci are defined as ‘the species that predominantly inhabit the oral cavities and upper respiratory tracts of humans and animals as commensal and cause opportunistic infections at oral or nonoral sites and in immunocompromised patients’. However, as VGS are also isolated from the intestinal and genitourinary tracts, ‘oral streptococcus’ is not really an accurate description for them. Therefore, the term VGS is used throughout this review.

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Members of the viridans group streptococci

In 1986, the genus Streptococcus consisted of 36 species and two subspecies, and after many revisions of taxonomic change, this number has now increased to 60 species and 12 subspecies. Much confusion remains regarding the taxonomic status of many members of the VGS. Some researchers rely on phenotypic characteristics, whereas others believe in molecular techniques. In this review, VGS organisms are separated into four groups based on 16S rDNA analysis, which has been accepted by the Subcommittee on the Taxonomy of Staphylococci and Streptococci. These four groups are designated as salivarius, mitis, anginosus and mutans. Although there are several members of VGS that have been isolated from farm animals, this review will focus on those species that are isolated from humans.

The salivarius group consists of two species. In contrast to other VGS, Streptococcus salivarius is basically nonhaemolytic and few are alpha- or beta-haemolytic. Their habitat is predominantly on buccal epithelium, dorsal epithelium and tongue. Streptococcus vestibularis is found in the vestibular mucosa of the human oral cavity. The mitis group includes the following 12 species. The species that inhabit the oral cavity and pharynx include Streptococcus mitis, Streptococcus oralis, Streptococcus infantis, Streptococcus peroris, Streptococcus gordonii, Streptococcus australis, Streptococcus sanguinis, Streptococcus oligofermentans and Streptococcus sinensis, whereas Streptococcus cristatus and Streptococcus parasanguinis are found in the throat and mouth. Streptococcus pseudomoniae has been isolated from the respiratory tract. The anginosus group, formally known as the Streptococcus milleri group (SMG), consists of three species, namely Streptococcus anginosus, Streptococcus intermedius and Streptococcus constellatus. Some of these species cause beta haemolysis or no haemolysis. The oral cavity, gastrointestinal tract and urogenital organs are common habitats of these three species. Streptococcus mutans and Streptococcus sobrinus are members of the mutans group. S. mutans is found in dental plaque and carious teeth, whereas S. sobrinus inhabits the tooth surface and carious lesions.

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Viridans group streptococci: an opportunistic pathogen

It has been well documented that species belonging to the VGS are commensal bacteria but they can also cause infections, such as infective endocarditis, because of bacteraemia. Infective endocarditis is commonly caused by S. oralis, S. gordonii and S. sanguinis, and there is a strong correlation between the oral environment and infective endocarditis. An association has been shown between poor oral hygiene and infective endocarditis; bleeding after toothbrushing increases the risk of bacteraemia eight-fold [1]. The effect of flossing on bacteraemia has also been speculated; flossing had a higher risk (40–41%) than toothbrushing shown in previous studies (0–8%) of developing bacteraemia [2,3].

VGS can survive sudden environmental change, especially pH. In dental plaque, pH normally ranges from 6.0 to 6.5 but it is 7.3 in the blood. Once VGS obtain access to the bloodstream, a pH-sensitive promoter is activated and methionine sulfoxide reductase (MsrA) is expressed. The role of MsrA is to give protection from oxidative stress, hence maximizing growth [2]. Then bacteria may attach to heart valves and form vegetations (platelet–fibrin thrombi), preferably on the damaged valve, leading to bacterial invasion to the tissue. Various extracellular binding proteins, such as the fibronectin-binding proteins, CshA and FbpA, have been described as contributors to bacterial cell adhesion and pathogenesis.

Another clinical aspect of bacteraemia caused by VGS is septic shock known as viridans-related septic shock syndrome (VSSS). This has been described mainly in neutropenic and cancer patients treated with chemotherapy and radiotherapy. It has been suggested that reduction of Gram-negative bacteria by prophylaxis using ciprofloxacin may contribute to the VSSS. The mortality rate is reported as 40–100%. Nevertheless, although S. mitis appears to be more virulent than other VGS, the mechanism of the VSSS is still unclear [4].

Species within the mutans group, especially S. mutans, play an important role in the aetiology of dental caries. They are acidogenic and acid-tolerant bacteria. Almost all sugars can be fermented, resulting in rapid production of short-chain carboxylic acids following metabolism of carbohydrates. These acids have the ability to demineralize tooth enamel, which then leads to dental caries. The primary step in tooth decay is bacterial cell adhesion to the tooth surface. Mutans streptococci produce extracellular polysaccharides from sucrose with the support of glucosyltransferase. The stickiness of polysaccharides promotes adhesion to tooth surfaces. Moreover, these extracellular polysaccharides are essential for the construction of biofilms, where bacteria show different phenotypic characteristics. In a biofilm, bacteria gain protection from environmental stresses; oxidase, antibiotics, etc. alter gene expression and exchange their genetic information [5].

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Streptococcus pneumoniae

Streptococcus pneumoniae is the most frequent cause of bacterial pneumonia among people of all ages, as well as an important cause of meningitis and otitis media. Although enormous efforts to develop antibiotics and vaccines have been made to overcome pneumococcal infection, S. pneumoniae infection has been a major cause of death especially in developing countries. It is also estimated that 100 million people are infected by S. pneumoniae every year and 1.6 million people lose their lives. It is also estimated that deaths among children due to pneumococcal infection are 70 000 to 1 million every year around the world.

S. pneumoniae is a facultative anaerobe, Gram-positive cocci and alpha-haemolytic when it is cultured on blood agar. After the cultured cell reaches the stationary phase, it produces an autolysin and collapsing in the centre of the colony is observed. S. pneumoniae is a part of normal flora and its habitat is the upper respiratory tract. It has a capsular structure composed of polysaccharides outside the cell wall, which confers protection against phagocytosis. The capsule type is distinguished immunologically, and more than 90 serotypes have been described. The level of virulence and antimicrobial resistance may vary among the serotypes. Although the existence of noncapsulated strains has been reported, they are relatively avirulent. There are geographical differences in the distribution of pneumococcal serotypes or serogroups. In developed countries 14, 6, 19, 18, 9, 23, 7, 4, 1 and 15 are common with prevalence in descending order, whereas the same are 6, 14, 8, 5, 1, 19, 9, 23, 18, 15 and 7 in developing countries. The reason for differences between serotypes or serogroups and geographical distribution is yet to be investigated.

S. pneumoniae remains a very common causative agent of community-acquired pneumonia, otitis media and meningitis. Colonization of the nasopharynx is the key step in the development of pneumococcal infection, although many individuals stay asymptomatic as their pneumococci remain benignly part of their commensal flora. Colonization in infants begins as early as 1 month of age and carriage rates in adults, children and infants are 10, 20–40 and 60%, respectively [6]. Certain risk factors, including age extremes, crowding, HIV infection and smoking, are considered to increase the chance of pneumococcal infection. Colonization of S. pneumoniae in HIV-positive females is common, and an enhanced ability to adhere to epithelial cells in smokers has been demonstrated. Adhesion to epithelial cells is mediated by several cell-wall proteins. Pneumococcal surface adhesion A (PsaA) binds to N-acetyl-glucosamine on epithelium when asymptomatic colonization occurs, whereas when inflammation is invoked by cytokine stimulation, choline-binding protein (CbpA) interacts with cialic acid, lacto-N-neotetraose and also polymeric immunoglobulin receptor (plgR) on epithelial cells. In addition, unregulated platelet-activating factor (PAFr) on epithelial cells binds to cell-wall phosphocholine (ChoP) of pneumococci. Inflammation is caused by many factors, such as viral infection, malnutrition and damage to the mucosa. Such inflammation converts S. pneumoniae from mere colonisation status to becoming invasive [7].

Virulence factors of S. pneumoniae have been studied extensively. The most characterized virulence factor is pneumolysin (Ply), and it has multiple interactions to host cells and the immune system. Haemolysin was the first pneumolysin activity observed in 1905 with haemolytic reactions with rabbit, sheep, guinea pig and human blood cells. The study also revealed that the substance causing haemolysis was a protein that was inactivated by trypsin treatment. There is current evidence that pneumolysin is produced by almost all clinical isolates during the late log phase, and this haemolysin is located in the cytoplasm and causes cell lysis by binding cholesterol in the plasma membrane of host cells. Besides direct cytotoxicity, pneumolysin prevents opsonophagocytosis by binding IgG, increasing survival in the face of the host immune system in the early stages of infection. This activity is independent of haemolytic activity as it was stable even in pneumolysin-negative mutants [8]. Pneumolysin also can inhibit bactericidal activities of polymorphonuclear leukocytes by reducing their respiratory burst. One puzzle remains and is that pneumolysin has been identified in its close relatives S. mitis, S. oralis and S. pseudomoniae but they are considered to be less pathogenic than pneumococci.

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Prevention of pneumococcal disease by pneumococcal vaccine

In 1946, the first pneumococcal vaccine was introduced in the USA. Unfortunately, this was at the same time when antibiotics emerged for the global treatment of penicillin-susceptible bacterial infections. The vaccine was overshadowed by the then global success of antibiotics and disappeared from the market for a while. As antibiotic-resistant pneumococci began to emerge, a 14-valent pneumococcal polysaccharide vaccine (PPSV) was reintroduced in 1977, followed by the development of a 23-valent PPSV in 1983. The 23-valent PPSV contained purified polysaccharides covering 85–90% of pneumococcal infection-related serotypes in the USA and Europe. However, because of differences in geographical distribution, the actual coverage rate was less than 60% in other parts of the world. Another problem is its limited immunogenicity. It has been demonstrated that PPSV is highly immunogenic to the healthy adult population, whereas its efficiency is much poorer in the elderly, immunocompromised patients and young children, who are the most vulnerable to pneumococcal infection. Therefore, the development of the pneumococcal conjugative vaccine (PCV) was required.

The seven-valent PCV Prevnar was introduced by Wyeth in 2000. The advantage of PCV is that it is a T-cell-dependent antigen as it is conjugated to protein carriers, overcoming limited immunogenicity to high-risk populations. In contrast, one of its disadvantages is that it has less coverage of serotypes. The more the serotypes to cover, the larger the amount of protein carrier required. Klein's estimation in 1995 [9] was 345 μg of polysaccharides and 5750 mg of protein carrier to be administered, if the 23-valent PCV were produced. There was another concern that the more the carrier antigen that was included, the lower the antibody response produced to polysaccharides. That is why, current PCV is covering less serotypes than PPSV. The seven-valent PCV contains pneumococcal polysaccharides of serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, which cover more than 80% of pneumococcal-infection-associated serotypes in the USA, 60–70% in Europe and 55% in Asia.

A wide-ranging surveillance report from the Centers for Disease Control and Prevention (CDC) showed that invasive pneumococcal infection caused by vaccine-covered serotypes among children less than 5 years old has decreased by 94% followed by 64% reduction in elderly and 58% reduction in those aged between 5 and 64, when they compared the incidence between 1998–1999 and 2003. They have suggested that herd immunity results in large decreases of invasive pneumococcal infection in a nonvaccine target population [10]. Herd immunity is defined as a reduction of certain diseases in a nonvaccine target population as a result of decreased transmission from the immunized population. In another study, invasive pneumococcal infection decreased in children (<5) and adults (<65) 96 and 75%, respectively. One concern raised from this study was the emergence of nonvaccine-covered serotypes, especially 19A. In the elderly, the incidence of infection caused by 19A was two times higher than before the seven-valent PVC was introduced, and in children less than 5 years old, it was three times higher. ‘Vaccine escape’ was demonstrated by sequencing analysis of capsular gene of vaccine-covered serotype 4 and nonvaccine-covered serotype 19A. The study [11] concluded that the recently emerged 19A may have switched its capsule from serotype 4 through recombinational events. If capsular switching is occurring naturally, the current development of the 13-valent PVC will not be the definitive solution for pneumococcal infection.

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Identification of species within the genus Streptococcus

Identification of VGS within the genus Streptococcus appears to be one of the toughest tasks in contemporary clinical microbiology, even when employing refined molecular techniques. Lancefield's discovery in 1933 gave major impact to the classification of the genus Streptococcus, as only a few tests, such as the haemolytic reaction and fermentation tests, were available to distinguish streptococci. The classification system used group-specific carbohydrate antigens in beta-haemolytic streptococci and is still in use more than 70 years later. The development of molecular techniques resulted in some reclassification of this genus. New genera including Enterococcus, Lactococcus and Abiotrophla were proposed to replace faecal streptococci, lactic streptococci and nutritionally variant streptococci, respectively. Streptococcus parvulus, Streptococcus morbillorum, and Streptococcus hansenii were reassigned into genus Atopobium, Gemella and Ruminococcus, respectively. The difficulty of speciation of streptococci is mainly due to phenotypic and genetic heterogeneity.

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Conventional methods

Traditionally, species within the genus Streptococcus were distinguished by possession of a different class of Lancefield antigens and the haemolytic reaction. For example, Streptococcus pyogenes is beta-haemolytic and reacts to Lancefield antigen group A. They are indeed useful phenotypic characteristics to distinguish streptococci, as long as each single species falls into the specific criteria or each species shows unified results. Unfortunately, this is not the case for the genus Streptococcus. S. anginosus is a good example of this discontinuity, as S. anginosus can be beta-haemolytic, alpha-haemolytic and nonhaemolytic. Moreover, five of the Lancefield groups, including A, C, F, G and N, may be reactive depending on strain variability.

Biochemical tests have the longest history as an identification method and are the most accepted method to identify bacteria, at least in clinical microbiology laboratories. In fact, the Streptococcal Reference Laboratory in the UK (personal communication) relies on biochemical tests to identify streptococci. The biochemical tests used for speciation of streptococci are, basically, fermentation of sugars and enzyme production tests. The results obtained from each isolate are compared to standard criteria and assigned to certain species. Nowadays, individual tests are packed into convenient kit format and are commercially available, such as Rapid ID 32 STREP (bioMérieux, La Balme les Grottes, France) and STREPTOGRAM (Wako Pure Chemicals, Osaka, Japan). Each test result is converted to a number, a specific code that may be compared with a database of known species. The advantages of these tests are their easy handling, and involvement of less labour, whereas the disadvantages are phenotypic variability of streptococci and an imperfect database. It has been shown that results obtained by biochemical tests are variable even within each species in the case of the genus Streptococcus. In addition, it has been suggested that there is not enough information available about phenotypic variability of newly proposed species. Some typical biochemical tests for the VGS species and the results are summarized in Table 1 [12–14].

Table 1

Table 1

Conventionally, VGS and S. pneumoniae are distinguished by optochin sensitivity and bile solubility. VGS are optochin resistant and not solubilized by bile but pneumococci are sensitive and positive. Although this is now a well-established method, there have been many counter reports describing optochin-susceptible VGS and resistant pneumococci [15]. This is indeed an interesting phenomenon, as optochin is an antimicrobial agent and antibiotic resistance is what many investigators are concerned about. S. pseudomoniae is a newly described species, which has variable susceptibility to optochin when it is cultured in aerobic and microaerophilic conditions. Although culture conditions also affect optochin susceptibility in S. pneumoniae and VGS, the extent is much less, suggesting that previously reported atypical pneumococci may be categorized within this novel species. Arbique et al. [16] have recommended using optochin disc under microaerophilic conditions to avoid misidentification of S. pneumoniae. Taking into account that the mitis group streptococci cannot be differentiated reliably from each other when commercial identification kits are used, optochin and bile solubility tests are still useful techniques to identify S. pneumoniae in the clinical microbiology laboratory.

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Molecular methods

One of the earliest attempts employing DNA for identification was DNA–DNA hybridization. Weissman et al. [17] tried to compare the genetic relationships between reference strains and Lancefield antigen group D and N streptococci, which are now classified as the genus Enterococcus and the genus Lactococcus, respectively. Their results showed that species in different Lancefield groups were easily distinguishable by DNA–DNA hybridization techniques and such techniques may be useful to discriminate species within the same Lancefield group with support of phenotypic characteristics. Along with the development of other molecular techniques, DNA–DNA hybridization techniques were refined in the 1980s. Fluorometric detection allowed more sensitive and precise quantification, besides overcoming safety issues relating to radioisotope usage. This improvement empowered DNA–DNA hybridization to distinguish between the streptococcal species, even between closely related species, such as S. anginosus, S. constellatus and S. intermedius [18]. One taxonomic issue remained and was that a single species could possess more than one Lancefield antigen and present different haemolytic reactions. It was also uncertain which type of strain should be considered representative of each species.

The introduction of sequence analysis of the 16S ribosomal RNA (rRNA) gene has had a large impact on bacterial taxonomy. All bacteria possess 16S rRNA as it is part of the rRNA complex. rRNA plays an essential role in the production of amino acids from DNA via mRNA. Therefore, any bacterial cell should contain the 16S rRNA gene universally. Because the gene plays an essential role in bacterial life and mutations in the gene occur at a very slow rate, it was suggested that it may be used as a molecular clock. The 16S rRNA gene is composed of conserved regions and diverse regions. Broad-range PCR can be achieved by designing a primer set in conserved regions, which allows the 16S rRNA sequence to be obtained from all bacteria. The diverse regions are useful to distinguish bacteria to species level.

Even after improvements in DNA–DNA hybridization techniques, it was still difficult to differentiate some species within the VGS, namely S. mitis, S. oralis and S. pneumoniae, as cross-reactions were observed when clinical isolates were subjected to DNA–DNA hybridization techniques. Bentley et al. [19] and Kawamura et al. [20] conducted comprehensive analyses of 16S rRNA sequences obtained from streptococcal species. In Kawamura's study, 16S rRNA gene sequences from 34 streptococci were used to construct distinctive clusters by the neighbour-joining method. Each cluster was designated into a specific group, namely the anginosus group, the mitis group, the mutans group, the salivarius group, the bovis group and the pyogenic group. These group names are now widely accepted, although within certain groups, discrimination remains a problem, such as S. mitis, S. oralis and S. pneumoniae.

The sequence similarities of the 16S rRNA gene of S. pneumoniae and its close phylogenetic relatives, S. mitis and S. oralis, are more than 99%. This problem has led to the development of various molecular identification methods. Restriction fragment length polymorphism (RFLP) using chromosomal DNA of streptococci and several endonucleases has been applied to identifying streptococci. The results showed diverse RFLP pattern among different species but also within the species [21]. Schlegel et al. [22] have developed a PCR-RFLP method using about a 4500 bp sequence, containing the 16S rRNA gene, the 16S–23S rRNA interspacer (ITS) region and the 23S rRNA gene. Fragments digested by three cutting endonucleases, HhaI, MboII and Sau3AI, individually revealed distinctive patterns among species. Again, however, the fragment patterns were inconsistent within species. De Gheldre and Baele's groups used transfer DNA intergenic spacer length polymorphism (tDNA-ILP) to identify streptococci. In De Gheldre's study [23] few species were not distinguishable, and Baele's study [24] showed an inability to discriminate the mitis group streptococci. Random amplified polymorphic DNA (RAPD) was attempted primarily to distinguish S. mutans and S. sobrinus from other VGS simultaneously. However, none of the primers employed in this study yielded satisfactory results [25].

Species-specific PCR has also challenged the identification of streptococci. Garnier's method [26] consists of two-step PCR targeting the D-alanine: D-alanine ligase (ddl) gene, whereas Hoshino [27,28] used the glucosyltransferase (gtf) gene and 16S–23S rRNA intergenic spacer as a single-step PCR target. Their studies demonstrated that they could accurately detect expected species but a limited number of species were included in their studies. Species-specific PCR–DNA probe hybridizations attempted to identify species within the anginosus group. Jacobs's group [29] designed species-specific probes based on 16S rRNA sequences. When they applied the technique to 399 clinical isolates, 42 of them showed cross-reactivity with S. constellatus and S. intermedius. In Sultana's study [30], the type strains showed high sensitivity to probes based on 23S rRNA sequences but some of the clinical samples turned out to be undetectable either when using species-specific probes or with group-specific probes. They concluded that their unexpected results may be due to the species within the anginosus group being variable both phenotypically and genetically.

Probably, target gene sequence analysis is the most explored single method, as DNA sequencing quality, reduced cost and availability have increased. 16S rRNA sequence analysis was followed by target gene analysis, namely sodA, groESL, rnpB, rpoB, 16S–23S rRNA ITS, tuf, gyrB and dnaJ. The sodA gene, encoding manganese-dependent superoxide dismutase, is one of the most accepted molecular identification methods for streptococci [31]. The primer set constructed in this study was able to amplify a partial region of the sodA gene (480 bp) in 28 streptococcal species. They found that sequence variation was higher with sodA than those of 16S rRNA, which enables one to assign streptococci at an individual species level. Later, it was shown that intraspecies sequence diversity is higher than interspecies diversity in some cases when it was applied in a larger-scale study. The ability to distinguish S. pneumoniae from its close relatives is an advantage of this method.

Ribonuclease (RNase) P RNA gene (rnpB) sequences (370 bp) have been examined with clinical isolates [32]. Although it was difficult to distinguish S. anginosus and S. constellatus, the ability to distinguish S. mitis, S. oralis and S. pneumoniae appeared to be superior to other target gene analyses. One drawback of rnpB gene sequencing analysis is the limited amount of sequences that are available in GenBank. There is an rnpB sequence database, which is maintained at the Uppsala University Hospital in Sweden but, unfortunately, it is not an open resource. Therefore, it still remains to be seen as to whether or not this target gene is truly useful, until more sequence data have been accumulated.

16S–23S rRNA ITS sequences have also been examined as an identification tool. These sequence lengths are relatively short (<400 bp) but sequence similarities among the species are lower that those of 16S rRNA. The authors concluded that using molecular signatures, nucleotides specific to each species allow one to distinguish between these species [33]. However, the relative lack of 16S–23S sequence information, deposited in GenBank compared to 16S rRNA gene sequences, makes this form of sequence-based identification less attractive.

Other gene targets have been investigated as suitable loci for identification or speciation purposes. The groES and groEL proteins, encoding heat shock proteins, have been examined. Sequences (groES: 285 bp, groEL: 1623 bp) produced from 10 species in a previous study were unable to discriminate not only S. mitis and S. oralis but also S. gordonii and S. sanguinis [34]. These results suggest that there may be some difficulty in applying this method to a wide range of streptococcal species. The rpoB gene encodes the beta subunit of RNA polymerase. The sequences (740 bp) obtained from 23 streptococcal type strains revealed good separation with each species. In a previous study, pneumococcal-specific primers were designed from molecular signatures of partial rpoB gene fragments and resulted in two nonspecific amplifications with five of S. oralis, suggesting that identification of S. pneumoniae and its close relatives are not achievable with this gene [35]. Employment of partial sequence of the tuf gene (197 bp), which encodes the elongation factor Tu, has also demonstrated high similarity (more than 98.5%) of interspecies sequence diversity in some species, including S. mitis and S. pneumoniae [36]. The gyrB and dnaJ gene sequence (900 and 1040 bp) analyses have shown high-sequence diversity compared to 16S rRNA sequence, especially in the mitis group streptococci. Although the primer sets used in Itoh's study failed to obtain any amplicon from five species, these genes may be useful for the identification of streptococci [37].

The latest identification methods use multiple sequence analysis, the so-called multilocus sequencing analysis (MLSA). An earlier study conducted by Hoshino [38] demonstrated the importance of using multiple gene sequences to assign various streptococci to each species level. In his study, four housekeeping genes were used, namely ddl, gdh, rpoB and sodA, to construct 1630 bp of sequence information. Although some gene sequences were not obtained in some strains, the available sequence information made distinctive clusters between each species when they were subjected to phylogenetic analyses. This study was followed by Ip's work [39]. Ip's group used two housekeeping genes [i.e., gdh (zwf) and gki] to produce concatenated sequences. Their results demonstrated that multilocus sequencing is a powerful tool to separate S. pneumoniae from its close relatives. This improvement may be due to the inclusion of regions of two genes, which had been incorporated into the multilocus sequencing typing (MLST) scheme for S. pneumoniae.

In 2009, an MLSA scheme for VGS was introduced by Bishop's group. They screened six gene loci from 138 proteins sharing more than 80% amino acid homology among those streptococcal genomes currently available. An additional two genes were selected from Hoshino's study above. Only one of eight genes was unable to amplify even with several primer sets. Therefore, seven gene loci were incorporated into this scheme. The expected size of amplicons were 348–567 bp and each sequence trimmed to a given length and concatenated to make 3063 bp sequence in this order, map, pfl, ppaC, pyk, rpoB, sodA and tuf. When 420 concatenated sequences, including S. pneumoniae, S. pyogenes and S. agalactiae, were subjected to phylogenetic analyses, clearly separated clusters were observed [40]. The advantages of MLSA are that distinctive clusters are obtained even with closely related species and the result is less affected by recombination events. One disadvantage of MLSA relates to its lower resolution than MLST among S. mitis, S. oralis, S. pneumoniae and S. pseudomoniae, as the MLSA scheme focuses on broad-range identification rather than those closely related species.

Overall, MLSA appears to be the best method to identify unknown streptococcal species. However, it involves much sequencing; thus, it can be laborious especially in laboratories which do not have high-throughput sequencing availability, as this is the case with most clinical microbiology laboratories. Therefore, single-gene sequencing remains a feasible and reasonably reliable technique, preferably combined with phenotypic characteristics, to assign streptococcal isolates to the species level.

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Molecular identification and typing of Streptococcus pneumoniae

Two genes encoding pneumococcal virulence factors are often used to identify S. pneumoniae. Rudolph et al. [41] developed a nested PCR targeting the pneumolysin (ply) gene and the autolysin (lytA) gene in order to detect pneumococci from blood specimens. Since then, amplification of both genes has been widely used to detect S. pneumoniae. However, false positives have been reported. The CDC have described new primer sets which were designed to specifically amplify S. pneumoniae and have shown that with real-time PCR, employment of the lytA gene can successfully detect pneumococci but not its close relatives, with greater specificity, compared to the ply gene [42]. Accuprobe is an alternative method to detect S. pneumoniae. In a study by Greiner et al. [43], the sensitivity was 100%, whilst the specificity was 96%. However, after recognition of the closely related species, S. pseudomoniae, the value of this assay diminished, as this species also reacts to the probe [16].

For epidemiological studies, various typing methods have been developed for pneumococci. Two widely accepted methods are pulsed field gel electrophoresis (PFGE) and MLST. PFGE has been used in many studies and is traditionally considered to be the gold standard. The first attempts at PFGE analysis of pneumococci was made by Lefevre's group. Chromosomal DNA was digested by single endonuclease or multiple nucleases, ApaI and SmaI. Digested DNA was then subjected to PFGE, which basically relies on the same principle as ordinary agarose gel electrophoresis, where smaller DNA fragments migrate quicker than larger DNA fragments. In the case of PFGE, electronic field changes in three directions occur periodically and larger DNA takes much more time to migrate. Then, the resulting band patterns were compared to each other. Although it was a laborious and time-consuming method, some improvements have been made and this technique is currently still in use with universal uptake by the clinical microbiology community. However, the major disadvantage of PFGE is its reproducibility, as it has been pointed out that PFGE results may vary depending on the operator or method used; thus, interlaboratory comparison is difficult.

The MLST scheme for S. pneumoniae was introduced by Enright and Spratt in 1998 [44]. It incorporates seven gene sequences, namely aroE, gdh, gki, recP, spi, xpt and ddl. Later, the ddl gene was dropped as it had too high a degree of diversity especially with penicillin-resistant strains. Each unique sequence is assigned an allelic number with all respective genes, and the allelic profile is expressed as the sequence type. There is no direct correlation between serotypes and sequence types but strains having the same sequence type generally belong to the same serotype. Currently there are 4662 described sequence types in the MLST database and this total will increase, as more and more investigators start to rely on MLST typing of pneumococci, as this method lends itself to a high degree of reproducibility and as sequence types are easy to compare between different laboratories.

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Antibiotic resistance

Prevalence of antibiotic resistance in viridans group streptococci and pneumococci

Erythromycin-resistant VGS have been reported in various frequencies. Aracil's study [45] found that 76.4% of clinical isolates were macrolide nonsusceptible. This study also revealed a relatively high percentage (42%) of macrolide-nonsusceptible VGS within the healthy population. This result was supported by another study from Spain showing 79.3% of nonsusceptible clinical isolates [46]. Moreover, 70.0% of macrolide-nonsusceptible clinical isolates were presented in a Tunisian study [47]. Reports from Taiwan and the USA exhibited moderate resistance rates of 45.5 and 40.9%, whereas data from several countries showed relatively low resistance (Japan 17%, Sweden 19% and Turkey 27%).

Erythromycin-resistant S. pneumoniae also appears to exhibit geographical trends. A multicountry study from Asia [48] showed the lowest resistance of 1.5% in India compared to the highest rate (88.3%) in Vietnam, with an overall prevalence of 66.1% of macrolide-nonsusceptible isolates. According to the European Antimicrobial Resistance Surveillance System (EARSS) [49], five countries, including Cyprus, France, Hungary, Italy and Turkey, had more than 25% of prevalence of macrolide-nonsusceptible isolates (29, 31, 32, 27 and 29%, respectively), whereas the Czech Republic, Estonia and Bulgaria had a low prevalence (<5%) of macrolide-nonsusceptible isolates. Surveillance from the USA also described results similar to those of the European survey. The mean macrolide resistance rate during 2000–2004 was 29.3%. This result may be an underestimate of the true rate of nonsusceptibility, as these results showed only resistant isolates but not intermediately resistant isolates. Although there may be bias among reports, macrolide resistance in VGS and pneumococci is of major concern worldwide.

In a previous study by Kerr et al. [50], only 18% of VGS were ciprofloxacin sensitive in patients with a haematological malignancy. This might have been an extreme example of quinolone resistance in VGS. However, more and more evidence suggests the emergence of quinolone resistance in VGS. Although there are geographical and sampling target differences, three American studies have shown a periodical increase. In 1996, 2.6% of VGS from blood culture were found to be resistant, whereas in 2001, this resistance rate rose to 21.3% in 2001 and to 64% in 2006 [51–53]. Two studies from Germany and Poland also reported quinolone-resistant VGS, with resistance rates of 23 and 55.2%, respectively.

In general, the prevalence of quinolone resistance in S. pneumoniae remains low. The Asian Network for Surveillance of Resistant Pathogens (ANSORP) report demonstrated an overall resistance of 6%, with the lowest (2.6%) being reported in Saudi Arabia and the highest (11.8%) being in Hong Kong [54]. In Spain, 7.1% of clinical isolates were resistant to ciprofloxacin and 12% of multidrug-resistant S. pneumoniae in the USA were found to show resistance to ciprofloxacin. Additionally, a 10-year study from Canada showed a clear correlation between increased prescription use of ciprofloxacin and emerging resistance. Although in 1997, prescription rate per 1000 persons of the population was 38.2 and 59.4 in 2006, ciprofloxacin-resistant S. pneumoniae was found to be 0.6% in 1998 and 7.3% in 2006 [55]. One growing concern of quinolone-resistant pneumococci is hidden resistant strains. The Clinical and Laboratory Standards Institute (CLSI) defines the susceptibility breakpoint for levofloxacin as 2 μg/ml or less, whereas some S. pneumoniae strains having a ParC mutation often have a minimum inhibition concentration (MIC) of less than 2 μg/ml. In the same manner, about 30% of ciprofloxacin-susceptible strains possess ParC-associated resistance [56]. Therefore, quinolone resistance in VGS and pneumococci should be considered a major issue.

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Mechanism of macrolide resistance

Two major mechanisms are responsible for macrolide resistance in VGS and S. pneumoniae. The first mechanism is post-transcriptional target modification of 23S rRNA, by methylation. This is due to a methylase, encoded by the erythromycin ribosome methylase [erm(B)] gene. Possession of this gene confers high-level cross-resistance to macrolides, lincosamides and streptogramin B; therefore, it has been designated as the MLSB phenotype [57]. The other mechanism is an active efflux pump encoded by mef(A/E). Originally mef(A) was found in S. pyogenes and mef(E) was discovered from S. pneumoniae. Although the similarity is 90%, they are associated with two individual elements described below [58]. Acquisition of this gene leads to low-level resistance to 14-membered and 15-membered macrolides, such as erythromycin and azithromycin. This phenotype is known as the M phenotype. Both genes are located on mobile genetic elements, providing transformability of those resistant based upon these genes. The erm(B) gene is associated with the Tn916–Tn1545 family. The basic structure of this family is conjugation-related genes, tetracycline resistance genes, the integration (int) gene and the excionase (xis) gene. In addition to these genes, some classes possess the erm(B) gene and/or aphA-3 (kanamycin resistance gene) and mef(E) gene associated with Tn917 the macrolide–aminoglycoside–streptothricin element and the macrolide efflux genetic assembly (MEGA) element, respectively [59]. Tn1207 carries the mef(A) gene and the MEGA element for the mef(E) gene. Homologues of the msr(A) gene, which encodes the macrolide efflux pump in S. aureus, have been found in both elements, namely the open reading frame (ORF) 5 in Tn1207 and mel in MEGA [60]. Despite the lack of enzymes such as the int and xis genes, the insertion of MEGA into pneumococcal genomes has been described (116). In 2005, a new class of the mef gene was reported by Cochetti's group [61], which was designated as mef(I). This gene has 93.6% homology to mef(E) of MEGA and 91.4% mef(A) of TN1207. The other characteristic of this novel gene is that it carries the new element named 5216IQ complex. It contains IQ element having a homologue of msr(A), like the other two mef elements, and the catQ gene (Chloramphenicol resistance determinant), as well as the mef(I) gene.

Several in-vitro experiments have demonstrated the transformability within the genus Streptococcus and also between streptococci and other lineages [62]. These macrolide resistance determinant genes often show high similarity among a variety of Gram-positive bacterial species, suggesting that genetic exchange events of these genes are frequent occurrences in the natural environment.

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Mechanism of fluoroquinolone resistance

Mutations at the quinolone resistance determining regions (QRDRs) in DNA gyrase and DNA topoisomerase IV are responsible for fluoroquinolone resistance in VGS and S. pneumoniae. DNA gyrase is a tetramer and consists of A2B2 subunits encoded by the gyrA gene and gyrB gene, which unwinds ATP-dependent negative supercoiling DNA during DNA replication. DNA topoisomerase IV is also a tetramer and is composed of C2E2 subunits, encoded by the parC gene and parE gene, catalyzing segregation of the chromosome. Muñoz and De La Campa [63] have demonstrated that ParC is the primary target of ciprofloxacin, leading to low-level resistance. GyrA is the second target and mutations in both ParC and GyrA confer high-level resistance. Many mutations have been described in the QRDRs of four genes, especially in S. pneumoniae. On account of the nature of fluoroquinolone resistance, as detailed by mutations within the QRDRs, unlike macrolide resistance, it is difficult to define what mutations truly confer resistance. Those ‘true’ mutation positions frequently reported are Ser81in GyrA, Asp435 in GyrB, Ser79, Se81 and Asp83 in ParC and Asp435 in ParE. Compared to GyrA and ParC, GyrB and ParE gene loci are less important, or at least less commonly found, for the development of quinolone resistance. However, once the mutations occurred together with GyrA or ParC, these are likely to confer high-level resistance [64].

The other mechanism providing fluoroquinolone resistance is active efflux pump PmrA, which is encoded by the pmrA gene, a homologue of the norA gene in Staphylococcus aureus. This mechanism confers low-level resistance and the feature is inhibited by reserpine. On account of this feature, it is easily monitored during MIC tests. Jumbe et al. [65] have suggested that active efflux pumps play a key role during development of fluoroquinolone resistance. The low-level resistance caused by efflux pumps can increase the chance of obtaining target mutations. Although it used to be considered the only efflux system for fluoroquinolone, putative ATP-dependent efflux proteins PatA and PatB were described recently. The difference of this efflux system and the PmrA efflux system is its sensitivity to reserpine; PatA and PatB efflux systems were not influenced by reserpine [66]. There were some strains in which neither amino acid substitution nor efflux activity, assumed by reserpine sensitivity, was observed [67] suggesting the possibility that mutations outside the QRDR region conferred fluoroquinolone resistance. Comprehensive sequence analysis ruled out this possibility. However, there are still some puzzles in development of the resistance other than QRDR mutations and known efflux systems. Moreover, those resistance mechanisms in VGS have been rarely studied so far. Therefore, a comprehensive analysis of fluoroquinolone resistance of VGS would be beneficial to understanding those of S. pneumoniae.

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This work was supported financially by a project grant from the HPSS (NI) Research & Development Office commissioned Research in Antimicrobial Resistance Action Plan (AMRAP) (COM/2730/04).

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infective endocarditis; mouth; oral streptococci; pneumococci; viridans group streptococci

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