Anginosus Strains Research Articles

Diversity of CRISPR-Cas type II-A systems in Streptococcus anginosus.

Streptococcus anginosus is a commensal Streptococcal species that is often associated with invasive bacterial infections. However, little is known about its molecular genetic background. Many Streptococcal species, including S. anginosus, harbor clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. A CRISPR-Cas type II-A system as well as a type II-C system have been reported for this species. To characterize the CRISPR-Cas type II systems of S. anginosus in more detail, we conducted a phylogenetic analysis of Cas9 sequences from CRISPR-Cas type II systems with a special focus on streptococci and S. anginosus. In addition, a phylogenetic analysis of S. anginosus strains based on housekeeping genes included in MLST analysis, was performed. All analyzed Cas9 sequences of S. anginosus clustered with the Cas9 sequences of CRISPR type II-A systems, including the Cas9 sequences of S. anginosus strains reported to harbor a type II-C system. The Cas9 genes of the CRISPR-Cas type II-C systems of other bacterial species separated into a different cluster. Moreover, analyzing the CRISPR loci found in S. anginosus, two distinct csn2 genes could be detected, a short form showing high similarity to the canonical form of the csn2 gene present in S. pyogenes. The second CRISPR type II locus of S. anginosus contained a longer variant of csn2 with close similarities to a csn2 gene that has previously been described in Streptococcus thermophilus. Since CRISPR-Cas type II-C systems do not contain a csn2 gene, the S. anginosus strains reported to have a CRISPR-Cas type II-C system appear to carry a variation of CRISPR-Cas type II-A harboring a long variant of csn2.

Heterogeneity of Streptococcus anginosus ß-hemolysis in relation to CRISPR/Cas.

Streptococcus anginosus is a commensal of the oral mucosa that can cause severe invasive infections. A considerable proportion of Streptococcus anginosus strains are ß-hemolytic due to the presence of an SLS-like gene cluster. However, the majority of strains do not display ß-hemolysis. To investigate ß-hemolysin heterogeneity in S. anginosus, we determined the presence of sag genes and correlated it with the presence of CRISPR/Cas genes in a collection of ß-hemolytic and non-ß-hemolytic strains. All of the ß-hemolytic strains carried the sag gene cluster. In contrast to other streptococci, clinical S. anginosus strains that do not display ß-hemolysis do not harbor sag genes. Phylogenetic analysis of the ß-hemolytic strains revealed that they belong to two previously defined clusters within S. anginosus. Correlation with CRISPR/Cas genes showed a significant difference for the presence of CRISPR/Cas in ß-hemolytic versus non-ß-hemolytic isolates. The presence of the CRISPR/Cas type IIA or type IIC locus is associated with the absence of sag genes; in 65% of the non-ß-hemolytic strains a CRISPR/Cas locus was found, while only 24% of ß-hemolytic strains carry CRISPR/Cas genes. Further analysis of the spacer content of the CRISPR systems revealed the presence of multiple self-targeting sequences directed against S. anginosus genes. These results support the hypothesis that horizontal gene transfer is involved in the acquisition of ß-hemolysin genes and that CRISPR/Cas may limit DNA uptake in S. anginosus.

Nghiên cứu đặc điểm dịch tễ và di truyền của liên cầu khuẩn gây tan máu β lưu hành ở học sinh tiểu học một số vùng miền của Việt Nam

Beta hemolytic streptococci group A, C and G are bacteria that cause pharyngitis, lymphadenitis, pyoderma, erysipelas, toxic shock syndrome, etc which are commonly appeared in children of different ages. In order to study the epidemiological and genetical characteristics of β hemolytic steptococci in primary school pupils in some areas of Vietnam, a total of 1450 primary school pupils including 734 males (50.62%) and 716 females (49.38%) representing for six provinces of Vietnam was investigated. The results showed that the beta hemolytic streptococci infection rate in primary school pupils was 9.38%, in which group A, group C, and group G were 3.45%, 0.55%, and 5.38%, respectively. The highest and lowest rate of streptococcal infection rate was in Quang Tri (25.5%) and Tay Ninh (4.67%) respectively. Chronic pharyngitis and tonsillitis contributed to the increment of streptococcal infection. The 11 emm types of S. pyogenes strains were identified and it hypothesized that the different existence were caused by the diversity of genotypes in various geographical regions. Genetical diversity of S. anginosus strains has been detected based on toxic genes.

Members of a new subgroup of Streptococcus anginosus harbor virulence related genes previously observed in Streptococcus pyogenes

Conventionally categorized as commensals, the Streptococci of the species S. anginosus are facultative human pathogens that are difficult to diagnose and often overlooked. Furthermore, detailed investigation and diagnosis of S. anginosus infections is hampered by unexplored taxonomy and widely elusive molecular pathogenesis. To explore their pathogenic potential, S. anginosus isolates collected from patients of two geographical locations (Vellore, India and Leipzig, Germany) were subjected to multi-locus sequence analysis (MLSA). This analysis revealed the potential presence of a new distinct clade of the species S. anginosus, tentatively termed here as genomosubspecies vellorensis. A complementary PCR-based screening for S. pyogenes virulence factor as well as antibiotic resistance genes revealed not only the presence of superantigen- and extracellular DNase coding genes identical to corresponding genes of S. pyogenes, but also of erythromycin and tetracycline resistance genes in the genomes of the analyzed S. anginosus isolates, thus posing a matter of significant health concern. Identification of new pathogenic S. anginosus strains capable of causing difficult to treat infections may pose additional challenges to the diagnosis and treatment of Streptococcus based infections.

Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus

Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci.

Development of Real-Time PCR Assays for Detection of the Streptococcus milleri Group from Cystic Fibrosis Clinical Specimens by Targeting the cpn60 and 16S rRNA Genes

Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. intermedius), herein referred to as the "Streptococcus milleri group" (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed "McKay agar." McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The cpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10(4) genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.

Evaluation of genotypic and phenotypic methods for differentiation of the members of the Anginosus group streptococci

The terminology and classification of the Anginosus group streptococci has been inconsistent. We tested the utility of 16S rRNA gene and tuf gene sequencing and conventional biochemical tests for the reliable differentiation of the Anginosus group streptococci. Biochemical testing included Rapid ID 32 Strep, API Strep, Fluo-Card Milleri, Wee-tabs, and Lancefield antigen typing. Altogether, 61 Anginosus group isolates from skin and soft tissue infections and four reference strains were included. Our results showed a good agreement between 16S rRNA gene and tuf gene sequencing. Using the full sequence was less discriminatory than using the first part of the 16S rRNA gene. The three species could not be separated with the API 20 Strep test. Streptococcus intermedius could be differentiated from the other two species by β-galactosidase (ONPG) and β-N-acetyl-glucosaminidase reactions. Rapid ID 32 Strep β-glucosidase reaction was useful in separating S. anginosus strains from S. constellatus. In conclusion, both 16S rRNA gene and tuf gene sequencing can be used for the reliable identification of the Anginosus group streptococci. S. intermedius can be readily differentiated from the other two species by phenotypic tests; however, 16S rRNA gene or tuf gene sequencing may be needed for separating some strains of S. constellatus from S. anginosus.

The Streptococcus anginosus species comprises five 16S rRNA ribogroups with different phenotypic characteristics and clinical relevance.

A collection of 267 consecutively isolated Streptococcus anginosus strains was screened for the prevalence of previously described 'motile' strains by hybridization with oligonucleotide probes in a reverse line blot assay. The motile strains represented 101 (37.8%) of the S. anginosus strains. The vast majority of these strains fermented mannitol and raffinose, whereas most other S. anginosus strains did not (P<0.001). Most (83/101) of the motile strains were recovered from the urogenital tract (including five strains from neonatal surveillance cultures) and only a minority (36) of them were associated with infection-related samples (P<0.001). Strains that hybridized with the same oligonucleotide probes as the type strain S. anginosus ATCC 33397T (= NCTC 10713T) were designated ATCC-like strains. They accounted for 74 (27.7%) of the strains examined, were commonly distributed among the different body parts and were significantly more associated with infection-related samples. Three other hybridization patterns were recognized in the reverse line blot assay, ribogroup I (n = 51), ribogroup II (n = 21) and ribogroup III (n = 19). Ribogroup II strains were significantly more frequently recovered from the abdominal cavity and were associated with infection-related samples. Ribogroup I included the majority of the S. anginosus strains that carried Lancefield group C. Comparison of the nearly complete 16S rRNA sequence of two representative strains of each ribogroup revealed that all five ribogroups were closely related (>97% sequence similarities), and that most sequence divergences between the ribogroups occurred in the 1024-1064 bp region of the 16S rRNA gene. The present data confirm the heterogeneity within the S. anginosus species.

Experimental endocarditis induction and platelet aggregation by Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius

A total of 18 ` Streptococcus milleri' strains including the ATCC type strains of Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius were compared with Streptococcus oralis ATCC10557 for their ability to induce infective endocarditis in catheterized rats. Three days after intravenous injection of 10 8 colony-forming units all 8 S. anginosus strains tested produced infective vegetations and bacteremia in almost all rats whereas 5 S. constellatus strains and the S. oralis strain produced infective vegetations and bacteremia less frequently and 5 S. intermedius strains only occasionally. The vegetations infected with the S. anginosus strains harbored microbial cells in significantly higher numbers than those with the other strains. No strong correlation was found between the endocardial infectivity and platelet-aggregating capacity of these strains. The platelet-aggregating strains were members of the Lancefield groups F and G or ungroupable but not of group A or C.

Genotypic and Phenotypic Diversity within Streptococcus anginosus

Streptococcus anginosus is one of the three species currently included in the “anginosus group” of oral or viridans streptococci. In this study 21 strains of S. anginosus were examined in order to determine whether this species, as currently defined, is sufficiently heterogeneous to warrant further subdivision. Phenotypic strain characterization was carried out by performing biochemical tests with a commercial system (Rapid ID32 STREP kit; bioMerieux), by performing tests to determine hyaluronidase production, hemolysis on blood agar, and gliding motility on chocolate agar, by determining Lancefield groups, and by comparing whole-cell polypeptide patterns obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Variations in genotype were determined by studying 16S-23S rRNA intergenic spacer size polymorphisms by PCR amplification, by ribotyping, and by performing DNA-DNA base pairing studies. S. anginosus was found to be heterogeneous at both the species and intraspecies (subspecies) levels. Beta-hemolytic Lancefield group C strains that did not produce hyaluronidase formed a DNA homology group that was separate from the majority of the S. anginosus strains; the members of this group produced a 380-bp intergenic spacer PCR product, exhibited distinct ribotypes, produced an atypical SDS-PAGE pattern, and represented a previously undescribed species in the anginosus group. Two other strains (ATCC 9895 and 1007-77) remained ungrouped as determined by DNA-DNA hybridization and thus represented additional centers of variation at the species level. Hyaluronidase-producing, beta-hemolytic, Lancefield group C strains produced the same atypical SDS-PAGE pattern as beta-hemolytic Lancefield group C strains that did not produce hyaluronidase but differed from the latter organisms by producing a 600-bp intergenic spacer PCR product. In addition, both DNA homology data and ribotyping data suggested that these strains comprise a subspecies of S. anginosus. With the notable exception of the beta-hemolytic Lancefield group C strains that did not produce hyaluronidase, strains ATCC 9895 and 1007-77, and the beta-hemolytic Lancefield group C hyaluronidase-producing strains mentioned above, the strains studied formed a closely related group within which some additional genotypic and phenotypic heterogeneity was observed. The latter group included both strains that fermented mannitol and strains that did not ferment mannitol, as well as strains that exhibited so-called gliding motility. Although no clear-cut division of these organisms was possible, our results indicate that strain NCTC 10713 may not be the most suitable type strain for S. anginosus. We concluded that S. anginosus strains exhibit sufficient heterogeneity to warrant division at both the species and subspecies levels, although insufficient numbers of strains belonging to the putative new taxa have been characterized to allow formal taxonomic proposals to be made.

Bacteriologic characteristics and antimicrobial susceptibility of 70 clinically significant isolates of Streptococcus milleri group

The phenotypical characteristics of 70 clinical isolates of Streptococcus milleri group were analyzed. Association of the three species with particular sites of isolation could not be demonstrated in all cases, but S. anginosus strains predominated in abdominal area, while S. intermedius was isolated mainly in hepatic abscesses. Penicillin G and other β-lactams showed good in vitro activity against these streptococci, whereas >10% exhibited resistance to erythromycin and clindamycin. Resistance to tetracycline reached almost 40%. All of the isolates were uniformely susceptible to vancomycin, chloramphenicol, and trimethoprim.

Oral streptococcal colonization of infants

The objective of this study was to determine the prevalence and proportions of different streptococcal species among the streptococcal flora during infancy. A total of 60 oral samples were collected by oral swabbing of the buccal mucosa and alveolar ridges of 18 infants before tooth eruption and from buccal and lingual surfaces of teeth after tooth eruption. A total of 549 isolates on mitis salivarius agar were speciated, principally by recently revised biochemical criteria of Kilian et al. Streptococcus mitis biovar 1 predominated, both in prevalence (89%) and proportion of oral streptococci recovered in each sample (median = 87% of streptococcal flora). Streptococcus salivarius was also prevalent (94%) but generally represented a small percentage of the total streptococcal flora (median = 3%). Streptococcus oralis and Streptococcus anginosus strains were detected in approximately one third of predentate and dentate infants in the first year of life. Streptococcus sanguis strains were not detected before tooth eruption, but could be detected in 7/14 of the infants with teeth. Thus, S. mitis constitutes the major component of the initially colonizing streptococcal microbiota of the young infant.

Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus (the Streptococcus milleri group): association with different body sites and clinical infections

The associations of Streptococcus intermedius, S. constellatus, and S. anginosus (the three species of the S. milleri group) with clinical infections and sites of isolation were investigated by using a simple biochemical scheme to identify a collection of 153 clinical isolates. S. intermedius was associated with abscesses of the brain and liver, while both S. anginosus and S. constellatus were isolated from a wider range of sites and infections. S. anginosus strains predominated in both genitourinary and gastrointestinal sources and exhibited a wider range of phenotypes, particularly in the ability to ferment mannitol and/or raffinose.

Emended Descriptions and Recognition of Streptococcus constellatus, Streptococcus intermedius, and Streptococcus anginosus as Distinct Species

Strains currently classified as Streptococcus anginosus include strains previously identified as Streptococcus constellatus (Prevot 1924) Holdeman and Moore 1974, Streptococcus intermedius (Prevot 1925), and "Streptococcus milleri" (Guthof 1956) because these specific epithets were argued to be later synonyms of Streptococcus anginosus (Andrewes and Horder 1906) Smith and Sherman 1938 by Coykendall et al. (Int. J. Syst. Bacteriol. 37:222-228, 1987). However, recent data from DNA-DNA hybridization experiments, whole-cell-derived polypeptide patterns determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and data from phenotypic testing have demonstrated that Streptococcus anginosus strains represent three readily identifiable taxa to which the previously assigned type strains of Streptococcus constellatus (strain NCDO 2226 [= ATCC 27823], Streptococcus intermedius (strain NCDO 2227 [= ATCC 27335], and Streptococcus anginosus (strain NCTC 10713 [= ATCC 33397] have been shown to belong. Therefore, we propose recognition of Streptococcus constellatus (emend.) (type strain NCDO 2226 [= ATCC 27823]), Streptococcus intermedius (emend.) (type strain NCDO 2227 [= ATCC 27335]), and Streptococcus anginosus (emend.) (type strain NCTC 10713 [= ATCC 33397]) as distinct species and propose an emended description of each of these taxa.