Infecciones por estreptococos del grupo S. anginosus en pediatría / S. anginosus group streptococcal infections in children

Los estreptococos del grupo Streptococcus anginosus (EGA), también llamados "Streptococcus milleri", fueron reconocidos como parte de los estreptococos del grupo viridans (EGV) desde principios del siglo XX. Su rol como patógenos humanos, sin embargo comenzó a destacarse recién en la década de 1970. En esta actualización se describen aspectos microbiológicos y clínicos de los EGA. Los métodos fenotípicos de identificacón e incluso algunos genotípicos carecen de precisión para reconocer las tres especies del grupo (Streptococcus anginosus, Streptococcus constellatus y Streptococcus intermedius) e incluso pueden fallar en su clasificación a nivel de grupo. La mayoría de ellos son sensibles a los antibióticos beta-lactámicos pero son considerables los porcentajes de resistencia a macrólidos, lincosamidas y tetraciclinas. Los EGA son colonizantes habituales de las mucosas orofaríngea, intestinal y genitourinaria, pero, cada vez más frecuentemente, son reconocidos como patógenos humanos. Es ampliamente conocida su capacidad de formar abscesos en órganos sólidos, especialmente abscesos cerebrales, pulmonares y hepáticos. También producen sinusitis, empiemas y colecciones en piel y tejidos blandos, hueso, articulaciones, etc. Se han encontrado asociados con exacerbaciones pulmonares en pacientes con fibrosis quística y con enfermedad pulmonar obstructiva crónica. Producen también infecciones posteriores a mordeduras humanas, infecciones diseminadas, bacteriemia sin foco aparente y, en menor medida, endocarditis infecciosa (AU)

Streptococci from the Streptococcus anginosus group (SAG), also termed "Streptococcus milleri", were recognized as members of the viridans group streptococci (VGS) in the early 20th century. Nevertheless, their role as human pathogens only became evident in the 1970s. In this update, microbiological and clinical aspects of the SAG are described. Phenotypic and even some genotypic identification methods lack accuracy in recognizing the three species of the group (Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius) and may fail to classify them at the group level. Most of them are sensitive to beta-lactam antibiotics but rates of resistance to macrolides, lincosamides, and tetracyclines are significant. SAGs are common colonizers of the oropharyngeal, intestinal, and genitourinary mucosa, but are increasingly recognized as human pathogens. Their ability to form abscesses in solid organs, especially brain, lung and liver, is widely known. They may produce sinusitis, empyemas, and collections in skin and soft tissues, bone, joints, etc. They have also been associated with pulmonary exacerbations in patients with cystic fibrosis and chronic obstructive pulmonary disease. In addition, they may cause infections following human bites, disseminated infections, bacteremia without apparent focus, and, to a lesser extent, infective endocarditis (AU)

Estreptococos del grupo Streptococcus anginosus Parte I. Taxonomía, características microbiológicas e identificación / Streptococcus anginosus groupb Part I. Taxonomy, microbiological characteristics and identification / Estreptococos do grupo Streptococcus anginosus Parte I. Taxonomia, características microbiológicas e identificação

Resumen Los estreptococos del grupo Streptococcus anginosus (EGA), también llamados "Streptococcus milleri" fueron reconocidos como parte de los estreptococos del grupo viridans (EGV) desde principios del siglo XX. Sin embargo, su rol como patógenos humanos comenzó a destacarse recién en la década de 1970. Esta actualización consta de tres partes: en esta primera parte se tratarán los aspectos taxonómicos y microbiológicos así como los métodos de identificación de los EGA. El crecimiento de estas bacterias es relativamente lento, las colonias son pequeñas, incluso a las 48-72 horas de incubación y la mayoría de las cepas despide un olor a caramelo característico cuando crecen en agar sangre. Su crecimiento es estimulado en una atmósfera con 5% de CO2. Últimamente, con el reconocimiento de la asociación de los EGA con episodios indeseables en pacientes con fibrosis quística se han desarrollado medios selectivos para poner de manifiesto su presencia en las vías aéreas. Los métodos fenotípicos e incluso algunos genotípicos carecen de precisión para identificar las tres especies del grupo (Streptococcus anginosus, Streptococcus constellatus y Streptococcus intermedius) e incluso pueden fallar en su clasificación a nivel de grupo. Dentro de los métodos moleculares, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) no puede ser tomado como referencia para llegar a subespecie, pero sí es muy eficiente en la identificación a nivel de especie. Para algunos autores la secuenciación del gen sodA podría ser una buena opción, pero el gold standard es el multilocus sequence analysis (MLSA).

Abstract Streptococci from the Streptococcus anginosus group (SAG), also called "Streptococcus milleri", have been recognized as belonging to the viridans group (VGS) since the beginning of the 20th century. Their role as human pathogens, however, only began to emerge in the 1970s. This review consists of three parts: the first part will deal with the taxonomic and microbiological aspects and the identification methods of SAGs. The growth of these bacteria is relatively slow; the colonies are small even after 48-72 hours of incubation and most of the strains give off a characteristic caramel odor when they grow on blood agar. Their growth is stimulated in an atmosphere with 5% CO2. Lately, with the recognition of the association of SAGs with undesirable episodes in patients with cystic fibrosis, selective media have been developed to reveal their presence in the airways. Phenotypic and even some genotypic methods lack precision in identifying the three species in the group (Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius) and may even fail to classify at the group level. Among the molecular methods, MALDI-TOF MS cannot be taken as a reference to arrive at subspecies, but it is very efficient to identify at the species level. For some authors, sequencing the sodA gene may be a good option, but the gold standard is multilocus sequence analysis (MLSA).

Resumo Os estreptococos do grupo Streptococcus anginosus (EGA), também chamados de "Streptococcus milleri", foram reconhecidos como pertencentes ao grupo viridans (EGV) desde o início do século XX. Seu papel como patógenos humanos, no entanto, só começou a surgir na década de 1970. Esta atualização consiste em três partes: nesta primeira parte, trataremos dos aspectos taxonômicos e microbiológicos e dos métodos de identificação dos EGAs. O crescimento dessas bactérias é relativamente lento, as colônias são pequenas mesmo após 48-72 horas de incubação e a maioria das cepas emitem um cheiro de caramelo característico quando crescem em ágar sangue. Seu crescimento é estimulado em uma atmosfera com 5% de CO2. Ultimamente, com o reconhecimento da associação dos EGAs com episódios indesejáveis em pacientes com fibrose cística, foram desenvolvidos meios seletivos para revelar sua presença nas vias aéreas. Os métodos fenotípicos e mesmo alguns genotípicos carecem de precisão na identificação das três espécies do grupo (Streptococcus anginosus, Streptococcus constellatus e Streptococcus intermedius) e podem até falhar em sua classificação em nível de grupo. Entre os métodos moleculares, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) não pode ser tomado como referência para chegar a subespécie, mas é muito eficiente na identificação em nível de spécie. Para alguns autores, o sequenciamento do gene sodA poderia ser uma boa opção, mas o padrão-ouro é a análise de sequência multilocus (MLSA).

Rol de los estreptococos del grupo Streptococcus anginosus en infecciones de piel y partes blandas: reporte de 3 casos / Role of streptococci of the Streptococcus anginosus group in skin and soft tissue infections: report of 3 cases

Los estreptococos del grupo Streptococcus anginosus (GSA) comprenden uno de los grupos que conforman el grupo viridans de estreptococos y son parte de la microbiota humana habitual de la cavidad oral, del tracto respiratorio superior y del tracto gastrointestinal. Pueden producir colecciones purulentas de órganos sólidos, abscesos cutáneos y celulitis, entre otras infecciones. Este grupo posee numerosos factores de virulencia como hialuronidasa, adhesinas, enzimas extracelulares y hemolisinas. El objetivo de este trabajo fue presentar tres casos clínicos de infecciones asociadas a traumatismos vinculados con la cavidad oral como golpes de puño o mordeduras humanas. En todos ellos, los GSA desarrollaron como germen único, lo que pone de manifiesto su importante rol en este tipo de infecciones.

The Streptococcus anginosus group (SAG) is part of the viridans group streptococci and they belong to the normal human microbiota of the oral cavity, the upper respiratory tract and the gastrointestinal tract. It can produce purulent collections of solid organs, cutaneous abscesses and cellulitis, among other infections. This group has numerous virulence factors such as hyaluronidase, adhesins, extracellular enzymes and hemolysins. The objective of this work is to present three clinical cases of infections associated with trauma related to the oral cavity such as fist bumps or human bites. In all of them, the SAG developed as a single organism, which highlights its important role in this type of infection.

Os estreptococos do grupo do Streptococcus anginosus (GSA) são parte de um dos grupos que constituem o grupo viridans e fazem parte da microbiota normal da cavidade oral, do trato respiratório superior e do trato gastrointestinal dos seres humanos. Podem produzir coleções purulentas de órgãos sólidos, abscessos cutâneos e celulite, entre outras infecções. Este grupo possui numerosos fatores de virulência, como hialuronidases, adesinas, enzimas extracelulares e hemolisinas. O objetivo desse trabalho foi apresentar três casos clínicos de infecções associadas a traumatismos associados à cavidade oral, decorrentes de socos ou mordidas humanas. Em todos eles os GSA se desenvolveram como germe único, o que põe de manifesto seu papel importante nesse tipo de infecções.

A novel plasmid, pSAA0430-08, from Streptococcus anginosus subsp. anginosus strain 0430-08.

Mobile genetic elements (MGEs) are the genetic material often involved in the interspecies and intraspecies genetic transduction in bacteria. However, little is known about MGEs in the Anginosus group of streptococci (AGS), one of the streptococcal groups found in the oral cavity of humans. We looked for the presence of MGEs in Streptococcus anginosus subsp. anginosus (SAA), a representative species belonging to AGS, and found a novel plasmid from SAA strain 0430-08. This plasmid was 7038bp and ~31% G/C content which we named pSAA0430-08, and examined its genetic structure and characteristics. Open reading frame (ORF) prediction revealed that pSAA0430-08 was composed of 10 ORFs including a putative plasmid replication protein (ORF1) and a putative toxin-antitoxin system (ORF9 and ORF10). Between ORF10 and ORF 1, four tandem repeats of 22bp each, generally termed as iteron, were also observed. Using variant plasmids of pSAA0430-08, we confirmed that both ORF1 and iteron were necessary for replication in host cells. Interestingly, the region from ORF4 to ORF7 showed homology with a genomic DNA segment of S. gordonii strains. Thus, this plasmid may travel between the different species in Streptococci, i.e., S. gordonii and S. anginosus.

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.

Clinical and microbiological outcomes in patients with Streptococcus anginosus group bacteraemia identified through use of a rapid microarray assay.

Limited data exist evaluating outcomes in patients with serious Streptococcus anginosus group infections, particularly bacteraemia. A retrospective, single-centre cohort study was conducted to characterize potential risk factors along with clinical and microbiological outcomes in patients with S. anginosus group bacteraemia (SAGB). Adult inpatients with SAGB identified using the Verigene Gram-positive blood culture assay between March 2013 and April 2014 were included. Patients aged ≤ 18 or >89 years, those with SAGB identified at an outside facility and those who were incarcerated were excluded. Differences between groups were explored using a Wilcoxon rank-sum test, χ2 test, Student's t-test or Fisher's exact test as appropriate and a two-tailed P value of ≤ 0.05 was considered statistically significant. The 34 patients who met the inclusion criteria were 57 ± 14 (mean ± SD) years old and had a median Charlson co-morbidity index of 4 [interquartile range (IQR) 1-6] and 10 (29%) were immunosuppressed at baseline. Almost half (47%) had received antibiotics in the previous 90 days. Twelve (35%) patients had gastrointestinal malignancies and the commonest source of bacteraemia was the gastrointestinal tract (53%). The primary species responsible for SAGB was S. anginosus (68%), and overall susceptibility to penicillin was 91%. Patients were most often treated with a ß-lactam/ß-lactamase inhibitor combination (36%) for a duration of 8 (IQR 4-13) days. Length of stay (LOS) and infection-related LOS were 10 (IQR 5-17) and 9 (IQR 4-12) days, respectively. Twenty [59%] patients achieved a clinical cure, while 29 (85%) achieved a microbiological cure. Four (12%) patients died and one patient was readmitted within 30  days. In the largest cohort of patients with SAGB to date, gastrointestinal malignancies may have been an important risk factor for SAGB, while rapid identification via a microarray assay likely contributed to improved disease recognition and timely pharmacological and non-pharmacological therapy.

Comparing Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Phenotypic and Molecular Methods for Identification of Species within the Streptococcus anginosus Group.

The heterogeneity of members of the Streptococcus anginosus group (SAG) has traditionally hampered their correct identification. Recently, the group was subdivided into 6 taxa whose prevalence among human infections is poorly described. We evaluated the accuracy of the Rapid ID32 Strep test, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and a PCR multiplex method to identify 212 SAG isolates recovered from human infections to the species and subspecies level by using multilocus sequence analysis (MLSA) as the gold standard. We also determined the antimicrobial susceptibilities of the isolates. Representatives of all SAG taxa were found among our collection. MALDI-TOF MS and the Rapid ID32 Strep test correctly identified 92% and 68% of the isolates to the species level, respectively, but showed poor performance at the subspecies level, and the latter was responsible for major identification errors. The multiplex PCR method results were in complete agreement with the MLSA identifications but failed to distinguish the subspecies Streptococcus constellatus subsp. pharyngis and S. constellatus subsp. viborgensis. A total of 145 MLSA sequence types were present in our collection, indicating that within each taxon a number of different lineages are capable of causing infection. Significant antibiotic resistance was observed only to tetracycline, erythromycin, and clindamycin and was present in most taxa. MALDI-TOF MS is a reliable method for routine SAG species identification, while the need for identification to the subspecies level is not clearly established.

Identification of the 'Streptococcus anginosus group' by matrix-assisted laser desorption ionization--time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides rapid, accurate and cost-effective identification of a range of bacteria and is rapidly changing the face of routine diagnostic microbiology. However, certain groups of bacteria, for example streptococci (in particular viridans or non-haemolytic streptococci), are less reliably identified by this method. We studied the performance of MALDI-TOF MS for identification of the 'Streptococcus anginosus group' (SAG) to species level. In total, 116 stored bacteraemia isolates identified by conventional methods as belonging to the SAG were analysed by MALDI-TOF MS. Partial 16S rRNA gene sequencing, supplemented with sialidase activity testing, was performed on all isolates to provide 'gold standard' identification against which to compare MALDI-TOF MS performance. Overall, 100 % of isolates were correctly identified to the genus level and 93.1 % to the species level by MALDI-TOF MS. However, only 77.6 % were correctly identified to the genus level and 59.5 % to the species level by a MALDI-TOF MS direct transfer method alone. Use of a rapid in situ extraction method significantly improved identification rates when compared with the direct transfer method (P<0.001). We recommend routine use of this method to reduce the number of time-consuming full extractions required for identification of this group of bacteria by MALDI-TOF MS in the routine diagnostic laboratory. Only 22 % (1/9) of Streptococcus intermedius isolates were reliably identified by MALDI-TOF MS to the species level, even after full extraction. MALDI-TOF MS reliably identifies S. anginosus and Streptococcus constellatus to the species level but does not reliably identify S. intermedius.

Invasive Streptococcus anginosus group infection-does the species predict the outcome?

OBJECTIVE: To determine whether there is an association between the species of Streptococcus anginosus group (SAG) bacteria and the clinical outcome. METHODS: Isolates from invasive infections caused by SAG bacteria at our institution between January 2004 and February 2009 were identified phenotypically to the taxonomic level of species. Clinical data from the medical records of the patients from whom these isolates were recovered were obtained retrospectively and analyzed. RESULTS: Patients with invasive Streptococcus intermedius infections had a significantly longer hospital stay than patients infected with S. anginosus (p = 0.024) and a significantly higher 30-day all-cause mortality than patients infected with Streptococcus constellatus (p = 0.049). CONCLUSION: Identification of SAG bacteria to the taxonomic level of species may be of prognostic importance.

Fatal Streptococcus anginosus-associated pneumonia in a captive Sumatran orangutan (Pongo abelii).

BACKGROUND: Bacterial infections commonly affect the lungs and air sacs of orangutans; culture and identification is rarely performed and may have clinical relevance. METHODS: Necropsy, histopathology and bacterial culture were performend on a captive adult male Sumatran orangutan with chronic air sacculitis. Bacterial speciation was confirmed by sequencing of the 16s-23s ribosomal DNA spacer region. RESULTS: Necropsy revealed severe suppurative pneumonia. Moderate growth of Streptoccocus anginosus was recovered from the lungs. CONCLUSIONS: This is the first report of S. anginosus as a cause of fatal suppurative pneumonia in a non-human primate.

Phylogenetic relationship and virulence inference of Streptococcus Anginosus Group: curated annotation and whole-genome comparative analysis support distinct species designation.

BACKGROUND: The Streptococcus Anginosus Group (SAG) represents three closely related species of the viridans group streptococci recognized as commensal bacteria of the oral, gastrointestinal and urogenital tracts. The SAG also cause severe invasive infections, and are pathogens during cystic fibrosis (CF) pulmonary exacerbation. Little genomic information or description of virulence mechanisms is currently available for SAG. We conducted intra and inter species whole-genome comparative analyses with 59 publically available Streptococcus genomes and seven in-house closed high quality finished SAG genomes; S. constellatus (3), S. intermedius (2), and S. anginosus (2). For each SAG species, we sequenced at least one numerically dominant strain from CF airways recovered during acute exacerbation and an invasive, non-lung isolate. We also evaluated microevolution that occurred within two isolates that were cultured from one individual one year apart. RESULTS: The SAG genomes were most closely related to S. gordonii and S. sanguinis, based on shared orthologs and harbor a similar number of proteins within each COG category as other Streptococcus species. Numerous characterized streptococcus virulence factor homologs were identified within the SAG genomes including; adherence, invasion, spreading factors, LPxTG cell wall proteins, and two component histidine kinases known to be involved in virulence gene regulation. Mobile elements, primarily integrative conjugative elements and bacteriophage, account for greater than 10% of the SAG genomes. S. anginosus was the most variable species sequenced in this study, yielding both the smallest and the largest SAG genomes containing multiple genomic rearrangements, insertions and deletions. In contrast, within the S. constellatus and S. intermedius species, there was extensive continuous synteny, with only slight differences in genome size between strains. Within S. constellatus we were able to determine important SNPs and changes in VNTR numbers that occurred over the course of one year. CONCLUSIONS: The comparative genomic analysis of the SAG clarifies the phylogenetics of these bacteria and supports the distinct species classification. Numerous potential virulence determinants were identified and provide a foundation for further studies into SAG pathogenesis. Furthermore, the data may be used to enable the development of rapid diagnostic assays and therapeutics for these pathogens.

Taxonomy of the Anginosus group of the genus Streptococcus and description of Streptococcus anginosus subsp. whileyi subsp. nov. and Streptococcus constellatus subsp. viborgensis subsp. nov.

The Anginosus group of the genus Streptococcus has been the subject of much taxonomic confusion, which has hampered the full appreciation of its clinical significance. The purpose of this study was to critically re-examine the taxonomy of the Anginosus group, with special attention to ß-haemolytic, Lancefield group C strains, using multilocus sequence analysis (MLSA) combined with 16S rRNA gene sequence and phenotypic analyses. Phylogenetic analysis of concatenated sequences of seven housekeeping genes previously used for examination of viridans streptococci distinguished seven distinct and coherent clusters in the Anginosus group. Analyses of 16S rRNA gene sequences and phenotypic characters supported the MLSA clustering and currently recognized taxa of the Anginosus group. Single gene analyses showed considerable allele sharing between species, thereby invalidating identification based on single-locus sequencing. Two novel clusters of ß-haemolytic, Lancefield group C strains within the Streptococcus constellatus and Streptococcus anginosus species and isolated from patients with sore throat showed sufficient phylogenetic distances from other clusters to warrant status as novel subspecies. The novel cluster within S. anginosus was identified as the previously recognized DNA homology cluster, DNA group 2. The names S. anginosus subsp. whileyi subsp. nov. (type strain CCUG 39159(T) = DSM 25818(T) = SK1267(T)) and S. constellatus subsp. viborgensis subsp. nov. (type strain SK1359(T) = CCUG 62387(T) = DSM 25819(T)) are proposed.

Milleri group streptococcus--a stepchild in the viridans family.

The purpose of this investigation was to provide a comprehensive review of the pathogenic role and spectrum of disease of milleri group streptococci, with special attention to bloodstream invasion and to possible differential roles among the three species. All consecutive isolates of milleri group streptococci from any anatomic source, during a 37-month period, in a tertiary care teaching hospital in Tel-Aviv, Israel, were thoroughly investigated. Identification to the species level was performed by an automated system.Streptococcus anginosus constituted 82% of the 245 patient-unique isolates from hospitalized patients. All nonurinary isolates were involved in pyogenic infections mostly originating from the gastrointestinal tract, with bacteremia in 28 cases. The 71 urinary isolates represented either urinary tract infection or nonsignificant bacteriuria. No specific association could be detected between species and the infection site, except for a higher relative representation of Streptococcus constellatus in bacteremia. Milleri group streptococci are common in clinical practice and play a different pathogenic role to other viridans streptococci. Due to their invariable association with pyogenic processes, their presence in blood warrants immediate focus identification. In addition, they have a previously unappreciated clinical niche concerning urinary tract infection. The identification of viridans streptococci to the species level is of paramount clinical significance.

Amplification of minute amounts of oral bacterial DNA for real-time quantitative PCR analysis.

BACKGROUND: High-throughput technologies for typing caries or health-associated bacterial populations including PCR, DNA microarrays and next-generation sequencing techniques require significant amounts of bacterial DNA. In clinical settings, the amount of sampled DNA is often limited and amplification is therefore essential. Protocols should be able to reproducibly amplify sequences in order to maintain initial sequence ratios and should not bias the representation of particular DNA sequence types. METHODS: A linear amplification protocol using DNA polymerase I was modified to permit the amplification and subsequent analysis of small amounts of bacterial DNA. The protocol was tested on human oral bacterial biofilms from different sources, including carious dentine and plaque, and compared to amplification by degenerate PCR of 16S rDNA sequences. Real-time quantitative PCR of 24 bacterial species was used as a readout system to test amplified DNA against unamplified DNA. RESULTS: The amplification protocol reliably yielded 5-10 µg DNA from as little as 12.5 ng of template DNA. Correlation coefficients between real-time quantitative PCR results from amplified and unamplified DNA were between 0.78 and 0.98. CONCLUSION: The optimized protocol consistently produced amplification products from minute amounts of bacterial DNA from caries and plaque; the amplification products are suitable for downstream genetic analyses.

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 beta-galactosidase (ONPG) and beta-N-acetyl-glucosaminidase reactions. Rapid ID 32 Strep beta-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.

Prevalence of erythromycin and clindamycin resistance among clinical isolates of the Streptococcus anginosus group in Germany.

Members of the Streptococcus anginosus group (SAG) are frequently involved in pyogenic infections in humans. In the present study, the antimicrobial susceptibility of 141 clinical SAG isolates to six antimicrobial agents was analysed by agar dilution. All isolates were susceptible to penicillin, cefotaxime and vancomycin. However, 12.8 % displayed increased MIC values (0.12 mg l(-1)) for penicillin. Resistance to erythromycin was detected in eight (5.7 %) isolates. Characterization of the erythromycin-resistant isolates with the double-disc diffusion test revealed Macrolide-Lincosamide-Streptogramin(B) and M-type resistance in six and two isolates, respectively. The erythromycin-resistant isolates were further characterized by PCR for the resistance genes ermA, ermB and mefA. Resistance and intermediate resistance to ciprofloxacin were detected in two and six isolates, respectively. Molecular typing by PFGE revealed a high genetic heterogeneity among the SAG isolates and no evidence for a clonal relationship between the erythromycin-resistant isolates. Our data show that resistance to erythromycin, clindamycin and ciprofloxacin has emerged among SAG isolates in Germany. The implications of these findings for susceptibility testing and antimicrobial therapy of SAG infections are discussed.

Evaluation of the microbiota of primary endodontic infections using checkerboard DNA-DNA hybridization.

BACKGROUND/AIMS: The aim of this study was to evaluate the composition of the microbiota of primary endodontic infections in 111 selected cases of single-rooted teeth with necrotic pulp. METHODS: Samples were collected from the root canals using #15 Hedströen-type files and two sterile paper points, which were introduced 1 mm short of the apical foramen. The presence, levels, and proportions of 40 different bacterial species in each sample were determined using DNA probes and checkerboard DNA-DNA hybridization techniques. RESULTS: The mean number of species per sample was 22. Enterococcus faecalis (89.3%), Campylobacter gracilis (89.3%), Leptotrichia buccalis (89.3%), Neisseria mucosa (87.5%), Prevotella melaninogenica (86.6%), Fusobacterium nucleatum ssp. vincentii (85.7%), Eubacterium saburreum (75.9%), Streptococcus anginosus (75%), and Veillonella parvula (74.1%) were the most prevalent species. The species found in highest mean counts (over 10(5)) were F. nucleatum ssp. vincentii (13.14 x 10(5)), E. saburreum (5.67 x 10(5)), E. faecalis (5.38 x 10(5)), N. mucosa (4.19 x 10(5)), V. parvula (3.63 x 10(5)), C. gracilis (3.46 x 10(5)), Treponema socranskii (3.34 x 10(5)), Porphyromonas endodontalis (2.96 x 10(5)), Porphyromonas gingivalis (2.85 x 10(5)), Micromonas micros (2.81 x 10(5)), Prevotella nigrescens (2.68 x 10(5)) and Fusobacterium nucleatum ssp. nucleatum (2.64 x 10(5)). Most of these species were also found in high proportions. CONCLUSIONS: Our results suggest that several bacterial species considered to be oral pathogens seem to be implicated in the etiology of primary endodontic infections.

Bacterial examination of endodontic infections by clonal analysis in concert with denaturing high-performance liquid chromatography.

BACKGROUND/AIMS: The aim of this study was to examine the diversity of bacterial species in the infected root canals of teeth associated with endodontic abscesses by cloning and sequencing techniques in concert with denaturing high-performance liquid chromatography. METHODS: Samples collected from five infected root canals were subjected to polymerase chain reaction (PCR) with universal 16S ribosomal DNA primers. Products of these PCRs were cloned and sequenced. Denaturing high-performance liquid chromatography (DHPLC) was used as a screening method to reduce the number of clones necessary for DNA sequencing. RESULTS: All samples were positive for the presence of bacteria and a range of 7-13 different bacteria were found per root canal sample. In total, 48 different oral clones were detected among the five root canal samples. Olsenella profusa was the only species present in all samples. Porphyromonas gingivalis, Dialister pneumosintes, Dialister invisus, Lachnospiraceae oral clone, Staphylococcus aureus, Pseudoramibacter alactolyticus, Peptostreptococcus micros and Enterococcus faecalis were found in two of the five samples. The majority of the taxa were present in only one sample, for example Tannerella forsythia, Shuttleworthia satelles and Filifactor alocis. Some facultative anaerobes that are frequently isolated from endodontic infections such as E. faecalis, Streptococcus anginosus and Lactobacillus spp. were also found in this study. CONCLUSION: Clonal analysis of the microflora associated with endodontic infections revealed a wide diversity of oral species.

Rapid differentiation between members of the anginosus group and Streptococcus dysgalactiae subsp. equisimilis within beta-hemolytic group C and G streptococci by PCR.

Based on a pair of primers developed initially for differentiating the anginosus group from other viridans streptococci, the PCR reported here can also differentiate between members of the anginosus group and Streptococcus dysgalactiae subsp. equisimilis among beta-hemolytic group C and G streptococci. The resulting 742-bp PCR product was specific for members of the anginosus group, although a smaller, nonspecific product (361 bp) was generated from S. dysgalactiae subsp. equisimilis. Restriction digestion of the amplicon with XbaI and BsmI further differentiated Streptococcus anginosus from Streptococcus constellatus within the anginosus group.