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).

Streptococcus constellatus as an aetiological factor of extensive neck phlegmon complicated by sepsis - case study.

Introduction. Streptococcus constellatus are opportunistic microorganisms. When immunocompromised patients with concomitant systemic diseases are infected with S.constellatus, the bacteria may cause sepsis. Case study. A patient was admitted to hospital due to septic shock and multi-organ dysfunction in the course of neck phlegmon. The microbiological system identified S. constellatus in the patient who worked as a dog groomer. These facts confirmed that this aetiological factor may have caused such a serious infection because S. constellatus is a bacterial species found in dogs. It is most likely that the bacteria colonised the patient. Zoonotic transmission of microorganisms is particularly important for the development of infections in dogs and humans. Knowledge about how to treat deep cervical infections is necessary in the daily practice of a maxillofacial surgeon. The right antibiotic can applied only when the strain causing the infection has been identified.

Development and pyrosequencing analysis of an in-vitro oral biofilm model.

BACKGROUND: Dental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biofilm device (CBD) seeded with a natural saliva inoculum and analysed by next generation sequencing. The specific objectives were to determine the reproducibility and stability of the model by comparing the composition of the biofilms over time derived from (i) the same volunteers at different time points, and (ii) different panels of volunteers. RESULTS: Pyrosequencing yielded 280,093 sequences with a mean length of 432 bases after filtering. A mean of 320 and 250 OTUs were detected in pooled saliva and biofilm samples, respectively. Principal coordinates analysis (PCoA) plots based on community membership and structure showed that replicate biofilm samples were highly similar and clustered together. In addition, there were no significant differences between biofilms derived from the same panel at different times using analysis of molecular variance (AMOVA). There were significant differences between biofilms from different panels (AMOVA, P < 0.002). PCoA revealed that there was a shift in biofilm composition between seven and 14 days (AMOVA, P < 0.001). Veillonella parvula, Veillonella atypica/dispar/parvula and Peptostreptococcus stomatis were the predominant OTUs detected in seven-day biofilms, whilst Prevotella oralis, V. parvula and Streptococcus constellatus were predominant in 14-day biofilms. CONCLUSIONS: Diverse oral biofilms were successfully grown and maintained using the CBD. Biofilms derived from the same panel of volunteers were highly reproducible. This model could be used to screen both antimicrobial-containing oral care products and also novel approaches aiming to modify plaque composition, such as pre- or probiotics.

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.

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.

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.

Phototargeting oral black-pigmented bacteria.

We have found that broadband light (380 to 520 nm) rapidly and selectively kills oral black-pigmented bacteria (BPB) in pure cultures and in dental plaque samples obtained from human subjects with chronic periodontitis. We hypothesize that this killing effect is a result of light excitation of their endogenous porphyrins. Cultures of Prevotella intermedia and P. nigrescens were killed by 4.2 J/cm2, whereas P. melaninogenica required 21 J/cm2. Exposure to light with a fluence of 42 J/cm2 produced 99% killing of P. gingivalis. High-performance liquid chromatography demonstrated the presence of various amounts of different porphyrin molecules in BPB. The amounts of endogenous porphyrin in BPB were 267 (P. intermedia), 47 (P. nigrescens), 41 (P. melaninogenica), and 2.2 (P. gingivalis) ng/mg. Analysis of bacteria in dental plaque samples by DNA-DNA hybridization for 40 taxa before and after phototherapy showed that the growth of the four BPB was decreased by 2 and 3 times after irradiation at energy fluences of 4.2 and 21 J/cm2, respectively, whereas the growth of the remaining 36 microorganisms was decreased by 1.5 times at both energy fluences. The present study suggests that intraoral light exposure may be used to control BPB growth and possibly benefit patients with periodontal disease.

Mycotic Aneurysm Caused by Streptococcus constellatus subsp. constellatus.

An infected mycotic aneurysm due to Streptococcus constellatus subsp. constellatus has not previously been reported. We report on this condition in an 87-year-old woman who had aggravating abdominal pain and a large fusiform aneurysm over the thoracic-abdominal aorta with mural thrombus. Isolates from two sets of blood cultures and the debrided tissue were identified as S. constellatus subsp. constellatus by their biochemical reaction profiles, compatible 16S rRNA gene sequencing results, and sequencing results for the partial groESL gene and the 16S-23S intergenic spacer region.

Identification of the anginosus group within the genus Streptococcus using polymerase chain reaction.

The aim of this study was to establish an identification method for the anginosus group within the genus Streptococcus by polymerase chain reaction (PCR). Using a primer pair based on the group-specific sequences of penicillin-binding protein 2B (pbp2b) gene, a 275-bp fragment was amplified from each species in the group but no size-matched products were obtained in other streptococci. Further identification in the species or subspecies level was possible by a multiplex PCR with primers for the 16S ribosomal RNA gene of Streptococcus anginosus, the hyaluronate lyase genes both of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus, and the intermedilysin (ily) gene of S. intermedius. In the case ofStreptococcus constellatus subsp. pharyngis, the amplified fragment from the S. intermedius-type hyaluronate lyase gene was obtained, while that from the ily gene was not. These results also indicate that two different hyaluronate lyase genes are distributed among the anginosus group.