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)

Identification of Streptococcus pseudopneumoniae and other mitis group streptococci using matrix assisted laser desorption/ionization - time of flight mass spectrometry.

This study evaluated the ability of the MALDI-ToF MS from Bruker Daltonics to identify clinical Mitis-Group-Streptococcus isolates with a focus on Streptococcus pseudopneumoniae. The results were analyzed using the standard log(score) and the previously published list(score). Importantly, using the log(score) no misidentifications occurred and 27 of 29 (93%) S. pneumoniae and 27 of 30 (90%) S. oralis strains were identified, but only 1 of 31 (3%) S. pseudopneumoniae and 1 of 13 (8%) S. mitis strains were identified. However, our results show that 30 of 31 S. pseudopneumoniae strains had a S. pseudopneumoniae Main Spectral Profiles within the 3 best matches. Using the list(score) all S. oralis and S. pneumoniae strains were identified correctly, but list(score) misidentified 10 S. pseudopneumoniae and 5 S. mitis. We propose to use the log(score) for identification of S. pneumoniae, S. pseudopneumoniae, S. mitis and S. oralis, but for some strains additional testing may be needed.

Antibiotic Resistance of Viridans Group Streptococci Isolated from Dental Plaques.

Viridans group streptococci (VGS) are a common cause of infective endocarditis, and dental plaque is the major source of these bacteria. The present study examined the antibiotic resistance of 635 VGS isolates obtained from dental plaques. Isolates from supragingival plaques were identified using the rapid ID 32 Strep and mini API reader (bioMérieux, France), and minimal inhibitory concentrations (MICs) were determined by a broth microdilution method. High rates of resistance to ampicillin and tetracycline were detected among the isolates. The most resistant species were Streptococcus sanguinis and Streptococcus salivarius. Among the 635 isolates, 9.1% were resistant to erythromycin, and 20.6% to tetracycline. All isolates were sensitive to vancomycin. Resistance to amoxicillin was observed in 0.2% of all isolates. In this study, we showed the incidence of antimicrobial resistance and the susceptibility patterns among 635 VGS isolates from dental plaque.

Strain-level analysis of gut-resident pro-inflammatory viridans group Streptococci suppressed by long-term cotrimoxazole prophylaxis among HIV-positive children in Zimbabwe.

Antimicrobials have become a mainstay of healthcare in the past century due to their activity against pathogens. More recently, it has become clear that they can also affect health via their impact on the microbiota and inflammation. This may explain some of their clinical benefits despite global increases in antimicrobial resistance (AMR) and reduced antimicrobial effectiveness. We showed in a randomized controlled trial of stopping versus continuing cotrimoxazole prophylaxis among HIV-positive Zimbabwean children taking antiretroviral therapy (ART), that continuation of cotrimoxazole persistently suppressed gut-resident viridans group streptococcal species (VGS) that were associated with intestinal inflammation. In this addendum, we provide a broader overview of how antibiotics can shape the microbiota and use high read-depth whole metagenome sequencing data from our published study to investigate whether (i) the impact of cotrimoxazole on gut VGS and (ii) VGS associated inflammation, is attributable to strain-level variability. We focus on S. salivarius, the VGS species that was most prevalent in the cohort and for which there was sufficient genome coverage to differentiate strains. We demonstrate that suppression of S. salivarius by cotrimoxazole is not strain specific, nor did stool concentration of the pro-inflammatory mediator myeloperoxidase vary by S. salivarius strain. We also show that gut-resident S. salivarius strains present in this study population are distinct from common oral strains. This is the first analysis of how cotrimoxazole prophylaxis used according to international treatment guidelines for children living with HIV influences the gut microbiome at the strain-level. We also provide a detailed review of the literature on the mechanisms by which suppression of VGS may act synergistically with cotrimoxazole's anti-inflammatory effects to reduce gut inflammation. A greater understanding of the sub-clinical effects of antibiotics offers new insights into their responsible clinical use.

Frequent colonization of betahaemolytic streptococci at various body sites including the perineum and anal canal during erysipelas and cellulitis.

Background: Erysipelas and cellulitis are usually caused by betahaemolytic streptococci but the aetiology is often difficult to verify in clinical practice. Methods: Patients with erysipelas or cellulitis were analysed for betahaemolytic streptococci in samples from multiple body sites, including the perineum and the anal canal, during the acute episode and at follow up. Healthy control persons were sampled from the same sites. Results: Betahaemolytic streptococci group A, C or G were identified in 23/28 (82%) patients, most commonly group G. A wound or ulcer, present in 16/28 (57%), was colonized in 8/16 (50%). The perineum and anal canal were colonized in 11/28 (39%) and 10/28 (36%), respectively. At follow-up after about 4 weeks, only 4/28 (14%) were colonized (p<.001). In 39 healthy control persons, no betahaemolytic streptococci group A were found, groups C or G were found in 4/39 (10%). Group B streptococci were more often identified in controls, than in patients,12/39 (31%). Conclusions: Acute episodes of erysipelas or cellulitis are associated with colonization of betahaemolytic streptococci at multiple sites including the perineum and anal canal, in particular serogroup G. This may be important for choice of primary antibiotic therapy and possibilities for prevention of relapses.

Epidemiological and clinical characteristics of Streptococcus tigurinus endocarditis.

BACKGROUND: Streptococcus tigurinus was recently described as a new streptococcal species within the viridans group streptococci (VGS). The objectives of the present work were to analyse the clinical and microbiological characteristics of S. tigurinus isolated from patients with bacteraemias, to determine the prevalence of S. tigurinus among VGS endocarditis in Spain, and to compare the clinical characteristics and outcomes of endocarditis caused by S. tigurinus and other VGS. METHODS: Retrospective nationwide study, performed between 2008 and 2016 in 9 Spanish hospitals from 7 different provinces comprising 237 cases of infective endocarditis. Streptococcal isolates were identified by sequencing fragments of their 16S rRNA, sodA and groEL genes. Clinical data of patients with streptococcal endocarditis were prospectively collected according to a pre-established protocol. RESULTS: Patients with endocarditis represented 7/9 (77.8%) and 26/86 (30.2%) of the bacteraemias caused by S. tigurinus and other VGS, respectively (p < 0.001), in two of the hospital participants. Among patients with streptococcal endocarditis, 12 different Streptococcus species were recognized being S. oralis, S. tigurinus and S. mitis the three more common. No relevant statistical differences were observed in the clinical characteristics and outcomes of endocarditis caused by the different VGS species. CONCLUSIONS: In this multicenter study performed in Spain, S. tigurinus showed a higher predilection for the endocardial endothelium as compared to other VGS. However, clinical characteristics and outcomes of endocarditis caused by S. tigurinus did not significantly differ from endocarditis caused by other oral streptococci.

Viridans group streptococci bloodstream infections in neutropenic adult patients with hematologic malignancy: Single center experience.

Viridans group streptococci bloodstream infections (VGS BSI) remain a significant cause of mortality and morbidity in patients with severe neutropenia. The goal of our study was to evaluate clinical course and microbiological susceptibility of VGS BSI at our center. Retrospective analysis of all microbiologically documented bloodstream infections caused by VGS during the 9-year time period (from January 2006 until December 2014) was carried out. Only patients with severe neutropenia (< 500/µL) were included in the study. Clinical outcome and microbiological susceptibility pattern of isolates were recorded. Fifty-one individual patients with episode of VGS BSI were identified. The most frequent agent was Streptococcus mitis (23/51 cases, 45.1%). 88.2% (45/51) of patients were on recommended ciprofloxacin prophylaxis. 20/51 (39.2%) of patients suffered from mucositis at the time of diagnosis (10 patients had oral mucositis, 2 patients had bowel mucositis, and 8 patients both). Twenty-six patients (51.0%) had clinically relevant lung damage caused by VGS BSI (i.e., acute lung injury or acute respiratory distress syndrome). Twenty-four (47.0%) patients presented with bilateral lung infiltrated upon chest imaging, and two (4.0%) patients had unilateral lung infiltrates. Three patients (5.9%) died due to VGS BSI until day 28 of observation. No difference in signs of shock syndrome was observed in the patients during transplantation procedures compared to patients without transplantation as well as in a group received previous high-dose chemotherapy with cytosinarabinoside or in patients with mucositis. Only 3/51 of isolates (5.9%) were resistant to penicillin. All isolates were susceptible to empirical treatment. While the penicillin resistance of VGS remains low in middle Europe, initial antibiotic therapy of febrile neutropenia are still effective in most cases. The mortality and complication rates of VGS BSI were comparable to other studies, and no specific risk factor of shock presence could be identified.

Molecular Characterization of Group B Streptococcus Serotypes By Multiplex Polymerase Chain Reaction / Caracterização molecular dos sorotipos de estreptococos do grupo b por reação multiplex pcr

OBJECTIVE: Group B Streptococcus (GBS) serotypes (Ia, Ib and II to IX) are classified based on variations in their capsular polysaccharide; their prevalence differs between different geographic areas. We examined the prevalence of all GBS serotypes in rectal and vaginal swab samples obtained from 363 pregnant women followed at a Brazilian referral center (Hospital da Mulher Professor Doutor José Aristodemo Pinotti); bacterial susceptibility to antibiotics was further determined. METHOD: Prevalence of positive GBS was evaluated by latex agglutination and by multiplex PCR analysis; bacterial susceptibility to antibiotics, such as clindamycin, erythromycin, levofloxacin, linezolid, penicillin and tetracycline was determined by the disk diffusion method. RESULTS: (a) standard GBS culture and the multiplex PCR analysis tested positive for 83 swabs, collected from 72 women (prevalence of GBS colonization: 72/363; 20%); the most prevalent Serotype was Ia (n=43/83; 52%), followed by serotype V (n=14/83; 17%); according to anatomical origin, serotype Ia accounted for 27/59 (46%) and 16/24 (67%) of the vaginal and rectal samples, respectively; PCR also identified serotypes Ib, II, III and VI. Serotype VI is rarely described and had not been previously reported in Brazil or in Latin America. (b) The latex agglutination test only identified 44 positive samples, all of which were serotyped: 34 of these samples (77%) had serotypes matching those identified by multiplex PCR. (c) Only one sample (serotype Ia) showed resistance to erythromycin and clindamycin. CONCLUSION: Regional studies on GBS serotypes prevalence are essential to guide immunoprophylactic interventions (vaccines) and the implementation of adequate antibiotic prophylaxis or treatment. In this study, the incidence of the serotype VI, a new and rare serotype of GBS was described for the first time in a Brazilian population.

OBJETIVO: Os sorotipos (Ia, Ib e II ao IX) do estreptococo do grupo B (GBS) são classificados baseado nas variações em seus polissacarídeos capsulares; sua prevalência difere entre diferentes áreas geográficas. Nós examinamos a prevalência de todos os sorotipos do estreptococo do grupo B em amostras de swabs vaginal e retal obtidas de 363 mulheres seguidas em um centro de referência brasileiro, o Hospital da Mulher Professor Doutor José Aristodemo Pinotti; a susceptibilidade bacteriana a antibióticos foi também determinada. MÉTODO A prevalência de estreptococo do grupo B positivo foi avaliada por aglutinação em látex e através de análise por multiplex PCR; susceptibilidade bacteriana a antibióticos, tais como clindamicina, eritromicina, levofloxacin, linezolide, penicilina e tetraciclina foi determinada pelo método de disco difusão. RESULTADOS: (a) Tanto a cultura padrão para estreptococo do grupo B quanto a análise por multiplex PCR testaram positivos para 83 swabs. A prevalência para colonização por GBS foi 20%. O sorotipo Ia foi o mais prevalente (n= 43/83; 52%), seguido pelo sorotipo V (n= 14/83; 17%); De acordo com a origem anatômica, o sorotipo Ia positivou 27/59 (46%) e 16/24 (67%) das amostras vaginais e retais, respectivamente; o teste de PCR também identificou os sorotipos Ib, II, III, VI. O sorotipo VI é raramente descrito e não reportado no Brasil ou na América Latina até esta data. (b) O teste de aglutinação em látex somente identificou 44 amostras positivas, todas das quais foram sorotipadas: 34 destas amostras (77%) tiveram os sorotipos coincidindo com aqueles identificados pela multiplex PCR. (c) Somente uma amostra (sorotipo Ia) mostrou resistência a eritromicina e clindamicina. CONCLUSÃO: Estudos regionais sobre a prevalência dos sorotipos do estreptococo do grupo B são essenciais para guiar medidas imunoprofiláticas (vacinas) e a implementação de adequada antibiótico profilaxia. Neste estudo, a incidência do sorotipo VI foi descrita pela primeira vez na população Brasileira, um novo e raro sorotipo do estreptococo do grupo B.

[Comparison of MALDI-TOF and 16S rRNA methods in identification of viridans group streptococci]. / Viridans grup streptokok tanimlamasinda MALDI-TOF ve 16S rRNA yöntemlerinin karsilastirilmasi.

Accurate identification of viridans group streptococci (VGS) frequently encountered as a causative agent of infective endocarditis is always a challenge for the clinical microbiology laboratory. Clinical microbiology laboratories generally use semi automatic/full automatic systems, molecular methods and also conventional methods for the identification of these bacteria. There are recent published studies that have used MALDI-TOF (Matrix Assisted Laser Ionization Mass Spectrometry-Time of Flight) systems in the identification of VGS. The aim of the study was to compare the performance of the conventional methods, semi automatic and MALDI-TOF MS system used in identification of VGS in oral microbiota of persons under the risk of infective endocarditis, with the gold standard method 16S rRNA sequence analysis and to create a diagnosis algorithm for the identification of VGS in clinical microbiology laboratories according to the obtained data.The study was conducted with 51 VGS strains isolated from oral microbiota of the patients with rheumatologic cardiac, valve and/or prosthetic valve diseases, under the risk of development of infective endocarditis, who have admitted to Ankara Numune Training and Research Hospital, Department of Cardiology, between February-June 2015. Standard microbiology procedures, optochin susceptibility and bile solubility tests were done for the isolation of bacteria. Bacteria were also identified with APISTREP (bioMérieux, France) and MALDI-TOF MS Bruker Microflex (Bruker Biotyper; Bruker Daltonics, Bremen, Germany) methods. BSF-8 (5´-AGAGTTTGATCCTGGCTCAG-3´) and BSR-534(5´-ATTACCGCGGCTGCTGGC-3´) primers were used in the 16S rRNA sequence analysis of bacteria. ABI PRISM 3100 Avan t Genetic Analyzer (Applied Biossytems, Foster City, CA, USA) were used for the sequence analysis. Electropherograms were analyzed in SeqScape Software (Applied Biosystems, Foster City, CA, USA) and compared with the reference sequences in GenBank with BLASTN (NCBI). According to the result of optochin and bile solubility tests, with API STREP system, 16 (31,37%) of the isolates were identified as Mitis group, 15 (29.41%) as Anginosus group, 9 (17.5%) as Salivarius group, 7 (13,73%) as Sanguinis group and 4 (7.84%) as Bovis group among optochin and bile resistant alpha hemolytic streptococci. Moreover, of the same isolates 20 (39.22%) were identified as Mitis group, 14 (27.45%) as Anginosus group, 13 (25.49%) as Salivarius group and 4 (7.84%) as Sanguinis group with MALDI-TOF system. In the identification with 16S rRNA, 25 (49.02%) of the isolates were identified as Mitis group, 13 (25.49%) as Anginosus group, 12 (23.53%) as Salivarius group and 1 (1.96%) as Sanguinis group. According to the results, it was determined that 33 (64.70%) of the isolates identified in MALDI-TOF MS system and 31 (60.78%) of the isolates identified in API STREP system were compatible with 16S rRNA sequence analysis method. For Mitis group, API STREP test sensitivity was 48.00% and specificity was 84.62% and MALDI-TOF system sensitivity was 80.00% and specificity was 100%. As VGS identification is a complicated process, we believe a single method will be insufficient for the identification of these isolates in clinical microbiology laboratories. We suggest that MALDI-TOF system can be used for VGS diagnosis, however, optochin test and/or molecular methods should also be included in the diagnosis algorithm when necessary.

Insight into the Diversity of Penicillin-Binding Protein 2x Alleles and Mutations in Viridans Streptococci.

The identification of commensal streptococci species is an everlasting problem due to their ability to genetically transform. A new challenge in this respect is the recent description of Streptococcus pseudopneumoniae as a new species, which was distinguished from closely related pathogenic S. pneumoniae and commensal S. mitis by a variety of physiological and molecular biological tests. Forty-one atypical S. pneumoniae isolates have been collected at the German National Reference Center for Streptococci (GNRCS). Multilocus sequence typing (MLST) confirmed 35 isolates as the species S. pseudopneumoniae A comparison with the pbp2x sequences from 120 commensal streptococci isolated from different continents revealed that pbp2x is distinct among penicillin-susceptible S. pseudopneumoniae isolates. Four penicillin-binding protein x (PBPx) alleles of penicillin-sensitive S. mitis account for most of the diverse sequence blocks in resistant S. pseudopneumoniae, S. pneumoniae, and S. mitis, and S. infantis and S. oralis sequences were found in S. pneumoniae from Japan. PBP2x genes of the family of mosaic genes related to pbp2x in the S. pneumoniae clone Spain23F-1 were observed in S. oralis and S. infantis as well, confirming its global distribution. Thirty-eight sites were altered within the PBP2x transpeptidase domains of penicillin-resistant strains, excluding another 37 sites present in the reference genes of sensitive strains. Specific mutational patterns were detected depending on the parental sequence blocks, in agreement with distinct mutational pathways during the development of beta-lactam resistance. The majority of the mutations clustered around the active site, whereas others are likely to affect stability or interactions with the C-terminal domain or partner proteins.

Understanding the viridians group streptococci: Are we there yet?

The viridans group streptococci are a heterogeneous group of organisms which exist as commensals in the oropharynx and the gut. They cause serious infections when they gain entry into sterile sites particularly in patients with predisposing conditions. Classification and species differentiation of these organisms has always been a challenge because of phenotypic differences between strains of the same species. Facklam's typing scheme based on six metabolic properties has been the most widely used and many commercial identification systems are based on it. Due to the ambiguity in species differentiation based on phenotypic tests, nucleic acid-based methods have been developed to improve the identification of these organisms. Results using genotypic methods such as 16S rRNA and sodA gene sequencing have been promising. Multilocus sequence analysis of seven house-keeping genes map, pfl, pyk, ppaC, rpoB, soda and tuf amplified by polymerase chain reaction was found to be an accurate alternative to other methods and could be useful in the characterisation of larger collections of isolates.

Rare and unusual isolates of viridans streptococci from the human oral cavity.

CONTEXT: The genus Streptococcus consists of more than 65 species. The taxonomic classification of these members is not well-defined. Among the viridans group streptococci (VGS), there are major taxonomic changes by the addition of many new species; whereas, most of the new strains are of animal origin and only a few have been reported to be isolated from humans. Rare and unusual species of VGS such as Streptococcus thoraltensis, S. pluranimalium and S. hyointestinalis are normally associated with different animals. Their isolation from human being is not yet reported. AIM: To find out the rare and unusual species of viridans group streptococci from human oral cavity. SETTINGS AND DESIGN: A case-controlled study carried out at hospital-based dental services in a tertiary care hospital. MATERIALS AND METHODS: Subgingival plaque samples of the tooth were collected from 80 patients (34 with periodontitis and 46 without periodontitis) undergoing tooth extraction. Cultures were subcultured onto special media such as Tryptone Soya blood Agar supplemented with strepto supplement and Mutans-Sanguis Agar. Identification of strains and antimicrobial susceptibilities were measured as minimum inhibitory concentration using Vitek 2 (BioMérieux, Paris, France) automated system. RESULTS: We have identified three strains of VGS - S. thoraltensis, S. pluranimalium and S. hyointestinalis from subgingival plaque samples from patients with periodontitis. S. thoraltensis and S. pluranimalium were found to be resistant to most of the antibiotics. CONCLUSIONS: To the best of our knowledge, this is the first report of isolation of these rare and unusual strains from the human oral cavity.

Viridans Group Streptococci clinical isolates: MALDI-TOF mass spectrometry versus gene sequence-based identification.

Viridans Group Streptococci (VGS) species-level identification is fundamental for patients management. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been used for VGS identification but discrimination within the Mitis group resulted difficult. In this study, VGS identifications with two MALDI-TOF instruments, the Biotyper (Bruker) and the VITEK MS (bioMérieux) have been compared to those derived from tuf, soda and rpoB genes sequencing. VGS isolates were clustered and a dendrogram constructed using the Biotyper 3.0 software (Bruker). RpoB gene sequencing resulted the most sensitive and specific molecular method for S. pneumonia identification and was used as reference method. The sensitivity and the specificity of the VITEK MS in S. pneumonia identification were 100%, while the Biotyper resulted less specific (92.4%). In non pneumococcal VGS strains, the group-level correlation between rpoB and the Biotyper was 100%, while the species-level correlation was 61% after database upgrading (than 37% before upgrading). The group-level correlation between rpoB and the VITEK MS was 100%, while the species-level correlation was 36% and increases at 69% if isolates identified as S. mitis/S. oralis are included. The less accurate performance of the VITEK MS in VGS identification within the Mitis group was due to the inability to discriminate between S. mitis and S. oralis. Conversely, the Biotyper, after the release of the upgraded database, was able to discriminate between the two species. In the dendrogram, VGS strains from the same group were grouped into the same cluster and had a good correspondence with the gene-based clustering reported by other authors, thus confirming the validity of the upgraded version of the database. Data from this study demonstrated that MALDI-TOF technique can represent a rapid and cost saving method for VGS identification even within the Mitis group but improvements of spectra database are still recommended.

GyrB polymorphisms accurately assign invasive viridans group streptococcal species.

Viridans group streptococci (VGS) are a heterogeneous group of medically important bacteria that cannot be accurately assigned to a particular species using conventional phenotypic methods. Although multilocus sequence analysis (MLSA) is considered the gold standard for VGS species-level identification, MLSA is not yet feasible in the clinical setting. Conversely, molecular methods, such as sodA and 16S rRNA gene sequencing, are clinically practical but not sufficiently accurate for VGS species-level identification. Here, we present data regarding the use of an ∼ 400-nucleotide internal fragment of the gene encoding DNA gyrase subunit B (GyrB) for VGS species-level identification. MLSA, internal gyrB, sodA, full-length, and 5' 16S gene sequences were used to characterize 102 unique VGS blood isolates collected from 2011 to 2012. When using the MLSA species assignment as a reference, full-length and 5' partial 16S gene and sodA sequence analyses failed to correctly assign all strains to a species. Precise species determination was particularly problematic for Streptococcus mitis and Streptococcus oralis isolates. However, the internal gyrB fragment allowed for accurate species designations for all 102 strains. We validated these findings using 54 VGS strains for which MLSA, 16S gene, sodA, and gyrB data are available at the NCBI, showing that gyrB is superior to 16S gene and sodA sequence analyses for VGS species identification. We also observed that specific polymorphisms in the 133-amino acid sequence of the internal GyrB fragment can be used to identify invasive VGS species. Thus, the GyrB amino acid sequence may offer a more practical and accurate method for classifying invasive VGS strains to the species level.

Evaluation of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for identification of viridans group streptococci.

In this study, the performances of two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, MALDI Biotyper (Bruker Daltonics) and VITEK MS (bioMérieux), were evaluated in the identification of viridans group streptococci. Two collections of isolates were tested with both methods. From a panel of type collection strains (n = 54), MALDI Biotyper gave correct species-level identification for 51/54 (94 %) strains and 37/54 (69 %) strains for the VITEK MS in vitro diagnostic (IVD) method. Additionally, a collection of blood cultures isolates which had been characterized earlier with partial sequencing of 16S rRNA (n = 97) was analyzed. MALDI Biotyper classified 89 % and VITEK MS 93 % of these correctly to the group level. Comparison of species-level identification from the blood culture collection was possible for 36 strains. MALDI Biotyper identified 75 % and VITEK MS 97 % of these strains consistently. Among the clinical isolates, MALDI Biotyper misidentified 36 strains as Streptococcus pneumoniae. Nevertheless, our results suggest that the current MALDI-TOF methods are a good alternative for the identification of viridans streptococci and do perform as well as or better than commercial phenotypical methods.

Population structure and characterization of viridans group streptococci (VGS) isolated from the upper respiratory tract of patients in the community.

A study was undertaken to examine the population structure of viridans group streptococci (VGS) isolated the upper respiratory tract of adult and paediatric patients within the community. VGS are common commensal bacterial inhabitants of the upper respiratory tract and valuable sentinel reporters of underlying antibiotic resistance (AR). Laboratory examination of the colonising VGS species may provide a valuable ecological description of the species isolated from the upper respiratory tract and their antibiotic susceptibility, including an estimation of the AR reservoir in this population. Freshly obtained nasal and oropharyngeal swabs from 84 patients were examined by selective conventional culture on Mitis-Salivarius agar and yielded 363 isolates of VGS. Sequence analyses of the rpnB and 16-23S rRNA ITS genes identified these isolates to belong to 10 species of VGS and included S. anginosus, S. australis, S. constellatus, S. infantis, S. mitis, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis and S. vestibularis. The most frequent VGS organisms isolated was S. salivarius (282/363; 78.0%), followed by S. sanguinis (23/363; 6.3%), S. parasanguinis (21/363; 5.8%), S. mitis (18/363; 5.0%), S. anginosus (5/363; 1.4%), S. vestibularis (5/363; 1.4%), S. australis (3/363; 0.8%), S. oralis (3/363; 0.8%), S. infantis (1/363; 0.3%) and S. constellatus (1/363; 0.3%). All patients examined carried at least one VGS organism, where there were 17 combination patterns of carriage of the 10 species of VGS species isolated, where 54.2%, 37.3%, 7.2% and 1.2% of patients harboured one, two, three and four different VGS species, respectively. Antibiotic susceptibility was determined by standard disk diffusion assay testing against four classes of antibiotics, including the b-lactams [cefotaxime, cefuroxime], the tetracyclines [doxycycline], the fluoroquinolones [levofloxacin] and the macrolides [erythromycin]. Overall, there was no resistance to levofloxacin and cefuroxime, with limited resistance to cefotaxime (3.3%) and doxycycline (9.8%). Antibiotic resistance was highest in erythromycin, where 40.9% of isolates were resistant. S. vestibularis was the most antibiotic resistance of all VGS species examined (S. vestibularis v S. salivarius p=0.011), followed by S. anginosis. S. salivarius was the most antibiotic susceptible VGS species examined. Overall, given their infrequency in causing infection, relatively few studies to date have attempted to examine their ecology in their preferred body niche, namely the upper respiratory tract. However, knowing their prevalence is becoming increasingly important in relation to their ability to exclude significant respiratory pathogens, including Streptococcus pneumoniae. In conclusion, these data indicate that VGS colonisation of the upper respiratory tract in individuals within the community is dominated mainly with relatively antibiotic susceptible S. salivarius.

Value of matrix-assisted laser desorption ionization-time of flight for routine identification of viridans group streptococci causing bloodstream infections.

Phenotypic tests do not always unequivocally identify some species of viridans group streptococci (VGS). sodA sequence analysis is the most accurate method for identification, although it requires specialized personnel and has not been applied systematically in clinical microbiology laboratory routines. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) is emerging as a rapid alternative for bacterial identification. This study assesses the ability of MALDI-TOF and the API 20 Strep system to identify VGS isolates recovered from blood cultures using sodA sequence analysis as the reference method. All clinically significant VGS isolates recovered from blood cultures between January 2007 and January 2010 were identified by sodA sequence analysis and API 20 Strep. The strains were then tested by MALDI-TOF. Agreement between API 20 Strep/MALDI-TOF and sodA sequence analysis was determined. We examined 124 clinical isolates. Sensitivities of API 20 strep and MALDI-TOF for the species level identification of VGS isolates were, respectively, as follows: 60.5% and 73.4%. Sensitivities of API 20 strep and MALDI-TOF for the group level identification were, respectively, as follows: 70% and 94.3%. The turnaround times to identify VGS isolates by sodA sequence analysis, API 20 Strep and MALDI-TOF were 12-24, 24-48 h and 15 min, respectively. API 20 Strep cannot accurately identify all isolates of VGS. MALDI-TOF appeared to be a rapid and reliable alternative for identification of VGS strains to group level, but was not able to discriminate closely related species of certain groups.

Synergistic interaction between Candida albicans and commensal oral streptococci in a novel in vitro mucosal model.

Candida albicans is a commensal colonizer of the gastrointestinal tract of humans, where it coexists with highly diverse bacterial communities. It is not clear whether this interaction limits or promotes the potential of C. albicans to become an opportunistic pathogen. Here we investigate the interaction between C. albicans and three species of streptococci from the viridans group, which are ubiquitous and abundant oral commensal bacteria. The ability of C. albicans to form biofilms with Streptococcus oralis, Streptococcus sanguinis, or Streptococcus gordonii was investigated using flow cell devices that allow abiotic biofilm formation under salivary flow. In addition, we designed a novel flow cell system that allows mucosal biofilm formation under conditions that mimic the environment in the oral and esophageal mucosae. It was observed that C. albicans and streptococci formed a synergistic partnership where C. albicans promoted the ability of streptococci to form biofilms on abiotic surfaces or on the surface of an oral mucosa analogue. The increased ability of streptococci to form biofilms in the presence of C. albicans could not be explained by a growth-stimulatory effect since the streptococci were unaffected in their growth in planktonic coculture with C. albicans. Conversely, the presence of streptococci increased the ability of C. albicans to invade organotypic models of the oral and esophageal mucosae under conditions of salivary flow. Moreover, characterization of mucosal invasion by the biofilm microorganisms suggested that the esophageal mucosa is more permissive to invasion than the oral mucosa. In summary, C. albicans and commensal oral streptococci display a synergistic interaction with implications for the pathogenic potential of C. albicans in the upper gastrointestinal tract.

Misidentification of alpha-hemolytic streptococci by routine tests in clinical practice.

Accurate species-level identification of viridans group streptococci (VGS) is very important for understanding of their pathogenicity and virulence. However, an extremely high level of the similarity between VGS, especially Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis and Streptococcus pseudopneumoniae, often results in misidentification of these organisms, so there is an urgent need of novel approaches to species identification. A set of 50 randomly selected clinical isolates of alpha-hemolytic streptococci from upper respiratory tract were characterized by the routine phenotypic methods (alpha-hemolysis, colony morphology, Gram stain and optochin susceptibility). Modern proteomic and genetic approaches - the direct bacterial profiling (DBP) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique and multilocus sequence analysis (MLSA) scheme (http://viridans.emlsa.net/) - were applied for the accurate species identification. After that all isolates were stored at -70°C. Later they were re-inoculated, and a number of additional tests (bile solubility, latex agglutination by commercial "Slidex® pneumo-kit" and repeated optochin test) were performed. A considerable discrepancy was discovered in the results of the different approaches. Looking in the future, one could say that MLSA-like schemes based on the analysis of the nucleotide sequences of seven or more loci of the bacterial genome, appeared to be the most useful instrument in the VGS discrimination, in contrast to the numerous one-target identification schemes, which have been introduced into practice by now.