Clinical isolates of Streptococcus mitis/oralis-related species with reduced carbapenem susceptibility, harboring amino acid substitutions in penicillin-binding proteins in Japan.

Streptococcus mitis/oralis group isolates with reduced carbapenem susceptibility have been reported, but its isolation rate in Japan is unknown. We collected 356 clinical α-hemolytic streptococcal isolates and identified 142 of them as S. mitis/oralis using partial sodA sequencing. The rate of meropenem non-susceptibility was 17.6% (25/142). All 25 carbapenem-non-susceptible isolates harbored amino acid substitutions in/near the conserved motifs in PBP1A, PBP2B, and PBP2X. Carbapenem non-susceptibility is common among S. mitis/oralis group isolates in Japan.

Performance assessment of the Bruker Biotyper MALDI-TOF MS for the identification of difficult-to-identify viridans group streptococci.

Differentiating Streptococcus pneumoniae among nonpneumococcal viridans group streptococci (VGS) is challenging in conventional laboratories. Therefore, we aimed to evaluate the performance of the latest Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system in identifying VGS by comparing the results to those of the specific gene sequencing approach. Clinical isolates were initially identified using the BD Phoenix system to identify Streptococcus species. The optochin test was used to distinguish nonpneumococcal VGS from S. pneumoniae. The species of individual reference strains and clinical isolates were determined by comparing the sequences of the 16S rDNA, gyrB, sodA, groESL, or coaE genes with those in the GenBank sequence databases. We evaluated the performance of the Bruker Biotyper MALDI-TOF MS in VGS identification using two different machines with three databases. We collected a total of 103 nonpneumococcal VGS and 29 S. pneumoniae blood isolates at a medical center in northern Taiwan. Among these isolates, only seven could not be identified at the species level by the specific gene sequencing approach. We found that none of the nonpneumococcal VGS isolates were misidentified as pneumococci by the latest Biotyper system, and vice versa. However, certain strains, especially those in the mitis and bovis groups, could still not be correctly identified. The latest Bruker Biotyper 4.1 (DB_10833) showed significant improvement in identifying VGS strains. However, a specific gene sequencing test is still needed to precisely differentiate the species of strains in the mitis and bovis groups.

Comparative Genomics of Streptococcus oralis Identifies Large Scale Homologous Recombination and a Genetic Variant Associated with Infection.

The viridans group streptococci (VGS) are a large consortium of commensal streptococci that colonize the human body. Many species within this group are opportunistic pathogens causing bacteremia and infective endocarditis (IE), yet little is known about why some strains cause invasive disease. Identification of virulence determinants is complicated by the difficulty of distinguishing between the closely related species of this group. Here, we analyzed genomic data from VGS that were isolated from blood cultures in patients with invasive infections and oral swabs of healthy volunteers and then determined the best-performing methods for species identification. Using whole-genome sequence data, we characterized the population structure of a diverse sample of Streptococcus oralis isolates and found evidence of frequent recombination. We used multiple genome-wide association study tools to identify candidate determinants of invasiveness. These tools gave consistent results, leading to the discovery of a single synonymous single nucleotide polymorphism (SNP) that was significantly associated with invasiveness. This SNP was within a previously undescribed gene that was conserved across the majority of VGS species. Using the growth in the presence of human serum and a simulated infective endocarditis vegetation model, we were unable to identify a phenotype for the enriched allele in laboratory assays, suggesting a phenotype may be specific to natural infection. These data highlighted the power of analyzing natural populations for gaining insight into pathogenicity, particularly for organisms with complex population structures like the VGS. IMPORTANCE The viridians group streptococci (VGS) are a large collection of closely related commensal streptococci, with many being opportunistic pathogens causing invasive diseases, such as bacteremia and infective endocarditis. Little is known about virulence determinants in these species, and there is a distinct lack of genomic information available for the VGS. In this study, we collected VGS isolates from invasive infections and healthy volunteers and performed whole-genome sequencing for a suite of downstream analyses. We focused on a diverse sample of Streptococcus oralis genomes and identified high rates of recombination in the population as well as a single genome variant highly enriched in invasive isolates. The variant lies within a previously uncharacterized gene, nrdM, which shared homology with the anaerobic ribonucleoside triphosphate reductase, nrdD, and was highly conserved among VGS. This work increased our knowledge of VGS genomics and indicated that differences in virulence potential among S. oralis isolates were, at least in part, genetically determined.

Genetic variability of the helix 54 of the 23S rDNA and its use as a molecular target for identification of species within the viridans group streptococci (VGS).

The aim of the present study is to establish a method, based on sequence analysis of the helix 54 of 23S rRNA gene, to identify clinical relevant strains belonging to viridans group streptococci (VGS). A set of 25 randomly selected clinical isolates of alpha-hemolytic streptococci from upper respiratory tract were characterized by the routine phenotypic methods (API 20 Strep test). Molecular characterization was assessed by genotypic analysis of the nucleotide sequence of the helix 54 of 23S rRNA and Intergenic spacer region 16S23S. Partial sequencing of the gdh gene was used on 10 strains of mitis group. Sequence variations of the helix 54 allowed the identification of strains to group level and even to species level for certain strains within sanguinis and anginosus groups. Infact, species identification was ambiguous for some strains belonged to the salivarius group (of VGS16 to VGS20) and the mitis group (of VGS1 to VGS14). These results are almost similar to those obtained by sequencing the 16S23S intergenic region. Thus, we use the gdh gene sequencing for the identification of strains, not recognized, within the mitis group. The results generated herein indicate that no single methodology can be used to provide an accurate identification to the species level of all VGS, although nucleotide sequence analysis of the helix 54 of 23S rRNA gene proved to be a reliable method for the identification of VGS to the group level and even to the species level within sanguinis and anginosus groups.

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.

Metabolic changes associated with adaptive resistance to daptomycin in Streptococcus mitis-oralis.

BACKGROUND: Viridans group streptococci of the Streptococcus mitis-oralis subgroup are important endovascular pathogens. They can rapidly develop high-level and durable non-susceptibility to daptomycin both in vitro and in vivo upon exposure to daptomycin. Two consistent genetic adaptations associated with this phenotype (i.e., mutations in cdsA and pgsA) lead to the depletion of the phospholipids, phosphatidylglycerol and cardiolipin, from the bacterial membrane. Such alterations in phospholipid biosynthesis will modify carbon flow and change the bacterial metabolic status. To determine the metabolic differences between daptomycin-susceptible and non-susceptible bacteria, the physiology and metabolomes of S. mitis-oralis strains 351 (daptomycin-susceptible) and 351-D10 (daptomycin non-susceptible) were analyzed. S. mitis-oralis strain 351-D10 was made daptomycin non-susceptible through serial passage in the presence of daptomycin. RESULTS: Daptomycin non-susceptible S. mitis-oralis had significant alterations in glucose catabolism and a re-balancing of the redox status through amino acid biosynthesis relative to daptomycin susceptible S. mitis-oralis. These changes were accompanied by a reduced capacity to generate biomass, creating a fitness cost in exchange for daptomycin non-susceptibility. CONCLUSIONS: S. mitis-oralis metabolism is altered in daptomycin non-susceptible bacteria relative to the daptomycin susceptible parent strain. As demonstrated in Staphylococcus aureus, inhibiting the metabolic changes that facilitate the transition from a daptomycin susceptible state to a non-susceptible one, inhibits daptomycin non-susceptibility. By preventing these metabolic adaptations in S. mitis-oralis, it should be possible to deter the formation of daptomycin non-susceptibility.

Associations between echocardiographic manifestations and bacterial species in patients with infective endocarditis: a cohort study.

BACKGROUND: The diagnosis of infective endocarditis (IE) is based on microbiological analyses and diagnostic imaging of cardiac manifestations. Echocardiography (ECHO) is preferred for visualization of IE-induced cardiac manifestations. We investigated associations between bacterial infections and IE manifestations diagnosed by ECHO. METHODS: In this cohort study, data from patients aged 18 years or above, with definite IE admitted at the Karolinska University Hospital between 2008 and 2017 were obtained from Swedish National Registry of Endocarditis. Bacteria registered as pathogen were primarily selected from positive blood culture and for patients with negative blood culture, bacteria found in culture or PCR from postoperative material was registered as pathogen. Patients with negative results from culture or PCR, and patients who did not undergo ECHO during hospital stay, were excluded. IE manifestations diagnosed by ECHO were obtained from the registry. Chi-squared test and two-sided Fisher's exact test was used for comparisons between categorical variables, and student's t test was used for continuous numerical variables. Multivariable analyses were performed using logistic regression. Secular trend analyses were performed using linear regression. Associations and the strength between the variables were estimated using odds ratios (ORs) with 95% confidence intervals (CIs). P < 0.05 was considered significant. RESULTS: The most common bacteria were Staphylococcus aureus (n = 239, 49%) and viridans group streptococci (n = 102, 21%). The most common manifestations were vegetation in the mitral (n = 195, 40%), aortic (n = 190, 39%), and tricuspid valves (n = 108, 22%). Associations were seen between aortic valve vegetations and Enterococcus faecalis among patients with native aortic valves, between mitral valve vegetations and streptococci of group B or viridans group, between tricuspid valve vegetations and S. aureus among patients with intravenous drug abuse, and between perivalvular abscesses as well as cardiovascular implantable electronic device (CIED)-associated IE and coagulase negative staphylococci (all P < 0.05). CONCLUSIONS: Associations were found between certain bacterial species and specific ECHO manifestations. Our study contributes to a better understanding of IE manifestations and their underlying bacterial etiology, which pathogens can cause severe infections and might require close follow-up and surgical treatment.

Oral streptococci show diversity in resistance to complement immunity.

PURPOSE: Mechanisms underlying systemic infections by oral species of Mitis (Streptococcus mitis, Streptococcus oralis) and Sanguinis (Streptococcus gordonii, Streptococcus sanguinis) commensal streptococci are poorly understood. This study investigates profiles of susceptibility to complement-mediated host immunity in representative strains of these four species, which were isolated from oral sites or from the bloodstream. METHODOLOGY: Deposition of complement opsonins (C3b/iC3b), and surface binding to C-reactive protein (CRP) and to IgG antibodies were quantified by flow cytometry in 34 strains treated with human serum (HS), and compared to rates of opsonophagocytosis by human PMN mediated by complement (CR1/3) and/or IgG Fc (FcγRII/III) receptors. RESULTS: S. sanguinis strains showed reduced susceptibility to complement opsonization and low binding to CRP and to IgG compared to other species. Surface levels of C3b/iC3b in S. sanguinis strains were 4.5- and 7.8-fold lower than that observed in S. gordonii and Mitis strains, respectively. Diversity in C3b/iC3b deposition was evident among Mitis species, in which C3b/iC3b deposition was significantly associated with CR/FcγR-dependent opsonophagocytosis by PMN (P<0.05). Importantly, S. gordonii and Mitis group strains isolated from systemic infections showed resistance to complement opsonization when compared to oral isolates of the respective species (P<0.05). CONCLUSIONS: This study establishes species-specific profiles of susceptibility to complement immunity in Mitis and Sanguinis streptococci, and indicates that strains associated with systemic infections have increased capacity to evade complement immunity. These findings highlight the need for studies identifying molecular functions involved in complement evasion in oral streptococci.

The bacteriology in adult patients with pneumonia and parapneumonic effusions: increased yield with DNA sequencing method.

The aim of this study was to use a 16S rDNA sequencing method in combination with conventional culture in patients with parapneumonic effusions (PPE) to evaluate the methods, study the microbiological spectrum, and examine the presence of bacteria within the different stages of PPE. Adults with community-acquired pneumonia (CAP) and PPE (n = 197) admitted to the Departments of Infectious Diseases at four hospitals in Stockholm County during 2011-2014 were prospectively studied. All patients underwent thoracentesis. Twenty-seven non-infectious pleural effusions were used as controls. The pleural samples were analyzed with culture, 16S rDNA sequencing, pH, glucose, and lactate dehydrogenase. Microbiological etiology was found in 99/197 (50%) of the patients with mixed infections in 20 cases. The most common pathogens were viridans streptococci (n = 37) and anaerobic bacteria (n = 40). Among the 152 patients with both methods performed, 26/152 (17%) and 94/152 (62%) had bacteria identified with culture and 16S rDNA sequencing respectively (p < 0.001). In 24/26 (92%) culture-positive cases, the same organism was identified by 16S rDNA. All controls were negative in both methods. Among the patients with complicated PPE and complete sampling, bacteria were found in 69/74 patients (93%), all detected with 16S rDNA sequencing, compared to 23/74 (31%) culture-positive samples (p < 0.001). Compared with culture, 16S rDNA sequencing substantially improved the microbiological yield, a microbiological diagnosis was achieved in almost all patients with complicated PPE, and the specificity seemed to be high. 16S rDNA sequencing should be used together with culture in patients with PPE to guide antibiotic therapy.

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.

Identification of viridans streptococci With Matrix-Assisted Laser Desorption & Ionization Time-of-flight Mass Spectrometry by an In-house Method and a Commercially Available System.

Two matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based methods were compared for their ability to identify viridans streptococci. One approach employed a reference database and software developed in-house. All inhouse measurements were performed using an Autoflex II Instrument (Bruker Daltonics GmbH, Germany). The other system, a VITEK-MS (BioMérieux, France) was operated on the commercially available V2.0 Knowledge Base for Clinical Use database. Clinical isolates of viridans streptococci (n=184) were examined. Discrepant results were resolved by 16S rDNA sequencing. Species-level identification percentages were compared by a chi-square test. The in-house method correctly identified 179 (97%) and 175 (95%) isolates to the group and species level respectively. In comparison, the VITEK-MS system correctly identified 145 (79%) isolates to the group and species level. The difference between the two methods was statistically significant at both group and species levels. Using the Autoflex II instrument combined with an extraction method instead of whole cell analysis resulted in more reliable viridans streptococci identification. Our results suggest that combining extraction with powerful analysis software and the careful choice of well-identified strains included into the database was useful for identifying viridans streptococci species.

[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.

Genotyping Oral Commensal Bacteria to Predict Social Contact and Structure.

Social network structure is a fundamental determinant of human health, from infectious to chronic diseases. However, quantitative and unbiased approaches to measuring social network structure are lacking. We hypothesized that genetic relatedness of oral commensal bacteria could be used to infer social contact between humans, just as genetic relatedness of pathogens can be used to determine transmission chains of pathogens. We used a traditional, questionnaire survey-based method to characterize the contact network of the School of Public Health at a large research university. We then collected saliva from a subset of individuals to analyze their oral microflora using a modified deep sequencing multilocus sequence typing (MLST) procedure. We examined micro-evolutionary changes in the S. viridans group to uncover transmission patterns reflecting social network structure. We amplified seven housekeeping gene loci from the Streptococcus viridans group, a group of ubiquitous commensal bacteria, and sequenced the PCR products using next-generation sequencing. By comparing the generated S. viridans reads between pairs of individuals, we reconstructed the social network of the sampled individuals and compared it to the network derived from the questionnaire survey-based method. The genetic relatedness significantly (p-value < 0.001) correlated with social distance in the questionnaire-based network, and the reconstructed network closely matched the network derived from the questionnaire survey-based method. Oral commensal bacterial are thus likely transmitted through routine physical contact or shared environment. Their genetic relatedness can be used to represent a combination of social contact and shared physical space, therefore reconstructing networks of contact. This study provides the first step in developing a method to measure direct social contact based on commensal organism genotyping, potentially capable of unmasking hidden social networks that contribute to pathogen transmission.

Rapid detection and identification of infectious pathogens based on high-throughput sequencing.

BACKGROUND: The dilemma of pathogens identification in patients with unidentified clinical symptoms such as fever of unknown origin exists, which not only poses a challenge to both the diagnostic and therapeutic process by itself, but also to expert physicians. METHODS: In this report, we have attempted to increase the awareness of unidentified pathogens by developing a method to investigate hitherto unidentified infectious pathogens based on unbiased high-throughput sequencing. RESULTS: Our observations show that this method supplements current diagnostic technology that predominantly relies on information derived five cases from the intensive care unit. This methodological approach detects viruses and corrects the incidence of false positive detection rates of pathogens in a much shorter period. Through our method is followed by polymerase chain reaction validation, we could identify infection with Epstein-Barr virus, and in another case, we could identify infection with Streptococcus viridians based on the culture, which was false positive. CONCLUSIONS: This technology is a promising approach to revolutionize rapid diagnosis of infectious pathogens and to guide therapy that might result in the improvement of personalized medicine.

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.

Comparison of species identification of endocarditis associated viridans streptococci using rnpB genotyping and 2 MALDI-TOF systems.

Streptococcus spp. are important causes of infective endocarditis but challenging in species identification. This study compared identification based on sequence determination of the rnpB gene with 2 systems of matrix-assisted laser desorption ionization-time of flight mass spectrometry, MALDI Biotyper (Bruker) and VITEK MS IVD (bioMérieux). Blood culture isolates of viridans streptococci from 63 patients with infective endocarditis were tested. The 3 methods showed full agreement for all 36 isolates identified in the Anginosus, Bovis, and Mutans groups or identified as Streptococcus cristatus, Streptococcus gordonii, or Streptococcus sanguinis. None of the methods could reliably identify the 23 isolates to the species level when designated as Streptococcus mitis, Streptococcus oralis, or Streptococcus tigurinus. In 7 isolates classified to the Mitis group, the rnpB sequences deviated strikingly from all reference sequences, and additional analysis of sodA and groEL genes indicated the occurrence of yet unidentified Streptococcus spp.

[THE DIAGNOSTIC APPROACHES TO VERIFICATION OF STREPTOCOCCUS INFECTION IN PATIENTS WITH INFECTIOUS MONONUCLEOSIS].

The Rostovskii state medical university of Minzdrav of Russia, 344022 Rostov-on-Don, Russia The analysis is applied concerning significance of laboratory techniques of verification of streptococcus infection (bacteriological analysis, detection of anti-streptolysin O in pair serums) in 148 patients with infectious mononucleosis aged from 3 to 15 years. The content of anti-streptolysin O exceeded standard in 41 ± 4.8% of patients with concomitant in acute period and in 49.5 ± 4.9% during period of re-convalescence. This data differed from analogous indicator in patients with negative result of examination on streptococcus infection independently of period of disease (9.3 ± 2.8%). The exceeding of standard of anti-streptolysin O was detected more frequently (t ≥ 2, P ≥ 95%) in patients with isolation of Streptococcus pyogenes (56.9 ± 5.8%) than in patients with Streptococcus viridans (31.2 ± 6.5%). The concentration of anti-streptolysin 0 in patients with concomitant streptococcus infection varied within limits 200-1800 IE/ml. The minimal level of anti-streptolysin O (C = 200 IE/mI) was detected independently of type of isolated Streptococcus and period of disease. The high levels of anti-streptolysin O were observed exclusively in patients with isolation of Streptococcus pyogenes. In blood serum ofpatient with concomitant streptococcus infection (Streptococcus pyogenes + Streptococcus viridans) increasing of level of anti-streptolysin O was detected in dynamics of diseases from minimal (C = 200 IE/ ml) to moderately high (200 < C < 400 IE/mI). It is demonstrated that to identify streptococcus infection in patients with infectious mononucleosis the anamnesis data is to be considered. The complex bacteriological and serological examination ofpatients is to be implemented This is necessary for early detection ofpatients with streptococcus infection and decreasing risk of formation of streptococcus carrier state.

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.

Genetic determinants and elements associated with antibiotic resistance in viridans group streptococci.

OBJECTIVES: To investigate the distribution of erythromycin, tetracycline and chloramphenicol resistance mechanisms and determinants and the relevant genetic environments and elements in viridans group streptococci (VGS). METHODS: A total of 263 VGS collected from routine throat swabs in 2010-12 and identified to the species level were studied. Antibiotic resistance determinants and the relevant genetic contexts and elements were determined using amplification and sequencing assays and restriction analysis. RESULTS: The investigation provided original information on the distribution of resistance mechanisms, determinants and genetic elements in VGS. Erythromycin-resistant isolates totalled 148 (56.3%; 37 belonging to the cMLS phenotype and 111 belonging to the M phenotype); there were 72 (27.4%) and 7 (2.7%) tetracycline- and chloramphenicol-resistant isolates, respectively. A number of variants of known genetic contexts and elements carrying determinants of resistance to these antibiotics were detected, including the mega element, Φ10394.4, Tn2009, Tn2010, the IQ element, Tn917, Tn3872, Tn6002, Tn916, Tn5801, a tet(O) fragment from ICE2096-RD.2 and ICESp23FST81. CONCLUSIONS: These findings shed new light on the distribution of antibiotic resistance mechanisms and determinants and their genetic environments in VGS, for which very few such data are currently available. The high frequency and broad variety of such elements supports the notion that VGS may be important reservoirs of resistance genes for the more pathogenic streptococci. The high rates of macrolide resistance confirm the persistence of a marked prevalence of resistant VGS in Europe, where macrolide resistance is, conversely, declining among the major streptococcal pathogens.