Characterization of erm(B)-Carrying Integrative and Conjugative Elements Transferred from Streptococcus anginosus to Other Streptococci and Enterococci.

Integrative and conjugative elements (ICEs) are important vectors of lateral gene transfer and contribute to the evolution of bacterial pathogens. However, studies on the transfer among species and the physiological consequences of ICEs are rare. The objective of this study was to investigate the cross-species transferability of newly identified erm(B)-carried ICE in Streptococcus anginosus San95 and its physiological consequences after transfer. The erm(B)-carried ICE, characterized by a triple serine integrase module, integrated into hsdM genes, thus designated ICESan95_hsdM. Analysis of ICESan95_hsdM revealed 32 additional ICESan95-like ICEs in the available NCBI genome (n = 24) and sequence of clinical isolates (n = 8). Polymerase chain reaction (PCR) was used to evaluate the 467 clinical isolates, of which 84 were positive for core genes (integrase, relaxase, and T4SS genes) of ICESan95_hsdM. Cross-species transfer experiments demonstrated that ICESan95_hsdM could transfer from S. anginosus to different streptococcal and enterococcal recipients. Growth and competitive culture assays showed acquisition of ICESan95_hsdM incurred no fitness cost. Our work discovered a group of ICEs in Streptococci and Enterococci. For the first time, we demonstrated the broad cross-species transferability to different species or genera of ICEs with no fitness cost that enables commensal S. anginosus to deliver antimicrobial resistance genes to other streptococci and enterococci.

Antimicrobial activity of eravacycline and other comparative agents on aerobic and anaerobic bacterial pathogens in Taiwan: A clinical microbiological study.

OBJECTIVES: Eravacycline, a new tetracycline derivative, exhibits broad-spectrum antimicrobial susceptibility. This study aimed to comprehensively investigate in vitro activities of eravacycline, tigecycline, and ertapenem against various Gram-positive, Gram-negative, and anaerobic bacteria. METHODS: Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. The following bacterial species were collected: vancomycin-sensitive (VS) Enterococci species, vancomycin-resistant Enterococci species (VRE), Staphylococcus aureus, Streptococcus anginosus, Bacteroides species, Clostridioides difficile, Clostridium innocuum, Clostridium perfringens, Parabacteroides distasonis, and Stenotrophomonas maltophilia. RESULTS: We found that eravacycline exhibited superior in vitro activity compared to tigecycline and ertapenem. Notably, it exhibited the lowest MIC90 for several bacterial species, including VS E. faecalis (0.12 µg/mL), VS E. faecium (0.12 µg/mL), and others. Besides, VRE was susceptible to eravacycline (MIC90:0.12 µg/mL) and tigecycline (MIC90:0.12 µg/mL), but was all resistant to ertapenem (MIC90 > 64 µg/mL). S. aureus was also susceptible to eravacycline (MIC90:0.5 µg/mL) as well as tigecycline (MIC90:1.0 µg/mL). Furthermore, S. anginosus showed higher susceptibility to eravacycline (MIC90:2.0 µg/mL) and tigecycline (MIC90:4.0 µg/mL), but lower to ertapenem (MIC90:32.0 µg/mL). Eravacycline and tigecycline also demonstrated good susceptibility to anaerobes, including Bacteroides species (susceptibility rate: 100%), P. distasonis (100%), C. difficile (94.1‒100%), C. innocuum (94.1‒96.1%), and C. perfringens (88.9‒96.3%). For S. maltophilia, both tigecycline and eravacycline showed an MIC90 of 2 µg/mL. A moderate-to-strong correlation (rho = 0.608-0.804, P < 0.001) was noted between the MIC values of eravacycline and tigecycline against various bacterial species. CONCLUSIONS: Our study highlights the potential of eravacycline as an effective treatment option for multidrug-resistant bacterial infections.

Proton pump inhibitors alter gut microbiota by promoting oral microbiota translocation: a prospective interventional study.

BACKGROUND: The mechanism by which proton pump inhibitors (PPIs) alter gut microbiota remains to be elucidated. We aimed to learn whether PPI induced gut microbiota alterations by promoting oral microbial translocation. METHODS: Healthy adult volunteers were randomly assigned: PP group (n=8, 40 mg esomeprazole daily for seven days) and PM group (n=8, 40 mg esomeprazole along with chlorhexidine mouthwash after each meal for seven days). Fecal and saliva samples were analysed using 16S ribosomal RNA sequencing. Mouse models were introduced to confirm the findings in vivo, while the effect of pH on oral bacteria proliferation activity was investigated in vitro. RESULTS: Taxon-based analysis indicated that PPI administration increased Streptococcus abundance in gut microbiota (P<0.001), and the increased species of Streptococcus were found to be from the oral site or oral/nasal sites, in which Streptococcus anginosus was identified as the significantly changed species (P<0.004). Microbial source tracker revealed that PPI significantly increased the contribution of oral bacteria to gut microbiota (P=0.026), and no significant difference was found in PM group (P=0.467). Compared to the baseline, there was a 42-fold increase in gut abundance of Streptococcus anginosus in PP group (P=0.002), and the times decreased to 16-fold in PM group (P=0.029). Mouse models showed that combination of PPI and Streptococcus anginosus significantly increased the gut abundance of Streptococcus anginosus compared with using PPI or Streptococcus anginosus only. Furthermore, Streptococcus anginosus cannot survive in vitro at a pH lower than 5. CONCLUSIONS: PPIs altered gut microbiota by promoting oral-originated Streptococcus translocation into gut.

Update on the in vitro activity of dalbavancin against indicated species (Staphylococcus aureus, Enterococcus faecalis, ß-hemolytic streptococci, and Streptococcus anginosus group) collected from United States hospitals in 2017-2019.

This study updates dalbavancin activity against contemporary (2017-2019) isolates of indicated species/groups (n = 16,451). Isolates from 71 hospitals were tested by broth microdilution method. All isolates were susceptible to dalbavancin. Dalbavancin MIC50/90 values remained stable for Staphylococcus aureus, vancomycin-susceptible Enterococcus faecalis, ß-hemolytic streptococci, and Streptococcus anginosus group since its clinical approval.

Streptococcus anginosus group infections: Management and outcome at a tertiary care hospital.

BACKGROUND: Data on patients with invasive Streptococcus anginosus group (SAG) infections is limited, as it's been considered commensal bacteria in the human microbiota. We conducted an analysis of SAG infections to assist clinicians in understanding their burden and clinical outcomes. METHODS: A retrospective study of medical records, identifying invasive SAG bacteria of sterile-site isolates that were managed from May 2015 to April 2017, at a tertiary care hospital in Riyadh, Saudi Arabia. Demographic data, clinical presentation, site of infection, antibiotic use, and outcome were recorded and analyzed to identify factors associated with poor outcome and/or polymicrobial growth. RESULTS: We identified 105 cases of SAG infections in adults, with 52% of the patients being male and the mean age of 52.4 years with comorbidities occurring in more than half of the cases such as diabetes (38%) and malignancy (15%). Overall mortality was 6%, and it was statistically associated with age older than 65 years, polymicrobial growth and a history of malignancy. The infection frequencies were skin and soft tissue infections (SSTI; 55%), intra-abdominal infections (24%), bacteremia (14%), genitourinary infections (8.5%), and pleuropulmonary infections (5%). Abscesses accounted for 68% of cases. Polymicrobial infection (46%) with Enterobacteriaceae and Gram-negative anaerobes coincided with SAG infection. Polymicrobial growth was significantly associated with abscess formation, intra-abdominal source of infections, and poor outcome. In addition, death in patients with SAG was statistically associated with patients older than 65 years of age and those with history of cancer or transplant. CONCLUSION: SSTIs and intra-abdominal infections are the most common clinical presentations in our cohort. Bacteremia was uncommon; however, the prognosis is less favorable. Overall susceptibility to penicillin was 91%, therefore ß-lactam antibiotics are the drug of choice and additional coverage for anaerobic and gram-negative bacteria should be considered for intra-abdominal collection and solid or organ abscesses.

Heterogeneous antimicrobial activity in broncho-alveolar aspirates from mechanically ventilated intensive care unit patients.

Pneumonia is an infection of the lungs, where the alveoli in the affected area are filled with pus and fluid. Although ventilated patients are at risk, not all ventilated patients develop pneumonia. This suggests that the sputum environment may possess antimicrobial activities. Despite the generally acknowledged importance of antimicrobial activity in protecting the human lung against infections, this has not been systematically assessed to date. Therefore, the objective of the present study was to measure antimicrobial activity in broncho-alveolar aspirate ('sputum") samples from patients in an intensive care unit (ICU) and to correlate the detected antimicrobial activity with antibiotic levels, the sputum microbiome, and the respective patients' characteristics. To this end, clinical metadata and sputum were collected from 53 mechanically ventilated ICU patients. The antimicrobial activity of sputum samples was tested against Streptococcus pneumoniae, Staphylococcus aureus and Streptococcus anginosus. Here we show that sputa collected from different patients presented a high degree of variation in antimicrobial activity, which can be partially attributed to antibiotic therapy. The sputum microbiome, although potentially capable of producing antimicrobial agents, seemed to contribute in a minor way, if any, to the antimicrobial activity of sputum. Remarkably, despite its potentially protective effect, the level of antimicrobial activity in the investigated sputa correlated inversely with patient outcome, most likely because disease severity outweighed the beneficial antimicrobial activities.

Bicarbonate and unsaturated fatty acids enhance capsular polysaccharide synthesis gene expression in oral streptococci, Streptococcus anginosus.

We recently reported on the capsular polysaccharide (CP) synthesis (cps) genes of the oral streptococci, Streptococcus anginosus. In this study, we investigate the effects of carbon dioxide (CO2), bicarbonate (HCO3-) and unsaturated fatty acids (UFAs) on CP synthesis of S. anginosus. We found that CP production increased when bacteria were exposed to high concentrations of CO2. This increase was similarly observed in the presence of sodium bicarbonate (NaHCO3) under atmospheric condition. Since ectopic expression of carbonic anhydrase, which converts CO2 to HCO3-, eliminated the requirement for CO2 in CP production and growth of S. anginosus lacking this enzyme, it seemed that HCO3- is an essential factor for CP production. Furthermore, UFAs also stimulated the CP production. Promoter-reporter assay and quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis confirmed that stimulation of CP production occurs at the transcription level. The results of the promoter assays suggest that the expression and stimulation of cps genes by HCO3- or UFAs require the cpsA gene, which is located in the first position of the cps operon. With respect to the relationship between HCO3-and UFAs, HCO3- may stimulate UFA synthesis pathway at transcription level. Therefore, it is possible that UFAs are definitive signals for the CP production in S. anginosus.

Identification of highly potent competence stimulating peptide-based quorum sensing activators in Streptococcus mutans through the utilization of N-methyl and reverse alanine scanning.

Quorum sensing (QS) controls the pathogenic behavior of Streptococcus mutans, a primary cause of dental caries. S. mutans uses the competence stimulating peptide (CSP) to control mutacin production, a bacteriocin utilized by S. mutans to outcompete different commensal bacteria in mixed biofilm environments. In this study, we performed an N-methyl scan of an 18-CSP-based scaffold lacking the first two amino acid residues that were shown to be dispensable, to gain important mechanistic insight as to the role of backbone amide protons in the interaction between CSP and the ComD receptor. We then utilized the reverse alanine approach to develop CSP-based analogs with enhanced activities. The two most potent analogs were found to induce bacteriocin production at sub-nanomolar concentration using an interspecies inhibition assay. Overall, our analysis revealed that the 18-CSP sequence is not optimized and can be improved by replacement of multiple positions with alanine. Our results further suggest that the hydrophobic residues in S. mutans 18-CSP are involved in both receptor binding and activation.

In vitro activity of lascufloxacin, a novel fluoroquinolone antibacterial agent, against various clinical isolates of anaerobes and Streptococcus anginosus group.

The in vitro activities of lascufloxacin were evaluated by comparison with seven reference compounds using 412 clinical isolates of anaerobes and Streptococcus anginosus group. Lascufloxacin showed potent and broad antibacterial activities greater than those of existing quinolones against the clinical isolates used in this study.

Decreased susceptibility of Streptococcus anginosus to vancomycin in a multispecies biofilm is due to increased thickness of the cell wall.

Background: Streptococcus anginosus, Pseudomonas aeruginosa and Staphylococcus aureus are often co-isolated from the sputum of cystic fibrosis patients. It was recently shown that S. anginosus is protected from the activity of vancomycin when it grows in a multispecies biofilm with P. aeruginosa and S. aureus. Objectives: Elucidating the underlying cause of the reduced susceptibility of S. anginosus to vancomycin when growing in a multispecies biofilm with P. aeruginosa and S. aureus. Methods: The transcriptome of S. anginosus growing in a multispecies biofilm was compared with that of a S. anginosus monospecies biofilm. Subsequently, transmission electron microscopy was performed to investigate changes in cell wall morphology in S. anginosus and S. aureus in response to growth in multispecies biofilm and to vancomycin treatment. Results: S. anginosus responds to growth in a multispecies biofilm with induction of genes involved in cell envelope biogenesis. Cell walls of S. anginosus cultured in a multispecies biofilm were thicker than in a monospecies biofilm, without antibiotic challenge. S. aureus, when cultured in a multispecies biofilm, does not respond to vancomycin treatment with cell wall thickening. Conclusions: Growth in multispecies biofilms can have an impact on the expression of genes related to cell wall synthesis and on the cell wall thickness of S. anginosus.

In Vitro Activity of Ceftazidime-Avibactam against Isolates from Patients in a Phase 3 Clinical Trial for Treatment of Complicated Intra-abdominal Infections.

The increasing prevalence of multidrug-resistant Gram-negative pathogens has generated a requirement for new treatment options. Avibactam, a novel non-ß-lactam-ß-lactamase inhibitor, restores the activity of ceftazidime against Ambler class A, C, and some class D ß-lactamase-producing strains of Enterobacteriaceae and Pseudomonas aeruginosa The in vitro activities of ceftazidime-avibactam versus comparators were evaluated against 1,440 clinical isolates obtained in a phase 3 clinical trial in patients with complicated intra-abdominal infections (cIAI; ClinicalTrials.gov identifier NCT01499290). Overall, in vitro activities were determined for 803 Enterobacteriaceae, 70 P. aeruginosa, 304 Gram-positive aerobic, and 255 anaerobic isolates obtained from 1,066 randomized patients at baseline. Susceptibility was determined by broth microdilution. The most commonly isolated Gram-negative, Gram-positive, and anaerobic pathogens were Escherichia coli (n = 549), Streptococcus anginosus (n = 130), and Bacteroides fragilis (n = 96), respectively. Ceftazidime-avibactam was highly active against isolates of Enterobacteriaceae, with an overall MIC90 of 0.25 mg/liter. In contrast, the MIC90 for ceftazidime alone was 32 mg/liter. The MIC90 value for ceftazidime-avibactam (4 mg/liter) was one dilution lower than that of ceftazidime alone (8 mg/liter) against isolates of Pseudomonas aeruginosa The ceftazidime-avibactam MIC90 for 109 ceftazidime-nonsusceptible Enterobacteriaceae isolates was 2 mg/liter, and the MIC range for 6 ceftazidime-nonsusceptible P. aeruginosa isolates was 8 to 32 mg/liter. The MIC90 values were within the range of susceptibility for the study drugs permitted per the protocol in the phase 3 study to provide coverage for aerobic Gram-positive and anaerobic pathogens. These findings demonstrate the in vitro activity of ceftazidime-avibactam against bacterial pathogens commonly observed in cIAI patients, including ceftazidime-nonsusceptible Enterobacteriaceae (This study has been registered at ClinicalTrials.gov under identifier NCT01499290.).

Aciduricity and acid tolerance mechanisms of Streptococcus anginosus.

Although Streptococcus anginosus constitutes a proportion of the normal flora of the gastrointestinal and genital tracts, and the oral cavity, it has been reported that S. anginosus infection could be closely associated with abscesses at various body sites, infective endocarditis, and upper gastrointestinal cancers. The colonization in an acidic environment due to the aciduricity of S. anginosus could be the etiology of the systemic infection of the bacteria. To elucidate the aciduricity and acid tolerance mechanisms of the microbe, we examined the viability and growth of S. anginosus under acidic conditions. The viabilities of S. anginosus NCTC 10713 and Streptococcus mutans ATCC 25175 at pH 4.0 showed as being markedly higher than those of Streptococcus sanguinis ATCC 10556, Streptococcus gordonii ATCC 10558, and Streptococcus mitis ATCC 49456; however, the viability was partially inhibited by dicyclohexylcarbodiimide, an H+-ATPase inhibitor, suggesting that H+-ATPase could play a role in the viability of S. anginosus under acidic conditions. In addition, S. anginosus NCTC 10713 could grow at pH 5.0 and showed a marked arginine deiminase (ADI) activity, unlike its ΔarcA mutant, deficient in the gene encoding ADI, and other streptococcal species, which indicated that ADI could also be associated with aciduricity. These results suggest that S. anginosus has significant aciduric properties, which can be attributed to these enzyme activities.

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

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

Sites of infection associated with Streptococcus anginosus group among children.

Streptococcus anginosus group (SAG) are parts of normal flora of the oral cavity and associated with abscess forming in various sites on the body. Although the clinical features of infections caused by each member of the SAG in adults has been reported, it has not well been known in children. The aim of this study was to clarify the site of infections associated with individual SAG species among children. Medical records from March 2010 to July 2016 were reviewed at Tokyo Metropolitan Children's Medical Center. Any SAG species (S. anginosus, S. constellatus, or S. intermedius) isolated from clinical samples and recorded in the microbiological database were included for analysis. Analysis of 52 infectious episodes found that S. anginosus was most frequently isolated from the genitourinary tract, and 73% of genitourinary tract infection was balanoposthitis. All genitourinary tract infections were associated with S. anginosus. These findings were different from those of a previous study of adults. Of all the patients, 45 patients (87%) had polymicrobial infections. More than 70% of patients infected by S. anginosus and S. constellatus were co-infected by obligate anaerobes, in comparison with only 21% of S. intermedius cases. Among the obligate anaerobes species, Bacteroides spp. was significantly accompanied with S. anginosus. Susceptibility to penicillin, ampicillin, cefotaxime, erythromycin, clindamycin, levofloxacin, and vancomycin was 100%, 100%, 100%, 77%, 89%, 97% and 100%, respectively. S. anginosus was often isolated from balanoposthitis among children.

Community Composition Determines Activity of Antibiotics against Multispecies Biofilms.

In young cystic fibrosis (CF) patients, Staphylococcus aureus is typically the most prevalent organism, while in adults, Pseudomonas aeruginosa is the major pathogen. More recently, it was observed that also Streptococcus anginosus plays an important role in exacerbations of respiratory symptoms. These species are often coisolated from CF lungs, yet little is known about whether antibiotic killing of one species is influenced by the presence of others. In the present study, we compared the activities of various antibiotics against S. anginosus, S. aureus, and P. aeruginosa when grown in monospecies biofilms with the activity observed in a multispecies biofilm. Our results show that differences in antibiotic activity against species grown in mono- and multispecies biofilms are species and antibiotic dependent. Fewer S. anginosus cells are killed by antibiotics that interfere with cell wall synthesis (amoxicillin plus sulbactam, cefepime, imipenem, meropenem, and vancomycin) in the presence of S. aureus and P. aeruginosa, while for ciprofloxacin, levofloxacin, and tobramycin, no difference was observed. In addition, we observed that the cell-free supernatant of S. aureus, but not that of P. aeruginosa biofilms, also caused this decrease in killing. Overall, S. aureus was more affected by antibiotic treatment in a multispecies biofilm, while for P. aeruginosa, no differences were observed between growth in mono- or multispecies biofilms. The results of the present study suggest that it is important to take the community composition into account when evaluating the effect of antimicrobial treatments against certain species in mixed biofilms.

Identification of Anion Channels Responsible for Fluoride Resistance in Oral Streptococci.

Recently, it has been reported that eriC and crcB are involved in bacterial fluoride resistance. However, the fluoride-resistance mechanism in oral streptococci remains unclear. BLAST studies showed that two types of eriCs (eriC1 and eriC2) and two types of crcBs (crcB1 and crcB2) are present across 18 oral streptococci, which were identified in ≥ 10% of 166 orally healthy subjects with ≥ 0.01% of the mean relative abundance. They were divided into three groups based on the distribution of these four genes: group I, only eriC1; group II, eriC1 and eriC2; and group III, eriC2, crcB1, and crcB2. Group I consisted of Streptococcus mutans, in which one of the two eriC1s predominantly affected fluoride resistance. Group II consisted of eight species, and eriC1 was responsible for fluoride resistance, but eriC2 was not, in Streptococcus anginosus as a representative species. Group III consisted of nine species, and both crcB1 and crcB2 were crucial for fluoride resistance, but eriC2 was not, in Streptococcus sanguinis as a representative species. Based on these results, either EriC1 or CrcBs play a role in fluoride resistance in oral streptococci. Complementation between S. mutans EriC1 and S. sanguinis CrcB1/CrcB2 was confirmed in both S. mutans and S. sanguinis. However, neither transfer of S. sanguinis CrcB1/CrcB2 into wild-type S. mutans nor S. mutans EriC1 into wild-type S. sanguinis increased the fluoride resistance of the wild-type strain. Co-existence of different F- channels (EriC and CrcB) did not cause the additive effect on fluoride resistance in oral Streptococcus species.

Effect of smokeless tobacco products on human oral bacteria growth and viability.

To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32-2.14 log10 fold, while 14 strains demonstrated enhanced growth of 0.3-1.76 log10 fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3-2.11 log10 fold, 18 strains showed enhanced growth of 0.3-0.97 log10 fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3-2.96 log10 fold, 8 strains showed enhanced growth of 0.3-1.0 log10 fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1-66.5% decrease for 4 strains at 1 mg/ml, 20.3-85.7% decrease for 10 strains at 10 mg/ml, 20.0-93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 µg/ml; while the growth of P. micros was enhanced 0.31-0.54 log10 fold; the growth of Veillonella parvula was repressed 0.33-0.36 log10 fold; and the cell viabilities of 2 strains decreased 56.6-69.9%. The results demonstrate that STAEs affected the growth of some types of oral bacteria, which may affect the healthy ecological balance of oral bacteria in humans. On the other hand, TSNAs did not significantly affect the growth of the oral bacteria.

Comparative Genome Analysis of the Daptomycin-Resistant Streptococcus anginosus Strain J4206 Associated with Breakthrough Bacteremia.

Streptococcus anginosus is a member of the normal oral flora that can become a pathogen causing pyogenic infections in humans. The genome of daptomycin-resistant strain J4206, originally isolated from a patient suffering from breakthrough bacteremia and septic shock at the University of Texas Health Science Center at San Antonio, was determined. The circular genome is 2,001,352 bp long with a GC content of 38.62% and contains multiple mobile genetic elements, including the phage-like chromosomal island SanCI that mediates a mutator phenotype, transposons, and integrative conjugative elements. Daptomycin resistance involves multiple alterations in the cell membrane and cell wall, and unique features were identified in J4206 that may contribute to resistance. A cluster of capsular polysaccharide (CPS) genes for choline metabolism and transport are present that may help neutralize cell surface charges, destabilizing daptomycin binding. Further, unique J4206 genes encoding sortases and LPXTG-target proteins that are involved in cell wall modification were present. The J4206 genome is phylogenetically closely related to the recently reported vancomycin-resistant SA1 strain; however, these genomes differ with SNPs in cardiolipin synthetase, histidine kinase yycG, teichoic acid modification genes, and other genes involved in cell surface modification. Transmission electron microscopy showed that the cell walls of both strains J4206 and SA1 were significantly thicker and more electron dense than daptomycin- and vancomycin-sensitive strain J4211. This comparative genomic study has identified unique genes as well as allelic variants in the J4206 genome that are involved in cell surface modification and thus might contribute to the acquisition of daptomycin resistance.

Streptococcus anginosus (milleri) Group Strains Isolated in Poland (1996-2012) and their Antibiotic Resistance Patterns.

Streptococcus anginosus, Streptococcus intermedius and Streptococcus constellatus form a group of related streptococcal species, namely the Streptococcus Anginosus Group (SAG). The group, previously called "milleri" had been rarely described until 1980/1990 as source of infections. Nowadays SAG bacteria are often described as pathogens causing predominantly purulent infections. The number of infections is highly underestimated, as SAG strains are often classified in the microbiology laboratory as less virulent "viridans streptococci" Epidemiological situation regarding SAG infections in Poland has been unrecognized, therefore we performed a retrospective analysis of strains isolated between 1996 and 2012. Strains suspected of belonging to SAG were re-identified using an automated biochemical approach (Vitek2) and MALDI-TOF MS. We performed first analysis of antibiotic resistance among SAG strains isolated in Poland using automated methods (Vitek2), disk diffusion tests and E-Tests. We also performed PCR detection of resistance determinants in antibiotic resistant strains. Clonal structure of analyzed strains was evaluated with PFGE and MLVF methods. All three species are difficult to distinguish using automated diagnostic methods and the same is true for automated MIC evaluation. Our analysis revealed SAG strains are rarely isolated in Poland, predominantly from purulent infections. All isolates are very diverse on the genomic level as estimated by PFGE and MLVF analyses. All analyzed strains are sensitive to penicillin, a substantial group of strains is resistant to macrolides and the majority of strains are resistant to tetracycline.