Tabernaemontana divaricata (L.) R. Br. flowers. T. divaricata flower methanolic extract (TDFME) was screened for antiulcer activity versus aspirin and ethanol induced gastric ulcers at three doses—125, 250, and 500 mg/kg—orally using misoprostol as a standard. Besides histopathological examination, seven parameters, that is, ulcer index, total protein, nonprotein sulphhydryls, mucin, catalase, malondialdehyde, and superoxide dismutase levels, were estimated. In addition to HPLC profiling, GC-MS analysis and electrospray ionization—high resolution mass spectral (ESI-HRMS) analysis of crude TDFME were carried out in an attempt to identify known phytochemicals present in the extract on the basis of m/z value. The results revealed a significant increase in the levels of catalase, superoxide dismutase, mucin, and nonprotein sulphhydryls, while they revealed a reduction in ulcer index, the levels of total protein, and malondialdehyde. Histopathological observations also demonstrated the protective effect. Though all the doses of TDFME exhibited gastroprotective function, higher doses were found to be more effective. Mass spectral analysis gave a few characteristic m/z values suggesting the presence of a few known indole alkaloids, while HPLC profiling highlighted the complexity of the extract. TDFME was found to exhibit its gastroprotective effect through antioxidant mechanism and by enhancing the production of gastric mucous.The present study was conducted to evaluate the antiulcerogenic effect and recognize the basic mechanism of action of  Tabernaemontana divaricata (L.) R. Br. ex Roemer and Schultes (Apocynaceae) has high medicinal value, and it is used in the ethnic systems of medicine all over the world. Traditionally, crepe jasmine flowers are used to treat corneal inflammation  w(d) Determination of Gastric Wall Mucin Content (GWM). Mucin content was estimated by the method described by Corne et al. (1974) + peaks and the list of probable compounds with their exact m/z values and molecular formulae as per the literature are summarized in m/z peaks and molecular formulae are shown in ESI-HR mass spectra in T. divaricata contains at least 66 characterized indole alkaloids  [A few characteristic e, 1991) , the obs., 2008) . The preT. divaricata flowers  [Enantia chlorantha and Voacanga africana at a dose of 50 mg/kg orally in rats [T. divaricata improved the blood supply to various organs and vascular tissue by vasodilation in dogs  [T. divaricata are reported to possess antiinflammatory activity  [Falcão et al. (2008) documented an extensive literature on antiulcer and antisecretory activities of various alkaloids . Taberso., 1981) . Tan et  in rats . In addi., 1984) . This al., 2006) , 13, 66.., 2006) . 2-receptor antagonists like cimetidine, famotidine, ranitidine, roxatidine) and anticholinergics (selective M1 muscarinic receptor antagonists like pirenzepine) that produce antisecretory and antispasmodic effects respectively  [T. divaricata contains isovoacristine, an indole alkaloid that exhibited antihistaminic, anticholinergic, and skeletal muscle relaxant properties in a study on isolated guinea pig ileum and rabbit, respectively  [in vivo screening of T. divaricata leaves and flowers conducted in rats by Taesotikul et al. (1989) suggests depressive effects on both peripheral and central nervous systems [+K+ATPase) through H2 receptors  [T. divaricata flowers can be attributed mostly to the indole alkaloids present in it. The present day treatment approach towards management of peptic ulcer disease employs antihistaminics  . T. divai, 1979) . Hence,  systems . The depi, 2003) . TherefoT. divaricata have shown antimicrobial activity in in vitro studies. Apparicine isolated by cell suspension culture of T. divaricata inhibited Polio III virus at a concentration of 250 μg/mL  [Corynebacterium, Escherichia, Proteus, Pseudomonas, Salmonella, Shigella, and Staphylococcus in an in vitro study at a concentration of 1.2%  [T. divaricata by Karawya and Aboutabl (1979) demonstrated strong antimicrobial activity against gram positive bacteria such as Bacillus subtilis and Staphylococcus aureus and moderate activity against gram negative bacteria like Escherichia coli and Pseudomonas aeruginosa  [T. divaricata extracts or its components can be effective against Helicobacter pylori. The biological activity of T. divaricata extracts against H. pylori is yet to be established [Furthermore, a few indole alkaloids from ., 1968) –71. It ez, 1977) . Voacami., 2008) , 73. As ablished . T. divaricata was also studied for antioxidant effects by various investigators. Mandal and Mukherji (2001) demonstrated that T. divaricata is a good scavenger of air pollutants [T. divaricata methanolic extract of leaves in carbon tetrachloride induced hepatotoxicity model. The extract produced significant dose dependent antioxidant and hepatoprotective effects by diminishing lipid peroxidation and elevating levels of antioxidant agents like Catalase, Glutathione and Superoxide dismutase [Earlier llutants . They alismutase . Among tismutase . It exerismutase , 78. TheT. divaricata (L.) R. Br. flower methanolic extract exhibits gastroprotective effect by enhancing the production of the gastric mucosa or preventing its depletion by aggressive factors while exhibiting antioxidant activity in chemically induced gastric ulceration in rats. The mass spectral analysis signifies the probable presence of few aspidospermatan, corynanthean, ibogan and plumeran sub-types of indole alkaloids in the flowers for the first time. The study also highlights the possible role of indole alkaloids in gastroprotective function of T. divaricata. It is concluded that


Today, exhaled nitric oxide has been studied the most, and most researches have now focusd on asthma. More than a thousand different volatile organic compounds have been observed in low concentrations in normal human breath. Alkanes and methylalkanes, the majority of breath volatile organic compounds, have been increasingly used by physicians as a novel method to diagnose many diseases without discomforts of invasive procedures. None of the individual exhaled volatile organic compound alone is specific for disease. Exhaled breath analysis techniques may be available to diagnose and monitor the diseases in home setting when their sensitivity and specificity are improved in the future. Acetaldehyde breath test has been used to measure the production of this compound after ingestion of ethanol, but its interpretation is affected by many factors: ALDH2 polymorphism, alcoholic drinking habits, and smoking which are correlated with higher breath and blood levels . The psy More than a thousand different volatile organic compounds (VOCs) have been observed in low concentrations in normal human breath . DespiteMycobacterium tuberculosis (M tbc), respectively, [M tbc cultures in vitro, predominantly derivatives of benzene, naphthalene, and alkanes [M tbc urease enzyme encoded by ureA (Rv1848), ureB (Rv1849), and ureC (Rv1850) hydrolyzes urea into carbon dioxide and ammonia [13C-urea tracer, thus preventing gastric Helicobacter species contaminations [Helicobacter pylori [M tbc in patients with active pulmonary TB with 80% accuracy, 71.2% sensitivity, and 72% specificity [M tbc, Aspergillus fumigatus, and Pseudomonas aeruginosa [A study for breath biomarkers of tuberculosis (TB) was conducted among 23 and 19 patients with positive and negative sputum cultures for  ammonia . An attr ammonia . Since 2 ammonia . The speinations . Anti-TBr pylori . Thus, Ur pylori , 13. A rcificity . In age-cificity . Two precificity , 16, wheruginosa .M. avium, M. gordonae, M. gastri, M. kansasii, M. szulgai, and M. flavescens can be identified by their distinctive patterns of volatile metabolites, including C14–C26 fatty acids and their methylated and hydroxylated derivatives [M. gordonae and M. kansasii were identified to species level [ M tbc was definitely identified in 85% of the cases [M. bovis and M. xenopi, both of which are rare causes of infection [ Other several species of Halic produce VOC metabolites that act as chemical “fingerprints”: ivatives –20. A stes level . M tbc whe cases . When itnfection . A recennfection . 2O2) level in patients with pulmonary TB revealed significantly decreased after 2 months of successful antituberculous treatment and its level did not correlate with circulating interleukin (IL)-18 in TB patients before or after treatment compared with control groups [A study of exhaled hydrogen peroxide  that are induced in patients with pulmonary cancer and result in measurable changes in the composition of the breath . In 2003 In 2007, a study concluded that a two-minute breath test can predict pulmonary cancer with accuracy comparable to screening chest computed tomography . The acc Alterations in breath VOCs in pulmonary cancer were consistent with a nonlinear pathophysiologic process, such as an off-on switch controlling high-risk CYP activity . VOCs brP < 0.0003) [KRAS oncogene in EBC [NO measurements during radiotherapy in patients with pulmonary cancer had a low ability to detect symptomatic radiation pneumonitis [Exhaled VOC analysis may therefore be useful in improving the specificity and sensitivity of conventional diagnostic approach to pulmonary cancer . A study 0.0003) . In pati 0.0003) . Exhaled 0.0003) . A recen 0.0003) . The dia 0.0003) . Cross-v 0.0003) . In 2007 0.0003)  and anot 0.0003) . Exhaled 0.0003) . A recen 0.0003) . Isopren 0.0003) . Some ree in EBC  and somee in EBC  after suumonitis . Pseudomonas aeruginosa (P. aeruginosa)-positive specimens than above other bacterial growths (P < 0.01), and levels of HCN greater than 100 part per billion were a sensitive (68%) and highly specific (100%) VOC of P. aeruginosa [Staphylococcus aureus [Streptococcus pneumoniae infections [ In 2005, a study reported that notably hydrogen cyanide (HCN) gas concentration was significantly higher above ruginosa . Increasruginosa . Elevateruginosa  and adulruginosa  with uppruginosa . Low exhs aureus . The incs aureus  and bacts aureus  and lowes aureus  respirats aureus . In norms aureus . A studyfections . Higher fections .NO) levels [NO concentrations between asthmatic and nonasthmatic patients with nasal polyposis [NO was reduced by effects of both soluble and suspension formulations of nebulized budesonide in asthmatic children [ Exhaled NO levels are increased in atopic asthma, normal in chronic obstructive pulmonary diseases (COPD) and asbestosis , 56, and) levels . A recenolyposis . FENO wachildren . Patientchildren . Exhaledchildren . A studychildren . In inhachildren . Raisingchildren . When cochildren . In 2007children .2 and EBC prostaglandin F2-alpha are markedly increased in COPD but not in asthma [2 . EBC concentrations of prostaglandin D-methoxime are the same in normal subjects and COPD patients [2-Ll is detectable in COPD patients [2 levels in EBC in COPD patients, whereas selective cyclooxygenase inhibitors have no effect [ Reduced EBC ammonium ions were observed in patients with acute asthma . Exhaledn asthma , 70. In n asthma  as well patients , whereaspatients  but is mpatients , 73, esppatients , but no patients . In patipatients . Elevatipatients , and stapatients  and was patients  and bronpatients  as well.patients . Higher patients . Nonseleo effect .NO, and measurement of breath VOCs with electronic nose application which can differentiate healthy smoking individuals from healthy nonsmoking subjects [2O2 [ In nonsmoking COPD patients, there is elevation of exhaled CO levels and also during exacerbations but decreases during recovery, respectively, , 81. Elesubjects , 100 aresubjects , MDA [10subjects , S-nitrosubjects , and nitsubjects  are elevsubjects , IL-1-besubjects , TNF-alpcts [2O2  during ects [2O2 . A studycts [2O2 . 1) for toluene in these patients, and no discrimination of CF patients from healthy subjects for ethane levels [ In stable and exacerbation CF patients, there are markedly elevation of exhaled CO levels and decrease after treatment. This indicates that exhaled CO level is a marker of CF severity . In 2000e levels . These Ve levels . EBC TNFe levels  with rede levels . EBC pH e levels  and its e levels . Elevatee levels . Glucosee levels . Use of e levels . 2O2, 3-NT, adenosine [2O2 [P. aeruginosa [ Increased concentrations of NO, CO, ammonia, pentane , ethane denosine , and 8-idenosine –127. Eledenosine . A studydenosine . A recendenosine . Small adenosine . Elevatedenosine  levels wine [2O2  levels iine [2O2 . Exhaledruginosa . Increasruginosa –138. Howruginosa –142. Incruginosa , 143 andruginosa . The eleruginosa . Either ruginosa  or increruginosa  exhaled ruginosa  were repruginosa  by steroruginosa . In contruginosa . A recenruginosa . A previruginosa . Some prruginosa , 151. NMruginosa . 2O2, hydrogen ions, and LT B4 levels was demonstrated in patients after pulmonary lobectomy [ A study among patients with head injury, ARDS, and those being at risk of developing ARDS concluded that plasma concentrations of MDA  and thioobectomy . SummarySeveral of the commonly occurring VOCs are derived from metabolic pathways that have been previously reported , for exaThe screening method must have acceptable technical performance parameters and must detect the disease at an earlier stage than would be possible without screening, while minimized false-positive and false-negative results . In addiBronchial biopsies provide mediators and inflammatory cells, as well as the spatial relationships between the inflammatory processes in the airway wall, but they may not reflect all pathologic changes in the lung periphery, and the bronchoscopic invasiveness precludes repeated measurements. BAL may provide more information on small airway and alveolar inflammation, but it has variable dilution and invasiveness. Induced-sputum examination can provide information about mediators, cells, and markers of oxidative/nitrative stress, but it has high variability of the biomarkers. NO measurements within a single day in both children and adults is superior to any conventional methods of airway inflammation in asthma that may allow physicians to perform two instead of three exhalations for obtaining the reliable results [NO does require an extra encouragement, compared with peak expiratory flow measurements [NO measurements are not found [NO levels are below or above a certain reference level, steroid treatment should be either increased or reduced [NO for the whole population was 16.6 parts per billion (ppb) and the upper limits of FENO ranged from 24.0 to 54.0 ppb [NO values are definitely higher than those reported in both normal adults and children in American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines [NO was significantly higher in patients with chronic rhinosinusitis and allergic rhinitis compared to patients with nonallergic rhinitis [NO values due to fluctuations of exposure to allergens have been reported [NO changes as recently reported in a random community survey of adults [NO reference values. NO chemiluminescence analyzers are mainly available in academic research laboratories and are presently expensive [NO measurement with a portable analyzer has been used beneficially for young adults with asthma screening [NO measurements in asthmatic children [NO measurements will not interfere with the system and be able to be used much more frequently, in contrast to the currently used semi-invasive or invasive procedures [High reproducibility of FE results  and redu results . This le results , asympto results , insensi results , their h results  which ma results , noninva results , and min results . FENO dourements . Systemiot found . The meaot found . A studyot found . When th reduced . A study54.0 ppb . Height 54.0 ppb . Such upidelines  and in aidelines . Recentlrhinitis . Seasonareported . Accordireported . With asf adults . Probablf adults  or in ref adults  of the hf adults . These cxpensive . Howeverxpensive . Recentlcreening  includinchildren . Therefoocedures . It is locedures . Flushinocedures . One of ocedures  measuremocedures . Today, ocedures , 164, buocedures . The maiocedures . The scrocedures .NO and EBC cysLTs in steroid-naïve asthmatics, and no correlation between EBC cysLTs and FEV1, or log PC20Arc [2O2 from neonates from a background with nasal continuous positive airway pressure alone or ventilator [2O2 levels lacks specificity in ARDS due to the presence of variable levels of sample contaminants [1 [NO to more fully elucidate the nitrogen oxide chemistry of the lungs and airways [2O2, and LT B4 elevated significantly in patients after pulmonary lobectomy, but not after the milder insult associated with cardiac surgery [2O2 in EBC and BAL, significantly higher BAL protein levels, higher EBC phospholipid levels, and no different levels of keratin in EBC and BAL [ There is strong evidence that EBC composition abnormalities may reflect biochemical changes of airway lining fluid and potentially be used to measure the targets of modern therapy in COPD and asthma clinical trials . Recentl PC20Arc . EBC LT  PC20Arc  and NT l PC20Arc  and CF [ PC20Arc  patientsntilator . The speaminants . An elevaminants  as well aminants . Identifaminants . TNF-alpaminants . Higher aminants . EBC IL-nants [1 . Althougnants [1 . EBC can airways . EBC ass airways . A study surgery . This in surgery . ATS/ERS surgery . A study surgery . EBC rep surgery . Compari and BAL . These i and BAL . Choice  and BAL . There i and BAL . EBC has and BAL . Thus, E and BAL . Both te and BAL  and EBC  and BAL . Solubil and BAL . The imp and BAL , and the and BAL . A study demonstrated a fundamental flaw in the breath pentane assays: the column employed in the gas chromatography did not separate pentane from isoprene, the most abundant compound in the breath . What MeNO. The use of FENO measurements may minimize the potential long-term side effects of ICS [NO analyzers are approved by the United States Food and Drug Administration for assessing and monitoring therapy in asthmatic patients, particularly with eosinophilia and patients with airway inflammation [NO measurement can reduce approximately 40% of daily dose of inhaled glucocorticoids with similar efficacy and reduce costs [NO is only one surrogate biomarker of airway inflammation, but unlikely to ruminate the whole complexity of the inflammatory process in respiratory diseases and its multiple expressions within a given syndrome or disease [NO measurement in treatment decision costs less than asthma management based on standard guidelines and has similar health benefits [Cryptococcus neoformans is a pathogenic fungus that produces large amounts of urease which catalyzes the hydrolysis of urea to ammonia that can be detected in exhaled breath [Currently, there is relative lack of information about how theses biomarkers are related to their reproducibility, disease severity, outcomes of concurrent therapies, and other clinical outcomes. None of the exhaled markers are diagnostic for a particular pulmonary disease, apart from very low exhaled and nasal NO in PCD. Single exhaled markers are usually evaluated in isolation, but markers are affected differently in different diseases, and their sensitivity varies to certain techniques. For example, as indicated above, COPD is characterized by normal or little elevation in exhaled NO, and by greater elevation in exhaled 8-isoprostane and CO levels . By conts of ICS , 214. FE disease . The usebenefits . Althougbenefits . Elevatebenefits . Cryptocd breath . It is pd breath . 2O2 was the most studied substance on numerous inflammatory diseases as a marker of oxidative stress [In the past, He stress . Today, e stress . There ie stress . In the e stress  and may e stress . 14C-urea in Helicobacter pylori infection [Measurement of exhaled breath temperature and humidity is another method which may serve as a simple, non-specific, and inexpensive method for home monitoring of rhinitis, COPD, CF, and asthma including assessment of anti-inflammatory treatment effects . A studynfection , 227. A nfection  and to mnfection . Polymernfection . The selnfection . NO reference values in the general population must consider dietary habits, age, weight, height, gender, asthma, atopy, smoking habits, current nasal and respiratory symptoms, and steroid assumption. The effect of various factors and different subpopulations on healthy subjects is also reported, in an effort to delineate future directions that need the establishment of EBC reference values.  Measurements of exhaled hydrocarbon and other exhaled markers are much more difficult using current technology. EBC value will depend on availability of reliable, fast, and inexpensive detector systems and standardized collection that will overcome its high cost and its present highly variable measurements. This could have far-reaching potential for the diagnosis and treatment of various respiratory diseases. There is little doubt that “inflammometry” will be a major step forward and will be useful in differentiating airway diseases and improving treatment. In the future, it is likely that smaller and more sensitive analyzers will extend the discriminatory value of exhaled breath analysis and that these techniques may be available to diagnose and monitor respiratory diseases in the general practice and home setting. More detailed insights into inflammatory processes can be obtained when the volatile and serum markers are considered together. The evaluation of FE


Mycoplasma hominis is mostly known for causing urogenital infections. However, it has rarely been described as an agent of brain abscess.M. hominis brain abscess in a 41-year-old Caucasian woman following uterus curettage. The diagnosis was obtained by 16S rDNA amplification, cloning and sequencing from the abscess pus, and confirmed by a specifically designed real-time polymerase chain reaction assay.We describe a case of M. hominis should be considered as a potential agent of brain abscess, especially following uterine manipulation.Findings from our patient's case suggest that  Mycoplasma hominis has rarely been reported [M. hominis is a fastidious and slow-growing bacterium, commensal of the genitourinary tract of healthy adults. It mostly causes urogenital infections but may also cause extra-genital infections [Mycoplasma sp. require specific antibiotic treatment. Lacking a cell wall and folic acid synthesis, they are resistant to antibiotics that target the cell wall or folic acid synthesis [M. hominis is sensitive to antibiotics that prevent the synthesis of proteins, including tetracyclines [M. hominis from human samples [Brain abscess is a life-threatening condition resulting from the invasion of brain tissues by microorganisms. Current microbiological documentation, mostly based on direct examination and culture of pus specimens, may underestimate the role of fastidious microorganisms in brain abscess . Among treported -7. M. hofections ,9. Infecynthesis . In partynthesis . In contcyclines . In addi samples .M. hominis (GenBank accession number AF443616). As a consequence, the antibiotic treatment was changed to doxycycline, 200 mg/day for 12 weeks. Our patient recovered rapidly. On follow-up, she remained asymptomatic six months after the discontinuation of antibiotics. In order to determine whether the infection was monomicrobial or polymicrobial, the PCR amplicon was subsequently cloned into Escherichia coli using the pGEM-T Easy Vector System . A total of 100 clones were analyzed by sequencing. Only 16S rDNA from M. hominis was detected in the 100 clones. The identification of M. hominis in our patient and the previously published cases motivated the development of a specific real time-PCR (RT-PCR) assay for this bacterium. 16S rDNA was selected as target. Using the Primer Express software (Applied Biosystems), specific primers and probes were designed as follows: MHMGB16Sd (5'-TGT TAT AAG GGA AGA ACA TTT GCA AT-3'), MHMGB16Sr (5'-GCC ATC GCT TTC TGA CAA GG-3') and MHMGB16S probe (FAM-AAA-TGA-TTG-CAG-ACT-GAC-MGB) respectively. RT-PCR was performed using a LightCycler (Roche). The PCR mix consisted of 4 μL of pus DNA, 10 μL of Quantitect Probe PCR Master Mix , 20 pM of each primer , 0.5 μL of Uracil DNA glycosylase (Invitrogen), 0.5 μL of 3.125 μM MHMGB probe (Applera), and 4 μL of water. DNA was amplified using the following cycling parameters: heating at 50°C for 2 minutes, and then at 95°C for 15 minutes, followed by 50 cycles of a two-stage temperature profile of 95°C for one second and 60°C for 45 seconds. The specificity of the primers and probes was tested using BLAST http://blast.ncbi.nlm.nih.gov/ and by tentatively amplifying DNA from 24 distinct Mycoplasma species. The system was found to be specific to M. hominis, as no amplification was obtained from any other mycoplasmal or human DNA. For our patient, positive amplification was obtained after 22 PCR cycles. Negative controls remained negative.In 2006, a previously healthy, 41-year-old Caucasian pregnant woman was admitted to our hospital with vertigo, severe headache, and left hemiparesis. She had no relevant medical history except two previous normal pregnancies and deliveries. A computed tomography (CT) scan and MRI scan of the brain identified a right fronto-parietal hematoma. The hematoma was surgically drained. Then 10 days later, at 22 weeks of gestation, our patient underwent early spontaneous miscarriage that required uterus curettage, complicated by important metrorrhagia. At three days following the miscarriage, our patient developed obnubilation, and subsequently coma. New cerebral CT and MRI scans revealed a fronto-parietal brain abscess. The abscess was surgically removed, and purulent material was sent to our laboratory. A nosocomial infection being suspected, an intravenous empirical treatment associating vancomycin (2 g/day) and meropenem (6 g/day) was started. Gram staining of the abscess specimen showed numerous polymorphonuclear leukocytes but no microorganism. The specimen was then plated onto 5% sheep blood agar and chocolate agar  and incubated at 37°C under aerobic, anaerobic, and microaerophilic conditions for 10 days. Plates were examined daily but no growth was observed. For molecular detection, DNA was extracted from the pus sample using the MagNA Pure LC DNA isolation kit II and the MagNA Pure LC instrument as recommended by the manufacturer . Amplification and sequencing of the 16S rDNA gene were performed using broad range primers as previously described . By compM. hominis frequently colonizes the lower genitourinary tract of women [M. hominis has previously been reported in only six patients as a cause of brain abscess [M. hominis infection complicated a traumatic or spontaneous brain hematoma in a context of normal vaginal or cesarean delivery [M. hominis infection complicated a head trauma in the context of urinary tract catheterization [M. hominis was the genital tract whereas it was the urinary tract in men. The most recent patient, a three-week-old baby, most likely acquired the M. hominis infection from passage through the maternal birth canal [M. hominis superinfected a brain hematoma. By searching the literature for other cases of M. hominis infection of hematomas, we found six articles describing patients who had developed infection of abdominal, peri-nephric, thigh or retroperitoneal hematomas following genitourinary invasive procedures [M. hominis appears to have a particular ability for superinfecting hematomas, in particular following genitourinary tract invasive procedures.of women . Host prof women . To the  abscess -7 (Tabledelivery ,3,7. In rization ,5. In feth canal . In our ocedures -22 (Tablocedures . TherefoM. hominis was only detected by PCR. In addition, in an effort to reduce the diagnostic delay, we developed a specific RT-PCR for M. hominis. This test provides a rapid alternative not only to culture but also to broad-range 16S rRNA PCR and sequencing detection, and may enable rapid antibiotic treatment adaptation.In addition, as previously reported , bacteriM. hominis should be suspected in patients developing brain abscess following genitourinary tract invasive procedures, notably uterine curettage. To facilitate the detection of this agent, we developed an accurate, sensitive, and specific RT-PCR assay for M. hominis that may enable the diagnosis to be obtained within one hour of DNA extraction.Our data suggest that Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.MAM and PEF wrote the manuscript while MD performed the microbiological identification. HD performed the surgical treatment and revised the manuscript. DR corrected the manuscript. All authors read and approved the final manuscript.


S. pneumoniae,S. mitis, andS. oralis. AStreptococcus species can be defined as a group of strains that share ≥ 95% DNA similarity in MLSA and AAI, and > 70% DNA identity in GGD. This approach allows an advanced understanding of bacterial diversity.The identification of the clinically relevant viridans streptococci group, at species level, is still problematic. The aim of this study was to extract taxonomic information from the complete genome sequences of 67 streptococci, comprising 19 species, by means of genomic analyses, multilocus sequence analysis (MLSA), average amino acid identity (AAI), genomic signatures, genome-to-genome distances (GGD) and codon usage bias. We then attempted to determine the usefulness of these genomic tools for species identification in streptococci. Our results showed that MLSA, AAI and GGD analyses are robust markers to identify streptococci at the species level, for instance, In such a scenario, the identification of bacteria isolates to the species level is a hard task2.Bacteria are subjected to numerous forces driving their diversification. As a consequence, different strains of a single bacterial species sometimes have the ability to explore distinct niches, to be pathogenic or non-pathogenic and to present different metabolic pathwaysStreptococcus comprises 99 recognized species, many of which are associated with disease in humans and animals (http://www.bacterio.net/s/streptococcus.html). The viridans group streptococci (VGS) encompass four phylogenetic clusters: Mitis, Mutans, Salivarius and Anginosus, which are part of the human microbiota, being isolated mainly from the oral cavity, gastrointestinal and genitourinary tracts3. The Mitis group currently includes the important pathogenS. pneumoniae and 12 other recognized species,S. australis,S. cristatus (formerlyS. crista),S. gordonii,S. infantis,S.mitis,S. oligofermentans,S. oralis,S. parasanguinis (formerlyS. parasanguis),S. peroris,S. pseudopneumoniae,S. sanguinis (formerlyS. sanguis) andS. sinensis. The Anginosus group includes three recognized species,S. anginosus,S. constellatus (including two subspeciesS. constellatus subsp.constellatus andS. constellatus pharyngis) andS. intermedius, and the Salivarius group includesS. salivarius,S. vestibularis, andS. thermophilus.Currently, the genus5. Identification of streptococci is based on the current taxonomic standards using a combination of 16S rRNA gene sequence analyses, DNA-DNA hybridization, serologic and phenotypic data; however, they have been strikingly resistant to satisfactory classification, reflected in frequently changing nomenclature7. For instance, the 16S rRNA gene sequences ofS. mitis andS. oralis are almost identical (> 99%) toS. pneumoniae, making the use of this information alone insufficient to distinguish these species8.Currently, bacterial species are considered to be a group of strains (including the type strain) that are characterized by a certain degree of phenotypic consistency, showing > 70% DNA-DNA hybridization values and over 97% 16S rRNA sequence similarity14. In order to determine the robustness of genomic markers in streptococci species delineation, we analyzed a collection of 67 complete genomes. The availability of whole genome sequences of several closely related species, for instance,S. mitis - S. oralis - S. pneumoniae, andS. salivarius - S. thermophilus - S. vestibularis, formed an ideal test case for the establishment of the genomic taxonomy of streptococci.Recent studies have used whole genome analysis to determine the taxonomic relationships among bacterial speciesnd, 2011 at the National Center for Biotechnology Information (NCBI) under the project accession number indicated inet al. (2009)13 and are briefly described below.The genomic sequences of 67 streptococci that were publicly available for download by June 2http://www.ncbi.nlm.nih.gov). The MLSA approach was based on the concatenated sequences of five house-keeping genes 16. The concatenated sequences were aligned with ClustalX program17. The phylogenetic inference was based on the neighbour-joining genetic distance method (NJ)18 using MEGA519. Distance estimations were obtained according to the Kimura-2-parameter20 for 16S rRNA gene and MLSA. The reliability of each tree topology was checked by 2000 bootstrap replications21.The 16S rRNA gene sequences and the gene sequences used for MLSA were obtained from GenBank  from one genome were searched against the genomic sequence of the other genome. The genetic relatedness between a pair of genomes was measured by the AAI of all conserved genes between the two genomes as computed by the BLAST algorithm. By this approach, a value of < 95% AAI of protein-coding genes indicates separate species.The AAI of all conserved protein-coding genes was calculated as described previouslyNc)24 was calculated using CHIPS (http://emboss.bioinformatics.nl/cgi-bin/emboss/chips) with the default parameters.Codon usage bias was calculated for each genome. The effective number of codons used in a sequence (http://emboss.bioinformatics.nl/cgi-bin/emboss/compseq) with default parameters. Dinucleotide relative abundances (ρ*XY) were calculated using the equation ρ*XY = fXY/fXfY where fXY denotes the frequency of dinucleotide XY, and fX and fY denote the frequencies of X and Y, respectively. The difference in genome signature between two sequences is expressed by the genomic dissimilarity (δ*), which is the average absolute dinucleotide of relative abundance difference between two sequences, and were calculated using the equation: δ* = 1/16Σ|ρ*XY (f) - ρ*XY (g)| (multiplied by 1000 for convenience), where the sum extends over all dinucleotides25.Mononucleotide and dinucleotide frequencies were calculated using COMPSEQ  for inferring intergenomic distances for species delimitation. The corresponding distance threshold can be used for species delimitation26.The genome distance was calculated using genome-to-genome distance calculator (GGDC)9. Overall our analysis, using a large data set, showed that genomic taxonomy is an accurate approach to clearly define the streptococci species. The taxonomic resolution of the 16S rRNA, AAI, MLSA, GGD and codon usage analysis for streptococci species definition is summarized inIn this work we compared complete genomes for 67 streptococci comprising 19 species to address their taxonomic position. A previous study with a small set of streptococci genomes (eight) and species (four), using a combination of several genomic analyses, showed the applicability of this approach in streptococci taxonomySimilarity values for the MLSA nucleotide sequences (lower left) and 16S rRNA gene sequences (upper right) for the Streptococcus strains.Click here for additional data file.Percentage of average amino acid identity (AAI) between Streptococcus strains.Click here for additional data file.Genomic dissimilarity [δ] values between Streptococcus strains.Click here for additional data file.S. infantis) to 2.3 Mb (S. sanguinis). The number of CDS varied from 1,700 (S. pyogenes) to 2,352 (S. pneumoniae)Streptococcus. One of the reasons for this variability could be associated with the frequent occurrence of horizontal gene transfer events29.The complete genome of the streptococci comprised a single chromosome. The estimated size of the genomes ranged from 1.7 Mb (http://spneumoniae.mlst.net/)16. Three genes,aroE,ddl andgki, are from the MLST scheme, andpheS andrecA were included in this work. The concatenation of these genes (7741 bp) allowed an accurate delineation of the streptococci species considered here. The nucleotide sequence similarities were much lower for MLSA than 16S rRNA gene. A pairwise comparison of MLSA among the species revealed sequence similarity between 67% and 100%, while the 16S rRNA gene sequence similarities varied from 92% to 100%. At the intraspecies level, the similarity values ranged from 95% to 100% for MLSA, and 99% to 100% for the 16S rRNA gene sequences. The closest species within the Mitis  and Salivarius groups  were clearly placed apart from each other by MLSA, while these species had almost identical 16S rRNA gene sequences (≥ 99% sequence similarity). A previously study showed thatrecA analysis is a valuable tool for proper identification of pneumococci in routine diagnostics, but limitations on discrimination of other members of the Mitis group were observed30.S. sanguinis ATCC 49296 showed a much closer relationship withS. oralis ATCC 35037T (95% similarity) than to otherS. sanguinis strains (77% similarity), suggesting it belongs to the speciesS. oralis. In addition,S. bovis ATCC 700338 was placed in theS. gallolyticus cluster with 98% MLSA sequence similarity. This work showed that MLSA, using this new combination of five concatenated genes , distinct from theStreptococcus MLST scheme, allowed a proper identification of most streptococci species, even within the VGS group.MLSA and 16S rRNA phylogenetic trees showed similar topologiesS. pneumoniae,S. mitis andS. oralis shared 89–93% AAI. The speciesS. salivarius,S. thermophilus andS. vestibularis showed a maximum AAI of 93%.S. sanguinis ATCC 49296 andS. oralis ATCC 35037 showed 96% identity andS. bovis ATCC 700338 andS. gallolyticus strains had 98% identity. These findings suggest that strains ATCC 49296 and ATCC 700338 belong to the speciesS. oralis andS. gallolyticus, respectively. According to our analyses the AAI and MLSA are the most useful genomic features for the elucidation of streptococci taxonomy.The percentage of average amino acid identity (AAI) among streptococci species ranges from 68% to 94%, while within species it varies from 95% to 100%. The VGS speciesS. salivarius,S. thermophilus andS. vestibularis species, showed dissimilarity values between 5 and 12 andS. pneumoniae, S. mitis andS. oralis species had dissimilarity values between 3 and 14. Thus, there was not a clear differentiation of these closely related species within the VGS group on the basis of the genomic dissimilarity values. This could be due to the extensive recombination and horizontal gene transfer events which occur between closely related streptococci species that share ecological niches30.The genomic dissimilarity values among streptococci were between 3 and 127, while the intraspecies values were between 0 and 17. Streptococci within the VGS group, for instance,33.On the other hand, species within the Pyogenic group had a distinct genomic signature, with values ranging from 13 to 85. However, genome signatures alone have significant limitations when used as phylogenetic markers for differentiating members of the VGS. The exact mechanisms that generate and maintain the genome signatures are complex, but possibly involve differences in species-specific compositional bias, i.e., G+C content, G+C and A+T skews, codon bias, and mutation biasNc values provide a meaningful measure of the extent of codon preference in a genome, values range between 20 (extremely biased genome where one codon is used per amino acid) and 61 . Within the set of 67 complete streptococci genomes examined in this study, theNc ranged from 44.0 to 54.5S. pneumoniae -S. oralis -S. mitis species hadNc values of 50, 51 and 50, respectively. The Salivarius group , andS. bovis ATCC 700338- S. gallolyticus showedNc values of 47 and 44.5, respectively. Overall, codon usage bias was very similar among the streptococci species investigated. However,S. sanguinis ATCC 49296 showed a much closerNc value with theS. oralis ATCC 35037  than otherS. sanguinis strains (54.5), which was in agreement with the other analyses used in this study.S. bovis ATCC 700338 andS. gallolyticus were identified as belonging to the same species by GGD.The GGD was calculated only for closely related species that were not differentiated by 16S rRNA gene sequence analysisS. bovis ATCC 700338 (biotype II) andS. gallolyticus as well asS. sanguinis ATCC 49296 andS. oralis ATCC 35037T were not separated and, therefore, according to this analysis would be classified as the same species, respectively. It was shown thatS. bovis biotype I and II/2 isolates were, in fact,S. gallolyticus34, andS. sanguinis ATCC 49296 was placed intoS. oralis species by GGD analysis. A misidentification ofS. sanguinis ATCC 49296 has already been shown by means of biochemical and serological properties by Narikawa and colleagues35.S. parasanguinis ATCC 15912 and F0405 strains were found to be at the upper limits for definition as members of the same species based on different genomic analyses. For instance, they shared 95% AAI, 94% identity by MLSA, a value of 17 on the basis of genomic signature and < 70% similarity in GGD. Therefore, based on these genomic markers, theseS. parasanguinis strains could, in fact, be separate species. This data reflects the complexity of bacterial species delineation, since these organisms are all under a constant evolutionary process.Another interesting result is that theNc values and genomic signatures. Based on the genomic analyses, aStreptococcus species can be defined as a group of strains that shares ≥ 95% identity in MLSA and AAI, and > 70% identity in GGD. This definition may be useful to advance the taxonomy ofStreptococcus. This approach allows an advanced understanding of bacterial diversity and identification.The delineation of closely related streptococci species was evident in this genomic study. Different methods produced different levels of taxonomic resolution. The methods with the higher resolution for species identification were MLSA and AAI, while closely related species had similar et al. 2006Rosello-Mora, would certainly improve the manuscript. The online analysis can be found here http://www.imedea.uib.es/jspecies/index.html.The conclusions are adequate and the data sufficient to replicate all the analyses.The data are openly accessible at GenBank.The article is well written with an appropriate title and abstract. The methods are adequate for the aims of the study, but I would suggest that including the Average Nucleotide Identity (ANI) analysis as suggested byI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Title and abstract are good enough to attract readers in the scientific community.Streptococcus, the authors carefully designed massive data genome analysis. The results are strong enough and supported by the results of the analysis.Genome based multi-gene sequence comparison is one of the promising tools to analyse bacterial populations. To achieve the analysis forStreptococcus bacteria.The conclusion is clear that authors proposed a threshold value on the basis of genome-based MLSA inI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Rickettsia parkeri in South America has been associated with Amblyomma triste ticks. The present study evaluated under laboratory conditions two colonies of A. triste: one started from engorged females that were naturally infected by R. parkeri (designated as infected group); the other started from noninfected females (designated as control group). Both colonies were reared in parallel for five consecutive generations. Tick-naïve domestic rabbits were used for feeding of each tick stage and generation. R. parkeri was preserved by transstadial maintenance and transovarial transmission in A. triste ticks for five consecutive generations, because all tested larvae, nymphs, and adults from the infected group were shown by PCR to contain rickettsial DNA. All rabbits infested by larvae, nymphs, and adults from the infected group seroconverted, indicating that these tick stages were all vector competent for R. parkeri. Expressive differences in mortality rates were observed between engorged nymphs from the infected and control groups, as indicated by 65.9% and 92.4% molting success, respectively. Our results indicate that A. triste can act as a natural reservoir for R. parkeri. However, due to deleterious effect caused by R. parkeri on engorged nymphs, amplifier vertebrate hosts might be necessary for natural long-term maintenance of R. parkeri in A. triste.The distribution of  Rickettsia parkeri was firstly reported infecting the Amblyomma maculatum tick in Texas >70 years ago [R. parkeri was recognized as a human tick-borne pathogen only in 2004 [R. parkeri was reported infecting Amblyomma triste ticks in Uruguay [R. parkeri-infected A. triste ticks were reported in Brazil [A. maculatum ticks were found infected by R. parkeri in Peru [R. parkeri rickettsiosis have been recently described in the United States, all associated with A. maculatum ticks [A. triste ticks [R. parkeri have been reported [R. parkeri sensu stricto have been reported in the country, despite the presence of the agent infecting A. triste ticks [The bacterium ears ago . Althougears ago , R. park in 2004 . At thatn Brazil  and Argen Brazil . More reum ticks –10, in Ste ticks , 12. In reported , 14; howte ticks . R. parkeri sensu stricto has been generally associated with A. maculatum in North America and A. triste ticks in South America, where usually ≈10% of field collected adult ticks have been found infected by R. parkeri [R. parkeri, which is the original strain first described by Parker in 1939. Therefore, it is possible that a single R. parkeri genotype  circulates among A. maculatum and A. triste populations in the New World, where both tick species use medium- to large-sized mammals as hosts for adult ticks, and chiefly small mammals and ground feeding birds for immature tick stages [R. parkeri rickettsiosis is still very incipient, the present study evaluated for the first time (i) the occurrence of transovarial transmission  and transstadial transmission (maintenance of the infection during tick ontogeny) of the agent in one of its natural vectors, (ii) if the infection with R. parkeri causes any deleterious effect on its tick host, and (iii) the vector competence of all feeding stages of A. triste.Reported information has indicated that the distribution of  parkeri , 10, 15.k stages –18. BecaA. triste adult ticks  were collected from a road-killed marsh deer (Blastocerus dichotomus) in the Km 177 of the BR 262 highway , within Agua Clara Municipality, state of Mato Grosso do Sul, Brazil. Ticks were brought alive to the laboratory, where female engorgement was completed by feeding on a tick-naïve uninfected New Zealand white rabbit (Oryctolagus cuniculus). A total of 14 females completed engorgement and were allowed to oviposit inside an incubator adjusted to 25°C and 90% relative humidity. Small samples of eggs  and the subsequent hatched larvae  derived from each of the 14 engorged females were subjected to DNA extraction and subsequently tested by PCR using primers CS-78 and CS-323, which target a 401 bp fragment of the citrate synthase gene (gltA) of possibly all Rickettsia species [R. parkeri previously reported in A. triste from Brazil, Uruguay, and Argentina [1) from two infected females were pooled to start the infected group of the present study. At the same time, the larval offspring from two PCR-negative noninfected females were pooled to start the control group. In September 1 2008, 1 unfed larvae of infected and control groups were reared separately in the laboratory for 5 consecutive generations until they were F5 unfed nymphs. Throughout the experiment, infestations with infected and control groups were done in parallel; all infested animals were held in the same room under the same environmental conditions. New Zealand white rabbits were infested with larvae, nymphs, and adult ticks, as previously described for A. triste [≈3,000 and 500 ticks, respectively, per host, whereas adult infestations consisted of 10 to 25 couples per rabbit. Infestation of each animal was performed inside a feeding chamber glued to its shaved dorsum, as previously described [The F. triste . In eachescribed . All infescribed . Naturalescribed , was det1 through F5, and 10 unfed adults of generations F1–F4 from both infected and control groups, were individually submitted to DNA extraction by using guanidine thiocyanate, as previously described [gltA gene [μL of water) and positive (5 μL of DNA extracted from R. parkeri strain NOD-infected cells) controls were included.During the experiment, random samples of 30 unfed larvae and 20 unfed nymphs of each of the generations Fescribed . A higheltA gene . PCR conR. parkeri antigens. For this purpose, blood samples were collected at 0 and 21 days after infestation; these samples were tested for anti-R. parkeri (strain At24) reactive antibodies by immunofluorescence assay (IFA), as previously described [All tick-infested rabbits were tested for seroconversion to escribed . Briefly3 and four F4 unfed adult ticks were processed by the shell vial technique for isolation of rickettsiae in Vero cell culture as described by Marrero and Raoult [gltA gene, as described above for ticks, and another protocol using primers Rr190.70p and Rr190.701n, targeting a 631 bp fragment of the rickettsial 190 kDa outer membrane protein gene (ompA), as previously described [http://blast.ncbi.nlm.nih.gov/Blast.cgi).Four Fd Raoult  and modid Raoult . Brieflyd Raoult . Third pescribed . PCR pro5 larval and F1–F4 nymphal molting success and F1–F4 female oviposition success  were compared by the chi-square test. In addition, weight of engorged females and their corresponding egg masses, percentage of egg hatching, and CEI values were compared by Student t test. Values were considered significantly different when P < 0.05. The study was approved by the Bioethical Committee in Animal Research of the Faculty of Veterinary Medicine of the University of Sao Paulo.During the experiment, tick biologic parameters were compared between infected and control groups. For this purpose, F5 unfed nymphs). In all infestations performed with ticks from this group, rabbits seroconverted to R. parkeri antigens with endpoint titers varying from 256 to 1024 (R. parkeri (serum dilution 1 : 64) at both 0 and 21 days after infestation. All rabbits from both infected and control groups remained afebrile during the study period.The infected tick group remained infected by rickettsiae through 5 consecutive generations, until the end of the experiment . These five isolates, designated as Agua Clara 1–5, have been deposited in the Rickettsial Collection of the Faculty of Veterinary Medicine of the University of São Paulo. All PCRs performed individually on unfed ticks  from five consecutive generations of the infected group resulted in amplicons compatible with  parkeri . In contP = 0.164).Significantly more control group-engorged nymphs successfully molted to adults than the engorged nymphs of the infected group ; that is1 and F2 females, which were significantly higher in the control group. However, when % egg hatching of the four generations was pooled, there was no significant difference between the groups. Reproductive data of engorged females were generally similar between control and infected groups . The onlR. parkeri was preserved by transstadial maintenance and transovarial transmission in A. triste ticks for 5 consecutive generations, because all tested larvae, nymphs, and adults from the infected group were shown by PCR to contain rickettsial DNA, from the first to the fifth generation. In addition, infestations of rabbits with larvae, nymphs, and adults from all generations confirmed that ticks of these 3 developmental stages were infected by R. parkeri because all infested rabbits seroconverted through IFA. These results also confirm that larvae, nymphs, and adults of A. triste ticks are competent vectors of R. parkeri. Our results strongly support clinical and epidemiologic data that have implicated the A. triste tick as the main vector of R. parkeri sensu stricto in South America [In the study reported here,  America , 12, 30.R. parkeri. Because all individual larvae of the infected group yielded rickettsial DNA by PCR, 100% transovarial transmission rate  and 100% filial infection rate  were observed. In contrast, no individual larva from the control group yielded rickettsial DNA by PCR. These results are in agreement with the larval infestations, which resulted in seroconversion in all rabbits infested by larvae from the infected group and with no seroconversion in animals infested with control group larvae. Although transovarial transmission of spotted fever rickettsiae in ticks seems to occur worldwide , such inR. parkeri-infected engorged nymphs was nearly 5 times higher than the mortality of uninfected nymphs. Similar to the present study, expressive mortality rates were reported for Rickettsia rickettsii-infected engorged nymphs of Dermacentor andersoni, and Rickettsia conorii-infected Rhipicephalus sanguineus nymphs, when compared with uninfected sibling ticks [R. parkeri-infected engorged larvae was observed, when compared to noninfected engorged larvae of the fifth generation. Unfortunately, we did not quantify larval molting success for generations F1–F4, precluding an overall comparison of data from the five larval generations.Expressive differences in mortality rates were observed between engorged nymphs from the infected and control groups, as indicated by 65.9% and 92.4% molting success, respectively. Overall, these rates mean that the mortality of ng ticks –34. ThesR. parkeri-infected and uninfected engorged females had overall similar survivorship and reproductive performance. These results contrast previous studies that reported higher mortality and lower reproductive performance of R. rickettsii-infected engorged females of Dermacentor variabilis, D. andersoni, and Amblyomma aureolatum ticks, when compared with uninfected sibling ticks [R. rickettsii-infection rates (<1%) usually reported among ticks in nature are related to the expressive mortality of the infected engorged females. On the other hand, the much higher R. parkeri-infection rates  generally reported for A. triste under natural conditions [On the other hand, our results demonstrated that both ng ticks , 36. Ticnditions –6 could R. parkeri were reared for five consecutive generations in the laboratory. The infection by R. parkeri was successfully maintained in the tick population by transstadial maintenance and transovarial transmission. These results indicate that R. parkeri could be maintained by A. triste in nature over years; therefore, A. triste can act as a natural reservoir of R. parkeri. However, because of a notable deleterious effect caused by R. parkeri on engorged nymphs, infected ticks would disappear from the tick population in a long-term scenario, unless new lineages of infected ticks are created through horizontal transmission via amplifier vertebrate hosts, which remain unknown for R. parkeri. Ticks naturally infected by


Paenibacillus vortex is notable for its advanced social behavior, which is reflected in development of colonies with highly intricate architectures. Prior to this study, only two other Paenibacillus species (Paenibacillus sp. JDR-2 and Paenibacillus larvae) have been sequenced. However, no genomic data is available on the Paenibacillus species with pattern-forming and complex social motility. Here we report the de novo genome sequence of this Gram-positive, soil-dwelling, sporulating bacterium.The pattern-forming bacterium P. vortex genome was sequenced by a hybrid approach using 454 Life Sciences and Illumina, achieving a total of 289× coverage, with 99.8% sequence identity between the two methods. The sequencing results were validated using a custom designed Agilent microarray expression chip which represented the coding and the non-coding regions. Analysis of the P. vortex genome revealed 6,437 open reading frames (ORFs) and 73 non-coding RNA genes. Comparative genomic analysis with 500 complete bacterial genomes revealed exceptionally high number of two-component system (TCS) genes, transcription factors (TFs), transport and defense related genes. Additionally, we have identified genes involved in the production of antimicrobial compounds and extracellular degrading enzymes.The complete P. vortex has advanced faculties to perceive and react to a wide range of signaling molecules and environmental conditions, which could be associated with its ability to reconfigure and replicate complex colony architectures. Additionally, P. vortex is likely to serve as a rich source of genes important for agricultural, medical and industrial applications and it has the potential to advance the study of social microbiology within Gram-positive bacteria.These findings suggest that  Paenibacillus vortex strain V453 . (GS 20)  and Illu (GS 20) . Using tP. vortex assembly is in several contigs, it provides complete genome coverage and with an extremely high accuracy  with an average G+C content of 48.7% , 32 local inverted repeats and 231 tandem repeats within the Repetitive sequences are the major reason for the difficulty we encountered in finishing the genome assembly into a complete sequence. Analysis of the scaffold ends (100 bp of each end) revealed that 78% of them have repetitive sequences that are on average 37 bp long and could be mapped on average onto 5 different scaffold ends.P. vortex genome have an extremely high coverage  of the total designed probes and no missed regions were found  and a subset of 50 soil bacteria genomes within this group , 97 histidine-kinases (HKs) and 10 hybrid kinases. The number of TCS genes was linear with genome size in agreement with .The DNA sequence was run through JCVI's prokaryotic annotation pipeline (JCVI Annotation Service), which includes gene finding by Glimmer, Blast-extend-repraze (BER) searches, HMM searches, TMHMM searches, SignalP predictions, and automatic annotations from AutoAnnotate. Additionally, the DNA sequence was annotated using NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) and the combined annotation was submitted to [GenBank: ftp://ftp.ddbj.nig.ac.jp/ddbj_database/16S/. The alignment of the chosen sequences was performed using ClustaX [The construction of the phylogenetic tree of 22 taxa was based on 16 S rRNA sequences downloaded in fasta format from DNA Data Bank of Japan (DDBJ)  ClustaX  and the  ClustaX . The per ClustaX . The evo ClustaX  utilizin ClustaX .http://pfam.sanger.ac.uk/[The approach used to identify putative TCS and TF genes utilized HMM (Hidden Markov Model) profiles found in Pfam database of protein families er.ac.uk/. TCS gener.ac.uk/ and [103er.ac.uk/. The comTo identify putative transport and defense related genes we utilized Cluster of Orthologous Groups (COG) profiles ,43. The The combined score was calculated as an average of the standard deviation (stdev) of two-component system, transcription factor, transport and defense genes for the dataset of 500 bacterial genomes. The combined score was calculated both as normalized and non-normalized to genome size.V. dahliae was grown on trypsin soy agar plates (TSA), at 28°C. A 10 day old stock plate was used to initiate the experiments as follows: A startup slice of 0.5 mm diameter was cut from the colony edge and placed on a fresh TSA plate. The fungal slice was positioned 1 cm away from the center of a 9 mm Petri dish. Plates were incubated untill V. dahliae colonies reached 1.5 cm diameter (6 days incubation). At this time-point an overnight P. vortex culture, grown in LB, 28°C, with shaking (200 rpm), was inoculated in a 6 cm long line, horizontal to V. dahliae. P. vortex was positioned 2.5 cm away from the V. dahliae colony center. V. dahliae colonies without the inoculation of P. vortex served as control. All tests were carried out in triplicate.P. vortex sp. nov. V453 was deposited at the Bacillus Genetic Stock Center (BGSC), Columbus, OH, USA, as strain 31A2T and at the Belgium Coordinated Collection of Microorganisms (BCCM/LMG) as strain LMG 25955.Isolate ORF: Open Reading Frames; TCS: Two-Component System; TF: Transcription Factor; COG: Cluster of Orthologous Groups; GS: Genome Sequencer; GA: Genome Analyzer; HR: Homologous Recombination; RR: Response Regulator; HK: Histidine Kinase; NRPS: Non-Ribosomal Peptide Synthetase; PKS: Polyketide Synthase; PBR: Plant Beneficial Rhizobacteria; HMM: Hidden Markov Model;The authors declare that they have no competing interests.ASM, TO, YH, CI, DLG, DL and EBJ were involved in study design. ASM, TO, YH, CI, IB, DR, EH, RCM, DL, EBJ performed the experiments. IB, RCM and SBZ contributed reagents, materials and analysis tools. ASM, TO, YH, CI, LB, DLE, VG, VK, RCM, DL, EBJ were involved in data analysis. ASM, TO, YH, CI, EBJ wrote the paper. All authors read and approved the final manuscript.Detailed supplementary information. This file includes additional information on P. vortex physiology, comparison of sequencing methods, validation of P. vortex annotation and materials and methods.Click here for fileMovement of a single vortex, 500× magnification and twice the real speed.Click here for fileEarly stage of colony organization including the formation of vortices and moving groups of bacteria. The magnification is 50× magnification and 60× rate.Click here for fileDynamic imaging of swarming by light microscopy. Single branch of a swarming culture moving on MH (1.5% w/v) agar is presented. Magnification 400× and 4 times the actual speed.Click here for fileP. vortex swarmingEffect of extracellular material derived from plates containing swarming cells on . Light microscopy of P. vortex moving on MH agar (0.3% w/v), extending into an area where extracellular material derived from washes of swarming cells was delivered by toothpick and allowed to soak into the agar. (A) Cell mass starts to disperse as it approaches the area of the extract. (B) Cell mass has dispersed into area of extract.Click here for fileP. vortex swarmingEffect of number of extracellular materials derived from plates containing swarming cells on . Light microscopy of P. vortex moving on MH agar (0.3% w/v), extending into an area where extracellular material derived from washes of swarming cells. (A) Cell mass is evaluating the gradient and starts move towards the area with the extract. (B) Cell mass starts to disperse as it approaches the area of the extract. (C) Additional cells are moving into this area from further back in the colony.Click here for fileP. vortex genomeA set of 500 complete bacterial genomes of 2-10 Mbp genome size, which were used in the detailed comparative genomic analysis with the .Click here for fileP. vortex genome size), that were used in the comparative genome analysisA subset of 50 soil bacterial genomes with genome size 4-8 Mbp (closer to the .Click here for fileP. vortex genome that are responsible for cell division, engulfment, cortex and coat synthesis, maturation and germination processesList of 153 sporulation genes encoded by the .Click here for fileP. vortexList of conserved PFAM domains, TIGR domains, COG categories or KEGG pathways associated with sporulation that were used in identification of sporularion genes in .Click here for fileA compiled list of Pfam domains that was used to identify Two-Component System genes is presented.Click here for fileA compiled list of Pfam domains that was used to identify Transcription Factor genes is presented.Click here for fileA compiled list of COG categories that was used to identify transport related genes is presented.Click here for fileA compiled list of COG categories that was used to identify defense related genes is presented.Click here for file


Leishmania tropica is a genetically divergent species. Amplification of entire internal transcribed spacer (ITS) region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis (ACL) in Iran, revealed a double-band pattern in agarose gel electrophoresis. This study explains how this pattern occurs.L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new reverse primer namely LITS-MG was designed to exclude this gap in PCR products.Twenty seven L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was corresponding to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG primer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis.Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles.Sequences analysis of GenBank  Leishmania genus, some are causative agents of several forms of leishmaniasis and others may induce special clinical forms of the disease , and new foci in south Sahara (Kenya and Namibia) to Middle East . The east border is restricted to Punjab and Rajasthan as north western states of India  focus .There are different methods and markers for discriminating cificity , 7. Kinecificity , 9, ITS cificity , 11, rancificity , amplificificity , and mulcificity , 15 have strains –19.In a L) focus , 21 in KL) focus . ElectroThe present study aims to explain how this two-band pattern occurs. The results of the study may help researchers to elucidate the delicate angles of molecular epidemiology of leishmaniasis.L. major were obtained from Shiraz, center of Fars province, southwest Iran. According to microscopy examination, 27 out of 40 samples from Bam and 13 out of 16 samples from Shiraz were finally selected for following molecular survey. One bone marrow aspirate loaded with leishman bodies was also taken from a patient in Shiraz Shahid Faghihi Hospital. Moreover, one reference strain of L. major (MRHO/IR/75/ER) and a previously defined L. infantum (MCAN/IR/07/ Moheb-gh) preserved in RPMI 1640 culture media were kindly provided by Prof. Gholamreza Hatam (5th author) from Shiraz University of Medical Sciences, Shiraz, Iran.Forty stained smear preparations from clinical samples were collected from typical CL patients who were referred to the Bam Cutaneous Leishmaniasis Control Center. Furthermore, 16 samples from patients suspected to be infected with Skin tissue on smears was scratched off and collected in 1.5 ml microtubes containing lysis buffer  and 10 µg /ml proteinase K was added and incubated at 56°C for one hour. The samples were extracted once with phenol/chloroform (25:24 v/v) and subsequently extracted once with chloroform. DNA was precipitated with equal volumes of isopropanol, washed with 70% ethanol, dried, and suspended in 50 µl ultrapure water and stored at -20 °C till use.For species identification, kDNA of all the samples were amplified by the primers 13Z (5’- ACT GGG GGT TGG GTG TAA AAT AG-3’) and LiR (5’- TCG CAG AAC GCC CCT -3’)  for specThe entire ITS1-5.8S-ITS2 in rDNA was amplified by LITSV (5- ACA CTC AGG TCT GTA AAC -3) and LITSR (5- CTG GAT CAT TTT CCG ATG -3) primers . The cycL. tropica, L. major, and L. infantum already deposited in GenBank were analyzed by Geneious Basic 5.5.6 software (ITS-sequences of software  and DNASL. tropica isolates were excised from the gel and purified by Bioneer gel purification kit (Cat.No.K-3035) according to the manufacturer's instruction. The yielded products were submitted to Bioneer Company (Korea) and subjected to sequencing by Applied Biosystem automated sequencer (3730 XL). Sequences analysis result was shown through Bio Edit software version 7.0.5.3  and five out of 13 samples were confirmed as L. tropica. In addition, amastigotes observed in bone marrow aspiration obtained from the single kala-azar sample taken from a hospitalized patient were identified as L. infantum ( 680 bp band size).The 27 smear samples collected from the patients in Bam district showed a 750 bp size band in agarose gel electrophoresis of kDNA amplification indicating that all were L. tropica isolates, while all L. major and L. infantum isolates had a sharp single band. L. infantum band size was similar to the larger band of L. tropica. Besides, the size of the single band of L. major species was about 1100 bp (L. major and with the size of 1040 bp for L. infantum (data not shown); however, analysis of L. tropica entire ITS sequences revealed two groups of sequences based on length size, the first group with the size of 950 bp and the second having fragments with the size of 1050 bp  in comparison to the second group (1050 bp length size)  .L. infantum nor L. major. Amplification of the same DNAs with the newly-designed primer, LITS-MG as the reverse primer against the forward primer LITSR, resulted in single band products (ITSMG amplicon) with about 800 bp band size  just before the gap resulted in expectedly single band products which reconfirmed our finding. Use of newly designed primer and amplification of ITS fragment resulted in one strong sharp band which is appropriate for following nucleotide sequencing or RFLP. Moreover, in comparison to studying ITS1 and ITS2 in apart runs, ITSMG amplicon sequences may show more variation due to covering larger fragments. This is also more economical due to the substitution of one step PCR rather than two apart PCR runs. In our study, the results of sequences analysis of ITSMG amplicon PCR products revealed the homogeneity of L. tropica sequences in Bam district, however, considering the low number of samples, this hypothesis should be checked in a larger analysis. This double-band pattern for whole ITS may also be applied in case of L. tropica in order to discriminate L. tropica from L. infantum or L. major in defined regions such as Bam districts..In this study, we focused on the double band electrophoretic pattern L. tropica ITS. Result showed existence of at least two alleles for ITS in ribosomal DNA. Mauricio et al.  with intermediate isoenzymatic pattern in Saudi Arabia (L. braziliensis and L. panamensis cause CL in Nicaragua (L. tropica(L. donovani (MON1 and non MON1) in Tunisia (L. donovani complex (L. tropica(Trypanosoma, another genus of Trypanosomatidae family (Leishmania, we emphasize that our evidence is only a clue and further studies are required for elucidation and verification of genetic basis of sequence alteration responsible for this pattern of ITS.Existence of different ITS electrophoretic patterns and difference in related alleles due to a 100 bp gap in one allele may propose probable recombination in i Arabia , 27 or aicaragua . Recombi tropica or hybri Tunisia . Another complex .Signific tropica and fusi tropica or sandf tropica are the e family –38. Impoe family . OverallL. tropica from L. infantum and L. major and can be used for confirmation of L. tropica species. Moreover, this evidence may empower findings of other researchers about probable recombination of Leishmania especially L. tropica. Using newly designed LITSMG primer a sharp single band was obtained which was suitable for following sequence analysis.Concomitant existence of at least two alleles with a 100 bp fragment difference within ITS sequence explained the entire ITS double band electrophoretic pattern. This whole ITS double band pattern may differentiate


A commentary onSearch-related suppression of hippocampus and default network activity during associative memory retrievalby Reas, E. T., Gimbel, S. I., Hales, J. B., and Brewer, J. B. (2011). Front. Hum. Neurosci. 5:112. doi: 10.3389/fnhum.2011.00112Recent work using an analysis technique employed in this study has led us to realize that a section of our results should be restated. The following revised paragraph should replace the originally published paragraph on page 7 of our article, under the heading “Activity Correlations with Response Time.”p < 0.05) during both the classify and the recall tasks in bilateral superior temporal cortex and PCC. During only the recall, but not the classify task, negative correlations with response time were additionally observed in bilateral anterior hippocampus, medial PFC, and inferior parietal cortex. Thus, greater activity in these regions was correlated with a faster response time.To examine a possible relationship between response fluency and hippocampal and default network suppression, correlations between BOLD signal and response time were computed. Activity was negatively correlated with response time (


Streptococcus gallolyticus subsp. gallolyticus is an important causative agent of infectious endocarditis, while the pathogenicity of this species is widely unclear. To gain insight into the pathomechanisms and the underlying genetic elements for lateral gene transfer, we sequenced the entire genome of this pathogen.S. gallolyticus subsp. gallolyticus strain ATCC BAA-2069, consisting of a 2,356,444 bp circular DNA molecule with a G+C-content of 37.65% and a novel 20,765 bp plasmid designated as pSGG1. Bioinformatic analysis predicted 2,309 ORFs and the presence of 80 tRNAs and 21 rRNAs in the chromosome. Furthermore, 21 ORFs were detected on the plasmid pSGG1, including tetracycline resistance genes telL and tet(O/W/32/O). Screening of 41 S. gallolyticus subsp. gallolyticus isolates revealed one plasmid (pSGG2) homologous to pSGG1. We further predicted 21 surface proteins containing the cell wall-sorting motif LPxTG, which were shown to play a functional role in the adhesion of bacteria to host cells. In addition, we performed a whole genome comparison to the recently sequenced S. gallolyticus subsp. gallolyticus strain UCN34, revealing significant differences.We sequenced the whole genome of S. gallolyticus subsp. gallolyticus promotes understanding of genetic factors concerning the pathogenesis and adhesion to ECM of this pathogen. For the first time we detected the presence of the mobilizable pSGG1 plasmid, which may play a functional role in lateral gene transfer and promote a selective advantage due to a tetracycline resistance.The analysis of the whole genome sequence of  Streptococcus gallolyticus subsp. gallolyticus (formerly known as S. bovis biotype I) is a gram-positive bacterium belonging to the Lancefield Group D streptococci. Over the last ten years, the classification of S. gallolyticus subsp. gallolyticus has been revised several times [S. bovis was previously divided into three biotypes, designated as biotype I, biotype II/1, and biotype II/2. The majority of isolates associated with human endocarditis have been assigned to biotype I, which was recently reclassified as Streptococcus gallolyticus subsp. gallolyticus [S. gallolyticus subsp. gallolyticus is a common member of the microflora and appears in approximately 2.5 to 15% of the gastrointestinal tract of healthy human [et al. estimated that S. gallolyticus subsp. gallolyticus is the causative agent in 24% of streptococcal endocarditis cases [S. gallolyticus subsp. gallolyticus infection [et al. suggest that premalignant and malignant lesions in the intestinal tract could facilitate translocation of S. gallolyticus subsp. gallolyticus through the disrupted mucosal barrier and provide access to blood circulation [S. bovis and colon carcinogenesis [S. gallolyticus subsp. gallolyticus [S. gallolyticus subsp. gallolyticus or S. bovis infections remain unclear.al times -4. S. boolyticus . Furtherhy human ,7. It ishy human -10. Receis cases . In addinfection , while tculation . Furtherogenesis . In addiolyticus . HoweverS. gallolyticus subsp. gallolyticus shares its environment with numerous other potentially pathogenic bacteria, such as S. agalactiae, Enterococcus faecalis or others. This implies the possibility of horizontal gene transfer of antimicrobial resistance genes or genomic islands, e.g. phage-related clusters, by transposons, plasmids or phages, within the human gut or the animal rumen [S. bovis [S. gallolyticus subsp. gallolyticus [al rumen . SeveralS. bovis . These fS. bovis , in partS. bovis . Recentlolyticus . Nevertholyticus ,24.In vitro studies have demonstrated the adhesion and invasion of S. gallolyticus subsp. gallolyticus to extracellular matrix proteins [S. gallolyticus subsp. gallolyticus [S. gallolyticus subsp. gallolyticus has 11 cell wall-anchored proteins with "microbial surface component recognizing matrix molecules" (MSCRAMM) characteristics, including a collagen-binding adhesin and proteins with similarities to pilus subunits [proteins ,25, viruproteins ,26,27, aproteins . Furtherproteins  and fimbolyticus . It has subunits .et al. published the first whole genome sequence of S. gallolyticus subsp. gallolyticus strain UCN34 and analyzed the main metabolic and cell surface features, particularly with regard to adaptation to the rumen and the virulence association of polysaccharide capsule, glucan mucopolysaccharides, different types of pili and collagen binding proteins [Recently, Rusinok proteins .S. gallolyticus subsp. gallolyticus strain. The strain under study was the tetracycline resistant strain ATCC BAA-2069, isolated from a patient with infectious endocarditis. We demonstrate the occurrence of a previously undescribed plasmid (pSGG1) which carries genes for tetracycline resistance ) and reveals strong sequence similarities to plasmids and chromosomes from several ruminal and gastrointestinal bacteria, indicating that pSGG1 may act as a native carrier for horizontal gene transfer.Here we present the whole genome sequence of a not described, considerably divergent S. gallolyticus subsp. gallolyticus was determined by pyrosequencing using the 454 GS FLX Titanium technique  and, after assembly of the 454 reads, remaining gaps were closed by PCR and conventional Sanger sequencing. The genome contains a 2,356,444 bp circular DNA molecule with a G+C-content of 37.65% and a previously undescribed 20,765 bp plasmid designated as pSGG1. Mapping of gene set was performed against S. gallolyticus subsp. gallolyticus genome UCN34 (GenBank Acc. No.: FN597254) [The whole genome sequence of N597254) . BioinfoS. sanguinis SK36 genome is larger , and the G+C-content is 1.7% lower than average . Altogether 2,309 ORFs were automatically annotated, which is 10% higher than the average of all complete sequenced Streptococcus genomes . In direct comparison to the S. gallolyticus subsp. gallolyticus genome UCN34, the BAA-2069 genome is 5.5 kb larger , has 70 fewer CDS  and contains the 20,765 bp plasmid pSGG1.The size of the BAA-2069 circular chromosome  exceeds the average of other previously published streptococcal genomes by 12%  .The sequences and annotations of chromosome and plasmid have been deposited at the NCBI GenBank  ORFs which are predicted to be common in BAA-2069 and UCN34. The arrangement of genetic information is very similar overall, based on alignment of the genomes and the synteny plot , including the putative major cell surface adhesin  mutants  and may S. mutants and S. ratti. Lanthionine is a lantibiotic (bacteriocin), a unique class of peptide antibiotic substances [Lactococcus lactis, resulting in a zone of clearing around BAA-2069 (data not shown).In addition, we found a unique 23 kb genetic island in the BAA-2069 genome, coding for bacteriocin-associated genes (SGGBAA2069_c00810-c00960). This region contains genes for lanthionine biosynthesis and for a bacteriocin/lanthionine exporter orthologous to genes described in bstances . ConductThree genes  are partly homologue to hemolysin A, hemolysin III and an undefined hemolysin-like protein, although group D streptococci are usually non-hemolytic or eventually display weak alpha hemolysis. Moreover, BAA-2069 does show alpha-hemolysis on Schaedler Agar with 5% sheep blood.cpsA - cpsM/SGGBAA2069_c09190 - c09300). The genes are located in a 13.5 kb region and are identical to the UCN34 genome.The polysaccharide capsule coding region, contains 12 genes  and to genes mentioned by Rusinok et al. regarding the UCN34 genome  [We predicted 21 proteins with C-terminal LPxTG motif by "-genes) ,30. All pac). The gene SGGBAA2069_c13900 and its paralog SGGBAA2069_c20580 have cell anchor characteristics but no similarities to functional genes. Furthermore, the unique protein SGGBAA2069_c22120 comprising the LPxTG motif is another gene with putative function in virulence.Within the analysis, we found three proteins containing the LPxTG motif carried by genomic islands specific to strain BAA-2069. The gene SGGBAA2069_c13880 and its paralog SGGBAA2069_c20560 have only very weak similarities to Gallo_1675 and code for a putative major cell surface adhesin  is located on a 9.9 kb unique island (SGGBAA2069_c10280 - c10350), together with the corresponding restriction-methylation subunit and an integrase gene. Another type II restriction endonuclease Eco47II and its modification methylase is encoded on a 9.7 kb region (SGGBAA2069-c22460 - c22570).In comparison to cas genes are for BAA-2069 SGGBAA2069_c14660 and c14670 (cas2), c14670 (cas1), respectively Gallo_1437, Gallo_1444 (cas2) and Gallo_1438, Gallo_1439 (cas1) for UCN34. CRISPR data of both genomes are also accessible by CRISPRs web server http://crispr.u-psud.fr.Mentionable regions missing in BAA-2069, but present in the UCN-34 genome, are a 46 kb phage-associated region containing a putative phage-associated cell wall hydrolase. A "cluster regulatory interspaced short palindromic repeats" (CRISPR) element is sited between 1,507,890 - 1,508,913 bp and containing 16 repetitions of a 36 bp consensus sequence. Another 5.6 kb CRISPR associated region is sited at 1,515,490 - 1,516,317 bp but mostly conserved between the two strains . A unique CRISPR locus for BAA-2069 is between 1,515,726 - 1,516,570 bp. Corresponding S. uberis 0140J and S. agalactiae 2603V_R was conducted. The analysis revealed a core genome consisting of 1118 genes common to all three species, whereas S. gallolyticus subsp. gallolyticus BAA-2069 has 804 unique genes . ComS. gallolyticus subsp. gallolyticus BAA-2069  and the mosaic tetracycline resistance gene tet(O/W/32/O), which are common in plasmids of gram-positive pathogens. Two insertion sequence IS1216 elements and a putative resolvase and a relaxase gene were identified. The relaxase gene has similarities to plasmid pTet35 from Campylobacter jejuni subsp. jejuni 81-176, which suggests its classification of the conjugative transfer system in clade MOBP4. Although, it is more likely that it belongs to the MOBV cluster, which is still ancestrally related to MOBP [rep elements, belonging to rep_1 superfamily (SGGBAA-2069_p00100) and rep_3 superfamily . The repA (SGGBAA-2069_p00140) element has 78% sequence identity to that of the cryptic plasmid pSBO1 isolated from S. equinus [rep gene by in silico analysis. The plasmid pSGG1 seems to be incapable of conjugal self-transfer since it encodes no tra protein and only a putative resolvase, although it was not tested experimentally. Moreover, a mobilization region orthologous to a mob gene in Streptococcus ferrus was found (SGGBAA2069_p00200), which is a necessary feature for transmissible plasmids and therefore promotes the ability for LGT transfer in presence of a helper conjugative plasmid. Five ORFs were assigned to encode proteins with unknown functions and no significant sequence similarities to previously described genes exist in these cases , we screened 41 strains by Southern blot hybridization analysis with a digoxygenin nick-labeled probe of pSGG1 , about 3.5% higher. This could be taken as a hint that, with the increasing number of sequenced strains, the pan-genome of S. gallolyticus subsp. gallolyticus is far larger by proportion. However, these data are preliminary, pending the sequencing of further S. gallolyticus subsp. gallolyticus strains.Within whole genome comparison analysis the "pan-genome" includes a core genome containing genes present in all strains of one species. This is complemented by an individual set of genes unique to a strain or not shared by all strains. With the growing number of sequenced strains, the increasing size of the pan-genome is evidence of the genomic diversity between different isolates of a distinct species. Tettelin  immense , whereaswas zero . The numS. gallolyticus subsp. gallolyticus is very diverse. The majority of exclusive sequences found in the UCN34 genome are located in three large regions representing 111 kb of sequence information (53%), whereas the three largest unique regions in BAA-2069 constitute only 87 kb (39%) of strain-specific sequence and mostly consist of smaller regions. However, the tendency of virulence factors to be located within genomic islands may lead to a higher ratio of exchangeability of such genes in comparison to other regions [In a direct comparison to the recently sequenced strain UCN34 , surpris regions . Further regions .Surface proteins and in particular proteins belonging to "microbial surface component recognizing matrix molecules" (MSCRAMM) were shown to play a functional role in the pathogenesis of all bacteria. Of specific interest is a group of proteins containing the C-terminal cell wall-sorting motif LPxTG, which serves as a recognition site for the membrane-associated sortase. After sortase-mediated cleavage of the protein, the polypeptide is covalently bound to the peptidoglycan of bacterial cell surface and can therefore promote the first step in bacterial adherence ,46. ThreIn silico analysis of genome data strongly indicated the presence of a multi-copy plasmid. The purification of plasmid DNA and further analysis of sequence data confirmed these hints and showed a localization of tetracycline resistance genes. Analysis of plasmid distribution shows only two mainly homologous plasmids in 41 strains overall. Therefore, the incidence of the pSGG plasmids among S. gallolyticus isolates does not seem to be widespread. The mosaic tetracycline resistance gene tet(O/W/32/O) is usually chromosomally located and mediates resistance by ribosome protection. It has been shown that the mosaic tet(O/W) genes have a higher level of resistance than the original genes [tetL gene is generally found on plasmids and coding for a tetracycline efflux protein [tetL and tetM, was located on the chromosome and adjacent to putative plasmid and transposon Tn916-associated genes [tetL and the occurrence of plasmids of the pSGG family.al genes . This co protein . In conted genes . This inS. gallolyticus subsp. gallolyticus is a commensal and facultative pathogen of animals, the intensive tetracycline treatments in animal husbandry, causes a general advantage regarding evolutionary fitness for pathogenic and natural habitants of the intestinal tract to accommodate resistance genes by LGT [Because of antibiotic treatment, gastrointestinal tract and rumen are well-known reservoirs of mobilizable antibiotic resistance genes . Furthers by LGT ,52. AlthS. gallolyticus subsp. gallolyticus strain BAA-2069, a causative agent of infective endocarditis. The results promote identification of genetic factors concerning the pathogenesis and adhesion to ECM. Novel candidate genes were detected probably contributing to the pathogenicity. The comparison to S. gallolyticus subsp. gallolyticus strain UCN34 revealed significant differences concerning virulence factors, surface proteins and protective elements.This study presented the analysis and comparison of the whole genome sequence of Furthermore, we detected for the first time the presence of the pSGG1 plasmid, containing 21 ORFs including mosaic tetracycline resistance genes and may play a functional role in lateral gene transfer.S. gallolyticus subsp. gallolyticus strain was isolated in 2003 at the Herz- und Diabeteszentrum Nordrhein-Westfalen from a blood culture from a 68-year-old woman with aortic heart valve endocarditis and deposited at the American Type Culture Collection  (BAA-2069). Strain BAA-2069 was confirmed by isolation of the same strain by lesion smear test of aortic heart valve and detection in valve excision material by culture and PCR. The strain was selected because it had been defined as virulent during earlier tests [The er tests  and shower tests .DNA sequencing was performed using 454 Life Science pyrosequencing technology [Curation and annotation of the genome were performed using the genome annotation system GenDB 2.4 . PredictS. gallolyticus subsp. gallolyticus BAA-2069 gene content was compared to S. gallolyticus subsp. gallolyticus UCN34, S. agalactiae A909, S. dysgalactiae subsp. equisimilis GGS_124, S. equi subsp. equi 4047, S. sanguinis SK36, S. suis BM407, S. uberis 0140J, S. pyogenes MGAS9429, S. pneumoniae ATCC 700669, S. mutans NN2025, S. mitis B6, S. thermophilus LMD-9, S. gordonii str. challis substr. CH1, S. oralis ATCC 35037, S. salivarius SK126 with EDGAR [et al. [ [et al. .Comparison of whole chromosome sequences was performed by MAUVE software using local collinear blocs (LCB). An LCB is defined as a collinear (consistent) set of multi-MUMs . The weight (the sum of the lengths of the included multi-MUMs) of an LCB serves as a measure of confidence that it is a true orthologous region instead of a random match and is set to 355. Therefore, the ORFs or sequences between the LCBs and any regions with low similarity (shown as white in LCB) are classified as strain-specific regions.For calculation of phylogenetic tree, EDGAR was used . In detath window.The GC skew measures the excess of Gs by calculating the difference between the number of Gs and Cs (G-C) in a sliding window of 1000 nucleotides. The skews were cumulated to obtain the cumulative GC skew that represents the sum of the GC skews from the first to the iS. gallolyticus subsp. gallolyticus strains for presence of pSGG1 plasmid or homologs was performed by Southern-hybridization analysis in accordance with standard protocols. The probe was prepared by nick translation DIG labeling of pSGG1 referring to DIG DNA Labeling Kit  [tetL gene by PCR using the whole genome sequence derived primer tet_f (5'-GCTATGGGAGAAGGTATCG-3') and tet_r (5'-GAGACAAACCCTGCTACTG-3'), or mob_f (5'-AAGCTGTACTTGGCTCTC-3') and mob_r (5'-CAGTGGCAGAACTATCTC-3') respectively, by standard methods.Screening of 41 different Germany) . FurtherS. gallolyticus subsp. gallolyticus was deposited at GenBank (Acc. no. FR824043). Sequence of the novel designated plasmid pSGG1 was deposited with accession no. FR824044.Whole genome sequence of S. gallolyticus subsp. gallolyticus strains and cultivated in 96 well plates at 37°C. After 16 h incubation, OD 600 was measured and growth was determined as OD 600 > 0.2. The assay was performed in triplicate.For each strain, 200 μL BHI broth  supplemented with indicated tetracycline concentration were inoculated with 1 μL of overnight culture of DH prepared the DNA and plasmid extraction, carried out the sequence analyses, participated in the gap closure and bioinformatics analysis and wrote the manuscript. TV participated in the design of figures and helped to draft the manuscript. CR performed sequencing and carried out the sequence alignment. JB worked on bioinformatics analysis. JK participated in the design and drafted the manuscript. CK and JD conceived, designed and coordinated the study and helped to draft the manuscript. All authors have read and approved the final manuscript.S. gallolyticus subsp. gallolyticus BAA-2069 and UCN34 genomePairwise synteny plot of the . Every CDS of the first contig is checked for a reziprocal best blast hit. If one is found, the stopposition of both CDS are read from the database and used as coordinates for a dot.Click here for fileS. gallolyticus subsp. gallolyticus BAA-2069 in relation to S. gallolyticus subsp. gallolyticus UCN34Unique genes of . Unique genes calculated by EDGAR analysis.Click here for fileS. gallolyticus subsp. gallolyticus UCN34 in relation to S. gallolyticus subsp. gallolyticus BAA-2069Unique genes of . Unique genes calculated by EDGAR analysis.Click here for fileS. gallolyticus subsp. gallolyticus BAA-2069 and three Enterococcus feacalis strainsCore genome set of . Following strains were used for calculation by EDGAR: E. faecalis 62 (Acc. No CP002491), E. faecalis OG1RF (Acc. no. CP002621) and E. faecalis V583 (Acc. no. NC_004668).Click here for fileNumber of unique or common ORFs. Numbers represent the common or unique ORFs in comparison to BAA-2069 and indicated species.Click here for fileAgarose gel electrophoresis of restriction fragment pattern. Pattern were obtained with seven different enzymes, regarding plasmid pSGG2 (left lane) and pSGG1 (right lane). Ladder marker: 1 kb Ladder plus .Click here for fileTetracycline susceptibility test. Minimum inhibitory concentration (MIC) was determined growth in liquid cultures with indicated tetracycline concentration.Click here for file