Helicobacter pylori is the causative agent for gastritis, and peptic and duodenal ulcers. The bacterium displays 5-6 polar sheathed flagella that are essential for colonisation and persistence in the gastric mucosa. The biochemistry and genetics of flagellar biogenesis in H. pylori has not been fully elucidated. Bioinformatics analysis suggested that the gene HP0256, annotated as hypothetical, was a FliJ homologue. In Salmonella, FliJ is a chaperone escort protein for FlgN and FliT, two proteins that themselves display chaperone activity for components of the hook, the rod and the filament.H. pylori significantly reduced motility. However, flagellin and hook protein synthesis was not affected in the HP0256 mutant. Transmission electron transmission microscopy revealed that the HP0256 mutant cells displayed a normal flagellum configuration, suggesting that HP0256 was not essential for assembly and polar localisation of the flagella in the cell. Interestingly, whole genome microarrays of an HP0256 mutant revealed transcriptional changes in a number of genes associated with the flagellar regulon and the cell envelope, such as outer membrane proteins and adhesins. Consistent with the array data, lack of the HP0256 gene significantly reduced adhesion and the inflammatory response in host cells.Ablation of the HP0256 gene in H. pylori. However, it is required for full motility and it is involved, possibly indirectly, in expression of outer membrane proteins and adhesins involved in pathogenesis and adhesion.We conclude that HP0256 is not a functional counterpart of FliJ in  Helicobacter pylori is a Gram-negative bacterium, colonising the human gastric mucosa. It is responsible for diverse duodenal- and stomach-related disorders, such as ulcers, B cell MALT lymphoma and gastric adenocarcinoma [H. pylori might help develop intervention strategies or therapeutics.arcinoma -4. Motilarcinoma ,6. Flagearcinoma . Since mH. pylori flagellar gene transcription is tightly controlled by three RNA polymerase sigma factors σ80, σ54 and σ28 [80 controls the transcription of class I genes (early flagellar genes). σ54 (RpoN) is responsible for the transcription of class II genes (middle flagellar genes). RpoN transcriptional activity is dependent on additional regulators, such the FlgR/FlgS system and the chaperone HP0958 [28 (FliA) and the anti-sigma factor FlgM [ and σ28 . σ80 cone HP0958 -12. Clastor FlgM ,14.Salmonella, several flagellar chaperones have been identified. FlgN has chaperone activity for the hook proteins FlgL and FlgK. The chaperone FliT is dedicated to the capping protein FliD, and FliS to the flagellin FliC [Salmonella and E. coli [H. pylori genomes identified homologues of most flagellar genes of the Salmonella/E. coli paradigm [H. pylori, presumably due to low sequence identity. Previous bioinformatics searches, targeting only functional domains, were successfully performed to identify the anti-sigma factor FlgM [The flagellar export system is recognized as a version of type III secretion systems , and durlin FliC -18. The lin FliC ,19. Flag E. coli ,20,21. Aparadigm -25. Howetor FlgM ,14, and tor FlgM .H. pylori genomes, bioinformatic analysis focusing on identification of specific and conserved domains of flagellar genes was performed. In Salmonella, FliJ is a 17 kDa protein with a relative abundance of charged residues. Fraser and colleagues showed that FliJ in Salmonella interacts with FliH (the presumptive inhibitor of the FliI ATPase) and FlhA (a flagellar biosynthesis protein) [H. pylori genomes, nor incidentally were homologues for FlgN or FliT [H. pylori homologues, a search using only the essential FliJ domain  did reveal a potential homologue . This analysis identified HP0256, encoding a hypothetical protein with unknown function and a predicted coiled coil domain.In an effort to identify novel flagellar genes in sequenced protein) . FliJ waprotein) . Howeverprotein) . FliJ bi or FliT ,23,25. AIn the present study, we phenotypically characterized a mutant lacking the HP0256 gene product and investigated the function of HP0256 in the flagellar regulon using global transcript analysis. The data suggest a novel role for HP0256 in motility but not flagellum assembly, and involvement in production of cell surface proteins.Salmonella did not identify any homologues in H. pylori. However, using only the FliJ N-terminal coiled-coil domain as a search query, HP0256 was identified as a potential FliJ homologue. The annotation of this H. pylori ORF indicates a hypothetical protein with an unknown function. Both FliJ and HP0256 proteins have a similar size  and have a high likelihood of forming N-terminal coiled-coils. They share 17% identity and 44% similarity. In contrast, FliJ from Salmonella and E. coli are 88% identical and 96% similar. Further searches identified potential HP0256 homologues in more related species  as well as two outer membrane protein genes (omp7/HP0252 and omp8/HP0254), and three hypothetical genes, one of which encodes a predicted secreted protein (HP0257) and the other a predicted integral membrane protein (HP0258). When comparing HP0256 with homologues from related species, it did not appear that any one domain of the protein was more or less conserved . We complemented the P79-derivative HP0256 mutant, by expressing the HP0256 gene, integrated into the chromosome, under the control of the flaA promoter .The partial loss of motility in the HP0256 mutant might have been due to altered production levels of major flagellar components. ntiserum . The antpe cells . In conts Figure . The norH. pylori CCUG17874 and P79 cells harboured 2-3 polar flagella . hopA was four fold down-regulated, whereas the other three Hop genes were up-regulated. HP1339 and HP1340, encoding respectively the biopolymer transport proteins ExbB and ExbD, were up-regulated in the HP0256 mutant. ExbB and ExbD in E. coli interact with the TonB-dependent energy transduction complex [E. coli, TonB is involved in the transduction of energy between the cytoplasmic membrane and the outer membrane [Interestingly, four genes encoding proteins of the Hop outer membrane family were identified as differentially expressed in the HP0256 mutant by microarray analysis ( complex . In E. cmembrane . Five gei.e. HP0115/flaB (encoding the minor flagellin FlaB), HP0870/flgE (encoding the hook protein FlgE) and HP1076 . Another RpoN-dependent gene HP1155/murG  was 1.955 fold up-regulated with a p-value of 0.034. However, RpoN and its associated regulators FlgR, HP0244 and HP0958 were transcribed at wild-type levels. As shown in Table hpaA3 (flagellar sheath associated protein) was significantly down-regulated. This gene is known to be essential for flagellar biogenesis, but its transcriptional regulation remains unclear. It has not yet been assigned to any flagellar gene class [fliN gene encoding a component of the switch was up-regulated (1.758 fold) with a p-value of 0.042.We identified a number of flagellar genes with altered expression levels Tables  and 2. TminD, coding for a protein involved in the correct localisation of the cell division site [E. coli, MinD (in synergy with MinC) inhibits the cell division protein FtsZ, that forms the FtsZ or Z ring at the septum [ftsZ was 1.9 fold up-regulated in the HP0256 mutant   secretion by gastric epithelial cells using an ) Figure . Superna) Figure . This isH. pylori. HP0256 encodes a hypothetical protein in H. pylori and shares common properties with FliJ, such as a similar size and a predicted N-terminal coiled coil. However, in comparison with the complete loss of motility reported in a Salmonella FliJ mutant [H. pylori HP0256 mutants retained some motility based on a motility plate assay. Complementation of a Salmonella FliJ mutant was attempted by introduction of the HP0256 gene product expressed under the control of an E. coli promoter, but this did not restore motility in the transformed Salmonella FliJ mutant (data not shown). Immunoblotting analysis revealed no significant differences in flagellin and hook protein synthesis between the wild-type and the HP0256 mutant. The partial loss of motility in the HP0256 mutant was therefore not due to impairment in filament and hook protein production. The increased degradation rate of flagellar proteins observed in the HP0256 mutant samples compared to the wild-type suggested a possible chaperone activity of HP0256. However, the apparently normal flagellum assembly and localisation at the pole in the HP0256 mutant cells suggested that HP0256 was not actually essential for flagellum protein stabilization or export apparatus positioning. In the HP0256 mutant, the significant reduction in motility still remained unclear. Quantitative data, e.g. average time and lengths of swimming runs, to characterize the motility phenotype of the HP0256 mutant would allow us to further comprehend the effect of HP0256 on Helicobacter pylori motility. Although this was not mechanistically wholly elucidated, the potential players in this phenotype were identified by array analysis.A focused bioinformatics analysis based on the functional domain of FliJ  suggested that HP0256 was a potential FliJ homologue in J mutant , H. pyloGlobal transcript analysis indicated that HP0256 interferes with the transcription of flagellar genes belonging to the RpoN regulon. Four RpoN-dependent genes were up-regulated in the HP0256 mutant, although transcription of RpoN and its associated regulators FlgR, HP0244 and HP0958 were at wild-type level. The different transcriptional profiles among RpoN-dependent genes suggested that some key RpoN-dependent genes may be under additional regulatory checkpoints, likely HP0256-dependent. However, we do not have data to explain the mechanistic links involved in this regulation. Among class II genes, the only known flagellar regulator HP0906/FliK controls the hook length and is involved in the hook-filament transition. HP0906 was transcribed at wild-type level, in agreement with the normal flagellar morphology in HP0256 mutants (i.e. absence of polyhooks). The up-regulation of four RpoN-dependent genes in the HP0256 mutant did not grossly interfere with flagellar assembly as demonstrated by transmission electron microscopy . However, a modification of the FlaA/FlaB ratio in flagella significantly affects motility  and thisSalmonella, the FliN protein is involved in the switch process and its interaction with FliH is crucial for the localisation of the FliI-FliH complex in the C-ring [Interestingly, HP0256 mutant cells were not predominantly swimming but tumbling, based on light microscopy observations. This abnormal motility behaviour, which may explain the reduced motility in the HP0256 mutant, underlined a probable disruption of the switch mechanism between swimming and tumbling. FliG, FliM and FliN are involved in switching between clockwise and counter-clockwise rotation of the motor and thus, of the filament ,42. It ie C-ring . We hypoi.e. adhesins, hop proteins, alpha-2-fucosyltransferase, may explain the reduced ability of the HP0256 mutant to adhere to host cells and to induce an inflammatory response, i.e. interleukin-8 secretion. The disruption of HP0256 and its effect on cell envelope architecture may modify the lipid profiles and/or membrane fluidity and therefore the function of the methyl-accepting chemotactic proteins. The biological significance of the alteration of expression of minD and ftsZ in the HP0256 mutant, two genes involved in the cell division process, remains unclear. A correlation with other membrane-associated protein expression, such as outer membrane proteins, cannot be excluded and additional experiments will be required to test this.A number of outer membrane proteins and LPS-related proteins were differentially expressed in the HP0256 mutant. BabA and BabB expression were both up-regulated in the HP0256 mutant. BabA binds to the blood group antigen Lewis b . The siaH. pylori based on bioinformatic analyses. Our data clearly show that HP0256 has a different function in H. pylori, compared to that of FliJ in Salmonella. Interestingly, HP0256 is still obviously involved in flagellum activity as its ablation caused a partial loss of motility. Its involvement with expression of some RpoN-dependent genes is noteworthy but did not result in major changes in the mutant phenotype . The partial loss of motility must therefore be due to effects upon other flagellar players. Based upon its observed up-regulation in the HP0256 mutant, FliN is a potential candidate responsible for the impaired motility we observed in the HP0256 mutant. Further investigation of FliN production in the HP0256 mutant or overexpression in the wild-type would confirm this hypothesis. The large number of membrane-associated proteins with an altered expression in the HP0256 mutant highlighted another aspect of the mutant phenotype: the alteration of the cell envelope architecture, likely responsible for the weak adhesion to, and the low inflammatory response induced in, host cells. We conclude that HP0256 is required for full motility of H. pylori, possibly through its involvement with the switch components, but that it also modulates directly or indirectly the normal expression of membrane proteins essential in pathogenesis.We initially hypothesized that HP0256 was a FliJ homologue in H. pylori strain P79 [H. pylori strains were cultured as previously described [H. pylori mutants lacking the HP0256 gene (one in CCUG17874 and one in P79) were generated as described below in Materials and Methods. Transformants were selected on CBA (Columbia agar base) plates supplemented with 10 μg/ml chloramphenicol (Sigma) and/or 50 μg/ml kanamycin (Sigma). One Shot TOP10 chemically competent E. coli cells  were propagated on Luria-Bertani (LB) agar plates or LB broth at 37°C supplemented with antibiotics: 50 μg/ml kanamycin (Sigma), 100 μg/ml ampicillin  and 10 μg/ml chloramphenicol (Sigma).Bacterial strains used in this study are listed in Table rain P79 , a strepescribed . Two H. Salmonella or other FliJ sequences did not result in significant hits with any HP0256 homologues. However, using one of the HP0256 homologues for a PSI-BLAST search often resulted in Bacillus FliJ sequences appearing just below the inclusion threshold with E-values ranging between 0.08 and 0.52. In addition, the alignments from these BLAST hits were deemed correct as judged by comparison to the multiple alignment presented in Figure PSI-BLAST was performed using bacterial sequences from the NCBI non-redundant protein databank at NCBI-BLAST. Three to four iterations were run and false-positives were edited from the output. Searching with Helicobacter DNA was isolated as previously described [BglII restriction site. The two amplicons were joined together by extension overlap PCR and the resulting DNA product was cloned into pUC18  following BamHI and EcoRI digestion. The resultant plasmid was cut with BglII and ligated with the chloramphenicol acetyl transferase (cat) gene which had been cut from the plasmid pRY109 [H. pylori cells were transformed with 1 μg of this plasmid for double-cross over gene disruption as previously described [escribed . Oligonud pRY109 . H. pyloescribed . PolymerE. coli [Salmonella fliJ mutant , the HP0256 gene was amplified with primer pairs HP0256-QF/HP0256-QR . For complementation of the HP0256 mutant, a full length copy of the gene was introduced into the HP0203-HP0204 chromosomal intergenic region of a P79 HP0256-KO mutant according to the method described by Langford et al. using the pIR203K04 plasmid [flaA promoter amplified using the primers FLA-F2 and FLA-R2, by overlap extension PCR. This composite fragment flaA promoter-HP0256 was then cloned into pIR203K04 as a Cla1/BamH1 fragment.Standard procedures were employed for plasmid cloning experiments in E. coli . For comprotocol . Electro plasmid . As exprH. pylori were grown on a plate containing brain heart infusion (BHI) supplemented with 10% foetal calf serum, for 24 h in a micro-aerobic atmosphere. Next, cells were harvested and carefully resuspended in 2% ammonium molybdate (Sigma) with 70 μg/ml bacitracin (Sigma), as a wetting agent. 5 μl cell preparation was applied to a copper grid overlaid with a carbon-coated Formvar film. The excess sample was carefully removed and the copper grid was dried. The copper grids were observed in a JEOL JEM-1200EX transmission electron microscope at an accelerating voltage of 80 kV.Cell samples were subjected to negative staining. Whole cells of H. pylori strains and mutants were grown for 2 days on CBA plates and then stab inoculated on Brucella soft agar plates containing 0.3% (w/v) agar and 5% (v/v) heat-inactivated foetal bovine serum (Sigma). Motility plates were incubated at 37°C in an atmosphere containing 5% CO2 and periodically observed for halo formation.H. pylori flagellin and hook protein was used as primary antibody [A standard protocol was used to perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis  and immuantibody . Anti-raH. pylori whole genome microarray was used in a Common Reference or Type II experimental design whereby Cy5-labelled RNA from each strain was co-hybridised to an array with a Cy3-labelled genomic DNA reference. The microarray experiment was performed as described previously by Douillard et al. [BμG@Sbase  and also ArrayExpress (Accession No. A-BUGS-18). Fully annotated microarray data have been deposited in BμG@Sbase  and also ArrayExpress (accession number E-BUGS-98).To compare the transcriptional profiles of the wild-type and HP0256 mutant strains, a d et al. . Four biQuantitative real-time PCR (qRT-PCR) was performed as a confirmatory test on selected genes following global transcript analysis by microarray. Real-time PCR primers were designed using the Primer3 software package  and are 5 cells per well for six days. H. pylori cells were harvested from two-day old plate cultures of wild-type strain or the HP0256 mutant using 1 ml sterile PBS. Bacteria were washed twice with HAMS F12 media  and adjusted to an OD600 of 0.3. Cells were added to three wells of pre-grown AGS cells at a multiplicity of infection of 500:1 and incubated at 37°C and 5% CO2 for 3 h. Next, 1 ml of medium was removed and stored at -20°C for ELISA analysis. Cell supernatants were tested for IL-8 protein using the commercially available DuoSet ELISA kit  as per manufacturer's instructions.AGS gastric epithelial cells were grown in six-well plates at 3.2 × 10H. pylori adhesion assay was performed to measure bacterial cells adhering to the AGS monolayer [H. pylori colonies were counted after 48 h and averaged. Adhesion of the HP0256 mutant was expressed as a percentage of the wild-type. Experiments were performed in triplicate.An onolayer . The remH. pylori type strains, participated in the transmission electron microscopy, performed protein electrophoresis and immunoblotting analyses, global transcript analysis, quantitative analysis of transcription by quantitative Real-time PCR, participated in motility plate assay and drafted the manuscript. KAR performed adhesion assay, participated in the generation of HP0256 mutants in two distinct H. pylori type strains, immunoblotting analyses, complementation of Salmonella FliJ mutant, motility plate assay, performed interleukin-8 ELISA and drafted the manuscript. MCL participated in the generation of HP0256 mutants in H. pylori type strains. DLC participated in complementation of Salmonella FliJ mutant. SAM performed bioinformatics analyses, participated in its design and coordination and helped to draft the manuscript. CWP performed transmission electron microscopy. JH designed and produced the microarrays, conceived the transcriptome experimental design, and helped analyze the array data. POT conceived the study, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.FPD participated in the generation of HP0256 mutants in two distinct


Different proteins are required in widely different quantities to build a living cell. In most organisms, transcription control makes a major contribution to differential expression. This is not the case in trypanosomatids where most genes are transcribed at an equivalent rate within large polycistronic clusters. Thus, trypanosomatids must use post-transcriptional control mechanisms to balance gene expression requirements.Trypanosoma cruzi, Trypanosoma brucei and Leishmania major. The tRNA complement reveals forty-five of the sixty-one possible anticodons indicating widespread use of 'wobble' tRNAs. Consistent with translational selection, cognate tRNA genes for favoured codons are over-represented. Importantly, codon usage (Codon Adaptation Index) correlates with predicted and observed expression level. In addition, relative codon bias is broadly conserved among syntenic genes from different trypanosomatids.Here, the evidence for translational selection, the enrichment of 'favoured' codons in more highly expressed genes, is explored. A set of highly expressed, tandem-repeated genes display codon bias in Synonymous codon bias is correlated with tRNA gene copy number and with protein expression level in trypanosomatids. Taken together, the results suggest that translational selection is the dominant mechanism underlying the control of differential protein expression in these organisms. The findings reveal how trypanosomatids may compensate for a paucity of canonical Pol II promoters and subsequent widespread constitutive RNA polymerase II transcription. Trypanosomatids have a devastating impact on the world's poor, causing African trypanosomiasis, Chagas disease and leishmaniasis . The conTrypanosoma brucei, the South American trypanosome, Trypanosoma cruzi, and Leishmania major [The protozoan parasites responsible branched early from the eukaryotic lineage and display a range of unusual molecular features. RNA polymerase II transcription of protein coding genes is polycistronic and constitutive and all mature mRNAs are trans-spliced to an identical leader sequence . Genome ia major . These tia major . Thus, tSaccharomyces cerevisiae [Cells must express different proteins over an enormous abundance-range, from fewer than 50 to more than a million molecules per cell reported in revisiae -8. Synonrevisiae -12. Therrevisiae . In contrevisiae -17. Evidrevisiae -20 and wrevisiae . The finT. cruzi [T. brucei assembly is excellent and most trypanosomatid genes share orthologues accessible through the GeneDB interface, the T. brucei genome was scanned for tandem duplicated protein-coding genes. Consistent with the first prediction above, sixty-four tandem-amplified genes in T. brucei encode proteins with orthologues among the 243 proteins over-represented (≥ 10 mass spectra) in the non-redundant T. cruzi proteome set [Most trypanosomatid genes are 'single copy'  but tandem gene amplification is thought to contribute to increased expression ,22. ThusT. cruzi  but the T. cruzi . Since teome set . This inT. brucei and for the orthologous sets from T. cruzi and L. major, >60,000 codons in total. Consistent with previous reports [L. major which uses only 40 of the 61 available codons. Figure T. brucei and L. major. What is clear from Figure L. major. This is likely explained by the higher 'background' GC-content; the intergenic and protein-coding GC-contents are 41% and 50.9% in T. brucei [T. cruzi [L. major [Codon usage was analysed for this tandem gene set from  reports ,11,12, t. brucei , 47% andT. cruzi  and 57.3L. major  respectiT. brucei shows little bias for Ala, Pro, Ser or Thr codons and GGGGly, AGGArg and CGGArg codons are more than two-fold under-represented in all three trypanosomatids . Indeed, there are several examples in Leishmania where tRNA genes that recognise favoured codons appear to have been specifically amplified since divergence from the trypanosomes  intergenic regions ; (b) 'Single-copy' genes; (c) 'Single-copy' genes detected through whole-cell proteome analysis (≥ 5 mass spectra) and (d) Tandem-arrayed genes detected through whole-cell proteome analysis (≥ 10 mass spectra). The analysis shown in Figure The codon adaptation index (CAI) is used to measure synonymous codon usage bias and can predict gene expression level if translational selection operates . Whole-cT. cruzi  and the T. cruzi proteome data [T. cruzi protein-coding sequences can predict relative steady-state protein expression level. The trend is remarkable because of the range of other parameters, both alternative modes of expression control and experimental sampling that could impact on the outcome. The findings are consistent with the idea that codon bias has a major impact on steady state protein levels in trypanosomatids.A more rigorous test of the contribution of translational selection to gene expression is whether expression level can be predicted for individual genes exclusively based on codon usage. Single-copy genes represented in the ome data  were useT. brucei and T. cruzi and the analysis indicates that relative scores are indeed conserved :473–476.2. Atwood JA, 3rd, Weatherly DB, Minning TA, Bundy B, Cavola C, Opperdoes FR, Orlando R, Tarleton RL: Table S1Trypanosoma brucei hsp83 gene clusterA transcriptional analysis of the . Mol Biochem Parasitol 1989, 37(1):115–127.3. Mottram JC, Murphy WJ, Agabian N: Trypanosoma bruceiCharacterization of the genes for fructose-bisphosphate aldolase in . Mol Biochem Parasitol 1988, 29(1):65–75.4. Marchand M, Poliszczak A, Gibson WC, Wierenga RK, Opperdoes FR, Michels PA: T. cruzi mass spectra values in additional tables S1 and S3 are from Atwood et al., [T. brucei GeneDB links are listed but further links to orthologous trypanosomatid genes can be found on each GeneDB page. Grey shading represents genes thought to be present in tandem arrays but not annotated (An) as such; HSP83 [T. brucei for example, but mostly due to incomplete assembly of T. cruzi genome sequence. Table S2 – Trypanosomatid tRNA genes. tRNA genes and cognate codons are indicated. Table S3 – T. cruzi 'single-copy' gene analysis. All these genes have orthologues annotated in the other trypanosomatids and are represented by ≥ 5 cognate mass spectra in the non-redundant proteome set. Table S4 – Realtive CAI analysis for three polycistronic gene clusters. The single-copy genes analysed are from: i. The 75 kbp polycistronic gene cluster on T. brucei chromosome 1 (GeneDB coordinates 815 – 985 kbp), syntenic with the 375 kbp cluster on L. major chromosome 12 (GeneDB coordinates 295 – 670 kbp). ii. The 120 kbp polycistronic gene cluster on T. brucei chromosome 6 , syntenic with the 170 kbp cluster on L. major chromosome 30 . iii. The 85 kbp polycistronic gene cluster on T. brucei chromosome 10 , syntenic with the 130 kbp cluster on L. major chromosome 36 (GeneDB coordinates 20 – 150 kbp). The syntenic chromosome numbers have not been determined in T. cruzi due to problems with sequence assembly.All genes listed are linked to the GeneDB database . The col et al., . Table Sh; HSP83  and aldoh; HSP83  in T. brClick here for file


Objectives: We performed a phase I trial to assess the safety and tolerance of a Lactobacillus vaginal suppository for prevention of recurrent UTI.Methods: Premenopausal women with a history of recurrent UTI were randomized to use L. crispatus CTV-05 or placebo vaginal suppositories daily for five days.Results: 30 women were randomized (15 to L. crispatus CTV-05). No severe adverse events occurred. Mild to moderate vaginal discharge and genital irritation were reported by women in both study arms. Seven women randomized to L. crispatus CTV-05 developed pyuria without associated symptoms. Most women had high concentrations of vaginal L. crispatus, L. jensenii, and L. gasseri were the most common Lactobacillus species identified, with stable prevalence over time.Conclusions: L. crispatus CTV-05 can be given as a vaginal suppository with minimal sideeffects to healthy women with a history of recurrent UTI. Mild inflammation of the urinary tract was noted in some women. Urinary tract infections (UTIs)affect millions of women each year, with an annual societal cost of billions ofdollars . Importa202-producinglactobacilli may have a protective effect against urogenital infections,including UTI [Lactobacillus species, mainly L. rhamnosus and  L. fermentum, can be given orally or vaginally with resultingcolonization of the vagina, reduction in vaginal coliform counts, and evenreduction in UTI recurrence [202, adhere to uroepithelial cells, interfere with uropathogen attachment and growth, and persist in the vagina [Lactobacillus species, L.crispatus [202 and possesses the adherence and inhibitory qualities essential for a good probioticcandidate [A growing body of evidence suggests that vaginal Hding UTI –11. It iurrence –18. Spece vagina .The mosrispatus , also prandidate , 23.L. crispatus strain CTV-05 has beendeveloped as a vaginal suppository (LACTIN-V) for the prevention of recurrentUTI in women. L. crispatus CTV-05 ishighly adherent to vaginal epithelial cells [L. crispatus CTV-05 has been reported to have a specific DNA fingerprint that distinguishesit from endogenous vaginal lactobacilli [L. crispatus CTV-05 and its effect onthe vaginal microbial flora, we conducted a phase I, randomized, double-blind,placebo-controlled trial of vaginal L.crispatus CTV-05 in premenopausal women with a history of recurrent UTI.al cells , and wheal cells .Furtherobacilli .To furtWe recruited premenopausal women aged 18–35years with a history of three or more uncomplicated UTIs diagnosed in the pastyear, or two uncomplicatedUTIs diagnosed in the past six months, from  Additional eligibility requirementsincluded regular menstrual cycles or amenorrhea for at least six monthssecondary to use of a hormonal contraceptive, a normal Pap smear documented inthe last year or at the baseline clinic visit, abstinencefrom sexual activity or participation ina mutually monogamous sexual relationship, use of birth control, agreement notto use other intravaginal products, agreement not to have sexual intercourse oruse tampons between the baseline and first follow-up visit, and capability tounderstand English and provide informed consent. Exclusion criteria included history ofurologic abnormality, recent urologic surgery or urinary catheterization,history of complicated pyelonephritis or renal calculi, hysterectomy, recent sexuallytransmitted infection (STI) or bacterial vaginosis, risk factors for STI andHIV, history of recurrent genital herpes, menses anticipated within ten days, pregnancy,lactation, recent antibiotic or antifungal use, diabetes or other immunocompromised state, drugor alcohol abuse, use of the (NuvaRing), prior use of the study drug or allergyto any of its components, and abnormal initial pelvic examination. The study was approved by , University of Washington, Wash,USA.)L. crispatus CTV-05 at adose of 5 × 108 colony forming units(cfu) or placebo vaginal suppository to be inserted daily for five days. L. crispatus CTV-05 and placebosuppositories were similar in appearance and consisted of a preservation matrixand maltodextran. Both were prepackagedby the manufacturer  according to a randomizationschedule and supplied to the study site sequentially labeled with a subjectnumber.Subjects were randomized in a double-blindedfashion to Subjectswere seen at three clinic visits over a period of one month. At the first (baseline) visit, eligible subjectsprovided informed consent before undergoing a structured medical andgynecologic history and physical examination.They provided urine, vaginal, and cervical specimens. Subjects inserted the first dose of studydrug in the clinic and were instructed to record any symptoms occurring duringthe first week of study on a prepared diary card. Subjects were seen in follow-up 6–8 days (1-week)visit and 26–34 days (4-week) visit after enrollment. During these visits, new symptoms, newurogenital infections, and any changes in clinical history were recorded. Follow-up visits were otherwise similar to thebaseline visit. Finally, subjects werecontacted by telephone six months after enrollment to assess symptoms,pregnancy, new diagnoses, or major medical events.Lactobacillus species at allvisits. Repetitive elementsequence-based polymerase chain reaction (rep-PCR) for L. crispatus CTV-05 was performed on three lactobacillus coloniesfrom culture at each visit [Neisseria gonorrhoeae and Chlamydiatrachomatis by DNA amplification was done at the baseline visit.Laboratorytesting included urine dipstick testing, urinalysis, urine culture, vaginalfluid wet preparation slides, gram stains, and culture for facultative isolatesand ch visit . UrinepL. crispatus CTV-05assessed by rep-PCR. The Fisher exacttest was used to test statistical significance.Asample size of 30 was chosen to evaluate the primary outcome of safety asassessed through self-reported symptoms, physical exam findings, and laboratorystudies. Secondary outcomes includedshifts in the vaginal flora assessed by vaginal culture and vaginalcolonization with L. crispatus CTV-05). All subjectstook the five planned doses of study drug.Two women randomized to L.crispatus CTV-05 completed treatment over six days, and one womanrandomized to placebo used six suppositories over six days. All women remained on study through the4-week visit . Thesesymptoms occurred with similar frequency in each treatment arm.L. crispatus CTV-05 and five randomized to placebo reported abnormalvaginal discharge at the 1-week visit. Abnormaldischarge was less frequently reported at the 4-week visit. External genital or vaginal irritation wasalso reported by six women in each treatment arm at follow-up visits. Vaginal odor was reported at the 1-week visitby one woman randomized to L. crispatus CTV-05,and at the 4-week visit by two women randomized to placebo. Dysuria was reported by one woman randomizedto L. crispatus CTV-05 at the 1-week visitand by one woman randomized to placebo at the 1- and 4-week visits. Headache, abdominal or pelvic cramps/abdominalpain, and low back pain were frequently reported at baseline as well as atfollow-up visits. There were nosignificant differences in reported symptoms at any visit between women in thetwo treatment arms.Abnormalvaginal discharge was the symptom most frequently reported by subjects atclinic visits following study drug use . While nL. crispatus CTV-05 than in women randomized to placebo , though this finding was also often apparent at thebaseline visit (6 women in each arm).One woman randomized to placebo had vaginal erythema noted at 1 week. No cervical, urethral, uterine, or adnexalabnormalities were noted.Physicalexam findings were benign in both treatment arms. External genital erythema was found at the 1-and 4-week visits in more women randomized to L. crispatus CTV-05 had nine episodes of pyuria with a urine white blood cell count ≥8 per mm3 [P = .01). Pyuria was positively correlated with urineleukocyte-esterase test results , lactobacillus (3), E. coli (1), enterococcus (1), group B streptococcus (1), and yeast(1). One subject had a negative urineculture, and none had symptomatic infection.Lactobacillus and mixed gram-positive rods were isolated at 103-104 cfu/ml from more women with pyuria (50% and 75%) than without  at the 1-week visit (L. crispatus CTV-05 versus placebo orin women with pyuria compared to those without pyuria at any visit. Two women randomized to L. crispatus CTV-05 and one randomized to placebo had hematuria atthe 4-week visit (3).Seven(47%) women randomized to  per mm3   growth.28 of 30 women had vaginalcolonization with Hp visits . Two womE. coli from one or more clinic visits. E. coli was isolated from vaginal cultures taken at the 1- and 4-week visits from 4women randomized to L. crispatus CTV-05 and from one women randomized to placebo . Threewomen had a positive assay for L.crispatus CTV-05 detected at the baseline visit before administration of thestudy drug. No women randomized toplacebo had a positive assay at any visit (P = .22 each visit).Using rep-PCR, L. crispatus, 37% had L. jensenii, 17% had L. gasseri, and 27% had other Lactobacillus species detected invaginal culture. The followingcombinations of Lactobacillus specieswere found: L. crispatus (27%), L. crispatus and L. jensenii (20%), L.crispatus and L. gasseri (7%), L. crispatus and other Lactobacillus species (13%), L. crispatus, L. jensenii, and L. gasseri (3%), L. crispatus, L. jensenii, and other Lactobacillus species (7%), L. crispatus, L. gasseri, and other Lactobacillus species (3%), L. jensenii (7%), L. gasseri (3%), other Lactobacillus species (3%), and no Lactobacillus (7%). The prevalences of individual species wererelatively stable over time .Wecompared our rep-PCR results to previver time . There wL. crispatus CTV-05 vaginal suppository, for the prevention of recurrent UTI was well tolerated with minimal side effects. There were no serious adverse events, anddespite the occurrence of abnormal vaginal discharge and external genital orvaginal irritation in several women, compliance was high. These mild to moderate symptoms appear to besecondary to the act of suppository use or a reaction to the preservationmatrix rather than a consequence of L.crispatus CTV-05 as they occurred with similar frequency among women ineach treatment arm. Several women alsoreported vaginal candidiasis. However,given the frequency of recurrent vaginal candidiasis experienced by subjectsprior to enrollment and the prevalence of Candida in baseline vaginal cultures, the relationship of this condition to study druguse is not compelling.In phase I, placebo-controlled trial, use of a L. crispatus CTV-05 use was associatedwith pyuria (detected by microscopy and urine leukocyte-esterase) in seven womenat either the 1- or 4-week visits (P = .04 at 4 weeks). Pyuria was not associated with urogenitalsymptoms, exam findings, or symptomatic UTI.While lactobacillus was not isolated from the urine during every episodeof pyuria, it is possible that some lactobacilli were misclassified as mixed gram-positiverods, as both were more frequently found in women with pyuria than in womenwithout. Our data suggest that vaginalinstillation of L. crispatus CTV-05 inducesa mild inflammatory response in the bladder or vaginal mucosa of some subjectswithout causing prolonged urogenital infection.The importance of this phenomenon with respect to safety or potential efficacyis unclear. It is possible that induction of an asymptomatic inflammatoryresponse by lactobacilli protects against uropathogen colonization of thevagina or infection in the bladder [ bladder .L. crispatus therapy is safe.Women surveyed after participation in a clinical trial of a vaginalcapsule formulation of L. crispatus CTV-05for treatment of bacterial vaginosis rarely reported adverse effects, and thosereported were largely related to a perceived difference in vaginaldischarge. Satisfaction with the vaginalcapsule was high [L. crispatus CTV-05 givenintravaginally to a small group of healthy women [L.crispatus strain GA198332 as a vaginal suppository every other day for oneyear [Lactobacillus haveindicated that they can be given to women safely with minimal or no sideeffects [Lactobacillus has not been previously reported, and should be evaluated further in futurestudies.Limitedexisting data from prior studies suggest that was high . No advehy women . Nine wooneyear . Studieseffects –18. To oL. crispatus CTV-05 use on vaginal Lactobacillus growth. However, most study participants had heavyvaginal growth of H2O2-producinglactobacilli at baseline, making detection of subsequent changes difficult. Data from a pilot study indicated that L. crispatus CTV-05 did not displace otherendogenous vaginal lactobacilli. Vaginalcolonization was most successful in women lacking vaginal H2O2-producinglactobacilli at the outset, suggesting that a lactobacillus probiotic may bemost effective at establishing vaginal colonization in women with abnormalflora, such as those with bacterial vaginosis or recurrent UTI [Wedid not see an effect of rent UTI .L. crispatus CTV-05 in a larger-scale clinical study. Although none of the placebo recipients werecolonized with lactobacillus strains having the characteristic rep-PCR pattern of L. crispatus CTV-05 at any time, threewomen randomized to L. crispatus CTV-05 had at least one isolate in their vaginal culture at baseline that borethe characteristic fingerprint. Therefore,colonization of the vagina following suppository administration was difficult to demonstrate. As there are nolarge-scale surveys of the prevalence and distribution of lactobacilli bearing this fingerprint, we do not know whether the rep-PCR technique failed todistinguish L. crispatus CTV-05 fromother genetically closely related strains of L. crispatus naturally colonizing the vagina at baseline, orwhether the identical strain was prevalent in our study population. Of note, subjects in our study had a muchhigher prevalence of vaginal H2O2-producinglactobacilli in baseline vaginal cultures than found in previous studies [L. crispatus, L. jensenii, and L. gasseri to be the most prevalent species, with relatively stableprevalences over time. Our prevalencedata are consistent with what has been previously documented [L. crispatus in women before and after the introduction of active suppositories or placebo.To our knowledge, our study represents the first attempt to use rep-PCR to identify the genetic fingerprint of the probiotic strain  studies . By comp studies , we charcumented , 21. AddL. crispatus CTV-05 on the rate of UTIrecurrence. Therefore, it is difficultto interpret the report of cystitis in two women randomized to L. crispatus CTV-05. Other small studies have suggested that use ofvaginally administered L. crispatus maybe associated with a lower rate of UTI recurrence [Our study was notdesigned nor statistically powered to evaluate the effect of currence , 29.The major strengthsof our study include the placebo-controlled, randomized study design, excellentcompliance, and extensive and complete followup. Use of a study diary provided finer detailsof our subjects’ experiences that confirmed adverse events and symptomsreported from memory at clinic visits. Weaknessesthat should be mentioned include the inability to accurately document vaginalcolonization with the probiotic strain, and the inherent lack of statisticalpower due to the small sample size typical of phase I studies. We are currently conducting a phase II trialthat will have more power to clarify some of the issues raised in thisstudy.L. crispatus CTV-05 iswell tolerated when given as a vaginal suppository to healthy women with ahistory of recurrent UTI. Mild tomoderate side effects related to suppository use occur but do not affectcompliance. L. crispatus CTV-05 maycause mild asymptomatic inflammation of the lower urinary tract. Timing of administration and efficacy inpreventing recurrent cystitis should be evaluated further in larger studies.Inconclusion,


Streptococcus pneumoniae is one of the most frequently encountered pathogens in humans but its differentiation from closely related but less pathogenic streptococci remains a challenge.S. pneumoniae, and its performance compared to other genotypic and phenotypic tests.This report describes a newly-developed PCR assay (Spne-PCR), amplifying a 217 bp product of the 16S rRNA gene of S. pneumoniae, not amplifying S. pseudopneumoniae or any other streptococcal strain or any strains from other upper airway pathogenic species. PCR assays  were previously described for the specific amplification of S. pneumoniae, but psaA-PCR was the only one found not to cross-react with S. pseudopneumoniae.The new PCR assay designed in this study, proved to be specific at 57°C for S. pseudopneumoniae as S. pneumoniae. Four other PCR assays and the AccuProbe assay were unable to distinguish between these species.Spne-PCR, developed for this study, and psaA-PCR were the only two assays which did not mis-identify  Streptococcus pneumoniae is one of the most pathogenic bacteria involved in human disease [S. pneumoniae from patient samples and to differentiate it from other less pathogenic viridans streptococci, frequently encountered in respiratory samples. Differentiation is also important with regard to resistance testing, since different antibiotic susceptibility breakpoints are applied for S. pneumoniae with regard to other viridans species [ disease , causing disease . Culture species . Culture species .S. pneumoniae variants [S. pseudopneumoniae is difficult to distinguish from S. pneumoniae and is e.g. positive with AccuProbe as well [However, straightforward phenotypic identification of pneumococci is hampered by the occurrence of optochin resistant variants -8 and. I as well .S. pneumoniae. Therefore several PCR assays have been developed over the past decades [It is of clinical relevance to rapidly and specifically detect  decades -14.S. pneumoniae PCR assays to that of a new approach, based on the 16S rRNA gene. One novelty of this approach regards the in-silico design of specific primers using published Streptococcus sequences, filtered for quality and annotation-reliability by profile-based methods . The approach used by this program relies on a systematic analysis of all published 16S rRNA gene sequences for all streptococci (and closely related organisms), using sequence profiles to eliminate obviously incomplete or erroneous submissions, which could induce wrong alignments.This study compared the specificity of four published S. pneumoniae and closely related relatives confirmed the consistency of the sequence pattern found. This method helped to detect discriminative species-specific sequence patterns for S. pneumoniae and S. pseudopneumoniae, thus saving time and effort through reduction of non-specific results in wet-lab testing.Sequences with a likely incorrect annotation (shown by low match scores to other sequences of the same species), with unexpected deletions/insertions, or with non useful annotations (e.g. "uncultured") were excluded, since they could induce misleading alignments. Thus, the most representative 16S rRNA sequences were determined for each species. Such a database of representative sequences was used to align closely related streptococcal species to identify specific positions in the 16S rRNA gene for the purpose of species-specific identification. Once these positions were identified, general searches on relevant published sequences of Streptococcus mitis [S. oralis [S. pneumoniae, including two reference strains and the type strain and including 10 optochin resistant strains, for which it was concluded in a previous study [S. pneumoniae. In addition, a total of 30 optochin resistant pneumococcus-like streptococci already well-characterized in an earlier study [S. pseudopneumoniae [Haemophilus influenzae (NCTC 8143T), Moraxella catarrhalis , Staphylococcus aureus (ATCC 29213 and NCTC 08530), S. epidermidis (CCM2 124T and CNRS N860069), Streptococcus agalactiae (LMG 14694T), S. anginosus (LMG 14502T), S. gallolyticus (LMG 16802T), S. mutans (LMG 14558T) and S. pyogenes (LMG 14237), i.e. species also present in the upper airway tract and/or other Streptococcus species, were used to test the specificity of primer set Spne1-Spne2Rb. In addition, S. parasanguinis (LMG 14537T and LMG 14538) and S. sanguinis  isolates were used to test the specificity of primer set Spne1-Spne2Rb.A total of 73 streptococcal strains were analyzed in this study, as listed in Table us mitis  and seve. oralis , includius study  that theer study , were aleumoniae  were incDNA-extraction was carried out by alkaline lysis as described previously [eviously . always The amplification reactions were performed as described previously , with miS. pneumoniae. Primers were designed to match exactly 2 positions within the 16S rRNA gene, which allow to distinguish S. pneumoniae from S. pseudopneumoniae , which allows rapid screening of validated published sequences of species of interest against other closely related species was used to design primers for the specific amplification of S. pneumoniae, and the stringent annealing temperature was determined by gradient PCR as 57°C , 0.5 μM of each of the two primers and 1 μl of the DNA extract. The 16S rRNA gene primers Spne1-Spne2Rb was designed to amplify S. pneumoniae.The cycling parameters were 94°C for 2 min, followed by 30 cycles of 10 sec at 94°C, 15 sec at 57°C, 1 min at 72°C, and final extension at 72°C for 5 min. The indicated primers are amplifying a 217 bp product of the 16S rRNA gene of Other PCR assays, i.e. ply- [psaA- [lytA-PCR [.e. ply- , psaA- [- [psaA-  and lytAlytA-PCR  were deslytA-PCR .Published streptococcal and other 16S rRNA sequences were extracted from EMBL using proprietary extraction methods based on sequence profiles and annotation searches; representative sequences for each species were determined using a proprietary algorithm developed by SmartGene for bacterial 16S rRNA sequences (SmartGene IDNS™ Bacteria Module). Sequences were analyzed and compared using search and alignment functions of the IDNS™ Bacteria Module of SmartGene. Results were exported as CLUSTAL-A or FASTA files for further analysis.psaA, lytA and ply-PCR results for a collection of 49 optochin resistant alpha-hemolytic streptococcal isolates, suspected of being atypical pneumococci [psaA-PCR to be the most specific genotypic technique for the identification of genuine pneumococci and optochin resistant pneumococci. In addition, in this study, 16S rRNA gene based primers Spne1 and Spne2Rb (Spne-PCR) were designed to amplify S. pneumoniae isolates and we tested these primers for specific amplification of S. pneumoniae using the same, previously well-studied selection of isolates [S. pseudopneumoniae and two S. parasanguinis isolates were added. Also, we tested the specificity of a PCR assay, i.e. spn9802-PCR [S. pneumoniae.Previously we reported on encapsulation, AccuProbe hybridization and umococci . We concisolates , to whicS. mitis group isolates, for 7 S. oralis isolates and for the 19 optochin R streptococcal isolates for which we had already concluded in the previous study [S. pneumoniae. In addition, Spne-PCR was negative for the three S. sanguinis and the two S. parasanguinis isolates.All five PCR assays were negative for seven commonly found respiratory tract species, for 8 us study  that theS. pneumoniae isolates included and for the ten optochin resistant streptococci, which had been considered as S. pneumoniae already, based on the PCR results from our previous study [All five PCR assays were positive for the nine optochin susceptible us study Table 1)S. pneumolytA-PCR, nine with ply-PCR and Spn9802-PCR (9), whereas none of these isolates yielded a positive result when tested with psaA-PCR and Spne-PCR.Thus far, all five PCR assays were found to be equally specific. However, for a total of 11 optochin R streptococcal isolates, designated during the previous study as group IIa and group IIb, four were positive with S. pseudopneumoniae type strain CCUG 49455T, the two S. pseudopneumoniae reference isolates CCUG 48465 and CCUG 50866, and one optochin-resistant pneumococcus-like isolate (KTL079) were positive with lytA-PCR, ply-PCR and spn9802-PCR, but negative with Spne-PCR and psaA-PCR. Sequence determination of the 16S rRNA gene (accession number: FJ827123) identified this clinical isolate unambiguously as S. pseudopneumoniae. This isolate was also positive with AccuProbe , thus possibly enhancing sensitivity when this PCR is applied for the direct detection and identification of S. pneumoniae in clinical samples.In addition, the advantage of a specific PCR test on the basis of the 16S rRNA gene is that there are several copies of this gene, i.e. 5 to 6 in other  species  and 4 coIn addition, 16S rRNA gene sequencing is a standard method in microbial taxonomy and can be applied directly on the amplified products of this PCR assay to to help resolve potentially ambiguous results.Spne-PCR, described here, and psaA-PCR [S. pseudopneumoniae as S. pneumoniae. The approach using representative sequences rather than unfiltered data from Genbank enabled us to select the correct sites for reliable species differentiation out of less relevant and consistent variations and allowed us to design highly specific primers. Future studies should enable us to develop an assay specifically for S. pseudopneumoniae and a real-time, multiplex assay for rapid discrimination of the most important viridans streptococci in bacterial cultures or patient samples.psaA-PCR  were theThe authors declare that they have no competing interests.NAE, SE, RV and MV participated in the development of the study design, the analysis of the study samples, the collection, analysis and interpretation of the data, and in the writing of the report. TK, TDB, BS, EA and PD participated in the analysis of the study samples and interpretation of the data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/104/prepub


Entamoeba gingivalis  is a parasitic protozoa of the oral cavity, most often found in gingival tissues around the teeth associated with poor oral hygiene. Here, we report a case of E. gingivalis in a pulmonary CT guided fine needle aspiration material, from a 60-year-old man with newly found lung mass. On site Diff-Quik® smear examination revealed the presence of marked acute inflammation, colonies of actinomyces, and a number of ‘large macrophages-like organisms’. Upon examination of the additional material, organisms morphologically consistent with E. gingivalis were identified. Pulmonary mass resolved after six weeks of treatment with antibiotics (Clindamycin followed by Penicillin). Proper recognition and distinction between E. gingivalis and other species of Entamoeba is important for the management of patients. A 60-year-old man was admitted for dehydration, weight loss, poor appetite and unstable gait. His past medical history was significant only for alcohol and tobacco use. A CT scan showed a large (50.0 × 50.0 mm) left lower lobe lung mass , during ® stained slides [E gingivalis. The patient was subsequently discharged, after he showed satisfactory improvement, and treated with antibiotics for six weeks (Clindamycin followed by Penicillin). A chest CT scan examined two months later revealed total resolution of the pulmonary mass, with only a small linear scar left in the left lower lobe. Microbial cultures of the pulmonary aspirate grew mixed Gram positive and negative rods.On-site cytological evaluation revealed marked acute inflammation and colonies of filamentous actinomyces. A few organisms, morphologically resembling large macrophages and embedded within the acute inflammatory exudates, especially along the edge of the filamentous structures, were also identified in the Diff-Quikd slides . They ocd slides . The orgE. gingivalis in sputum specimens was first reported in Western literature by Sutliff et al. in 1951[E gingivalis has been reported in the specimens obtained from the cervix, uterus, and neck lymph node[The occurrence of ymph node–5.E. gingivalis resembles E. histolytica-like amoebae and its trophozoite measures 10-35 µm in size. No cysts are observed in vivo, although cystic forms have been described in culture studies.[E. gingivalis. It was first documented in the year 1986, by the Johns Hopkins group, and their precise identification was made in the year 1992, using ribosomal RNA gene sequencing.[ studies. Transmis studies. Actinomyquencing.E. histolytica and E. gingivalis can be difficult. The morphologic appearances of E. histolytica and E. gingivalis are quite similar, although the trophozoites of E. gingivalis tend to be comparably larger (10-35 µm vs 15-20 µm).Distinction between Several important features that may help to distinguish these two parasites include the following:E. gingivalis has no cyst stage recognizable in the clinical specimens. It has multidirectional pseudopods, best seen in wet mount preparations and Diff-Quik® preparations [arations .E. gingivalis is the only species of amoeba that can phagocytose nuclear fragments of white blood cells, ingest bacteria, and an occasional red blood cell. Ingested material is often seen within the large food vacuoles [Figures 1 Figures , C, E.[1Figures 1E. gingivalis was seen to have coarser karyosome and a less delicate pattern of peripheral chromatin [E. histolytica. In addition, they are often seen in a markedly inflammed background, with abundant neutrophils. E. histolytica ingests erythrocytes, has a fine central karyosome, delicate peripheral chromatin and a single unidirectional pseudopod.hromatin , upon exE. histolytica serum. [E gingivalis. [Both organisms also stain with periodic acid schiff reaction, but it is nonspecific . Recognia serum.  In additgivalis. 7 The pregivalis. E. gingivalis and E. histolytica, for the management of patients. Treatment of associated bacteria infection with antibiotic might be enough for E. gingivalis infection. However, amebicidal agent (such as Iodoquinol or Paromomycin) or antiprotozoal agent (such as Timidazole) might be necessary to eliminate the E. histolytica infection.It is prudent to distinguish between


Two tbpB families have been documented in Neisseria meningitidis: an isotype I tbpB gene of 1.8 kb and an isotype II tbpB gene of 2.1 kb, the former expressed by meningococci in the disease-associated ST-11 clonal complex and the latter found among meningococci belonging to the hyper-invasive clonal complexes including ST-8, ST-18, ST-32, ST-41/44 as well as N. gonorrhoeae isolates. The origin of the isotype I tbpB gene is unknown, however several features in common with non-pathogenic Neisseria and the ST-11 clonal complex N. meningitidis isolate FAM18 have been documented leading to the hypothesis that the isotype I tbpB gene may also be shared between non-pathogenic Neisseria and ST-11 meningococci. As a result, the diversity of the tbpB gene was investigated in a defined collection of Neisseria species.Transferrin binding protein B (tbpB were identified: family A containing conserved genes belonging to ST-11 meningococci, N. polysaccharea and N. lactamica isolates and family B including more diverse isotype I tbpB genes from N. sicca, N. mucosa, N. flava, N. subflava as well as N. cinerea, N. flavescens and N. polysaccharea isolates. Three isotype II tbpB families were identified with: family C containing diverse tbpB genes belonging to N. polysaccharea, N. lactamica, N. gonorrhoeae and N. meningitidis isolates, family D including another subset of isotype II tbpB genes from N. lactamica isolates and family E solely composed of N. gonorrhoeae tbpB genes.Two families of isotype I Neisseria with the origin of the isotype I tbpB gene resulting from a horizontal genetic transfer event occurring between these two populations.This study reveals another instance of similarity between meningococci of the ST-11 clonal complex and non-pathogenic  Neisseria contains 12 species and biovars colonising humans most of which are non-pathogenic colonisers of the upper respiratory tract [Neisseria gonorrhoeae, the etiological agent of gonorrhoea and Neisseria meningitidis, a major cause of meningitis and septicaemia worldwide, regularly cause disease in humans [The genus n humans ,4.Neisseria within the human host is the ability to acquire iron, the majority of which is not circulating freely in the human body but is stored in ferritin and haemoglobin or is complexed with the glycoproteins transferrin and lactoferrin [Neisseria have devised ways to counteract this iron limitation through the evolution of several high-affinity receptor systems including the lactoferrin binding proteins A and B, the transferrin binding proteins A and B, and the haptoglobin-haemoglobin receptor HpuAB, each composed of an accessory lipoprotein subunit and a TonB-dependent gated porin [Neisseria can obtain iron through the expression of the surface-exposed haemoglobin receptor HmbR [A major determinant in the survival of toferrin . Neissered porin -11. In ator HmbR ,13.tbpB, N. meningitidis isolates can be classified into two major families: isotype I (tbpB gene of 1.8 kb and TbpB protein with a mass of approximately 68 kDa) and isotype II (tbpB gene of 2.1 kb and TbpB protein with a mass of approximately 80 to 90 kDa) [tbpB genes have been documented in several N. meningitidis clonal complexes including ST-8, ST-32, ST-18 and ST-41/44 as well as non-pathogenic Neisseria [tbpB genes, on the other hand, have solely been identified among N. meningitidis isolates belonging to the ST-11 clonal complex and have not been detected among other Neisseria species. Four tbpB families were recently described based on partial nucleotide sequences from serogroup A clonal complex ST-4 N. meningitidis and N. lactamica isolates collected in The Gambia [tbpB alleles from N. meningitidis and N. lactamica isolates and families two and three included isotype I tbpB alleles. Importantly, among the 138 isolates analysed  only three isotype I tbpB alleles were found, all of which belong to meningococci [Based on antigenic and genomic features of TbpB and  90 kDa) . Isotypeeisseria -17. Isote Gambia ,17. Famingococci ,17.tbpB gene, ST-11 meningococci can be distinguished from other hyper-virulent clonal complexes by the absence of an opcA gene and the possession of a class 2 porB gene [Neisseria spp. have been reported including the clustering of pilE sequences [opcA negative locus in two N. polysaccharea isolates [Neisseria and ST-11 meningococci which may have contributed to the described genetic isolation of this clonal complex [tbpB gene is unknown. Consequently, the distribution of the gene in a defined collection of Neisseria spp. was investigated with the hypothesis that the isotype I tbpB gene was present in other Neisseria spp.Meningococci from the ST-11 clonal complex have been a major cause of meningococcal disease worldwide throughout the last century and despite low carriage rates continue to be associated with disease . In addiorB gene -21. Furtequences  and the isolates . Taken t complex . The oritbpB genes were isolated from the non-pathogenic Neisseria spp. Two families of the gene became apparent. The first contained sequences closely related to meningococcal ST-11 tbpB genes belonging to three N. polysaccharea and two N. lactamica isolates. The second included more divergent isotype I tbpB genes from the non-pathogenic Neisseria spp. N. sicca, N. mucosa, N. flava, N. subflava, N. cinerea, N. flavescens as well as from another three N. polysaccharea isolates  displayed 100% sequence identity in both isotype I TbpB families, whereas six non-synonymous changes were observed among isotype II TbpB. Domain N4 (residues 409 to 422 in N. gonorrhoeae FA19) was also highly conserved among isotype I TbpB with the occurrence of three non-synonymous substitutions. Five variable sites were present among isotype II TbpB. Domain C3 (residues 703 to 713 in N. gonorrhoeae FA19) showed the most diversity with the occurrence of four non-synonymous substitutions among both TbpB isotypes.Functional assessment of the protein was beyond the scope of the present study. Nevertheless, previously documented putative transferrin binding sites were observed based on predicted translations of the nucleotide sequences ,27. In ptbpB sequences from isolates B16B6, M982, 8680, 8726, 2713, 2717 and FA19, used in previous analyses [N. meningitidis isolates FAM18, Z2491, MC58 and N. gonorrhoeae FA1090.All of the sequences were aligned manually with sequences starting from and ending at the same amino acid residue in each isolate. Published isotype I and II analyses ,16,17,27Phylogenetic analysis was undertaken using the software package ClonalFrame version 1.1, which is a statistical model-based method initially described for inferring bacterial clonal relationships using multilocus sequence data . InferentbpB genes indicated that these were a closely related group of sequences compared with the depth of the branches seen for isotype II tbpB genes where greater diversity is known to occur [tbpB genes observed by sequence analysis as well as three clusters of isotype II tbpB genes. For ease of interpretation, the two isotype I tbpB families have been named tbpBA and tbpBB with the isotype II clusters named tbpBC through to tbpBE  belonging to a variety of Neisseria spp. as well as containing a subset of N. polysaccharea isolates  with 85 polymorphic sites, the majority of which occur among N. polysaccharea and N. lactamica isolates. Overall, six fixed differences were observed between the genes of ST-11 meningococci and those of N. lactamica and N. polysaccharea with no shared polymorphisms between the two populations. Family B tbpB genes were more diverse  with 415 polymorphic sites. In a comparison of both gene families, there were 210 fixed differences and 54 shared mutations.Genes belonging to family tbpBC, D and E were more diverse . Genes belonging to the N. polysaccharea isolates shared 99% identity with 16 segregating sites, seven of which encoded non-synonymous changes. N. lactamica tbpB genes were more diverse with 696 polymorphic sites while 829 polymorphisms were observed among N. gonorrhoeae tbpB genes. A total of 445 shared mutations were observed between N. lactamica and N. meningitidis tbpBC genes indicative of recombination, while only six were apparent between N. polysaccharea and N. meningitidis.As expected, families tbpBA genes or between these and family tbpBB, however a mosaic gene structure was present among the latter indicating that recombination occurred frequently among tbpBB genes from N. sicca, N. flava, N. subflava, N. mucosa, N. flavescens, N. cinerea and N. polysaccharea  was included in the present study and harboured a tbpB gene similar to that of N. meningitidis isolate FAM18. Furthermore, opcA genes are absent among meningococci belonging the ST-11 clonal complex and were also undetectable among N. polysaccharea isolates 87043 and 90400 [tbpB genes. The identification of isotype I family A and B genes among Neisseria spp. is another characteristic shared with N. meningitidis isolates belonging to clonal complex ST-11 and is indicative of the occurrence of several horizontal genetic transfer events between non-pathogenic Neisseria, in particular N. polysaccharea and meningococci belonging to this clonal complex.A number of features are shared between clonal complex ST-11 rea pilE . The N. nd 90400 ,20, whictbpB isotypes among Neisseria are unknown. However, seclusion of isotype I tbpB to ST-11 clonal complex meningococci may be due to clonal expansion or selection for this isotype. Indeed, the isotype I TbpB protein has been shown to play an essential role in iron acquisition from human transferrin with isogenic mutants deficient in TbpB failing to grow on hTf as a sole iron source [tbpB genes belonging to families A (mean p-distance ranging from 0.001 to 0.040) and B (mean p-distance 0.117), highlighting the importance of this gene in contributing to the fitness of the organism. There has been selection for isotype I tbpB among meningococci belonging to the ST-11 clonal complex such that it has become restricted to this clonal complex. In contrast, isotype II tbpB genes have been found to provide a purely facilitating role such that TbpB-deficient mutants were only incapacitated with slower growth [N. gonorrhoeae, H. influenzae and M. catarrhalis  [tbpB gene has in iron acquisition may contribute to its hyper-variability. Indeed, Zhu et al found that the high rate of import among isotype II tbpB genes, although providing a temporary advantage because of antigenic composition, resulted in reduced fitness of the isolates [tbpB genes  -37. The isolates ,17. The nes Fig.  &2C combtbpB variants among Neisseria spp. and aimed to discover the origin of the isotype I tbpB gene. Results revealed this gene was found among diverse Neisseria spp. indicating the occurrence of a horizontal genetic transfer event between N. meningitidis and non-pathogenic Neisseria. Three features shared between ST-11 meningococci and non-pathogenic Neisseria have now been described: (i) the presence of isotype I tbpB genes (ii) the identical sequences upstream of the pilE gene and (iii) the analogous genetic organisation of the opcA negative locus.This work investigated the distribution of the two tbpB genes into two new families: tbpBA and tbpBB the former contained tbpB genes closely related to ST-11 clonal complex meningococci, the latter included the more distantly related tbpB genes belonging to many non-pathogenic Neisseria species. The scheme also separates isotype II tbpB genes into three new families: tbpBC comprising tbpB genes from N. meningitidis, N. lactamica, N. polysaccharea and N. gonorrhoeae isolates, tbpBD consisting of tbpB genes from N. lactamica and N. meningitidis isolates and finally, tbpBE containing N. gonorrhoeae tbpB genes.A revised nomenclature was proposed according to the published guidelines . The schNeisseria and N. gonorrhoeae isolates used in this study are listed in Table 2. Boiled cell suspensions were prepared for each isolate. Briefly, a PBS solution of overnight GC grown bacteria was boiled for 5 minutes, centrifuged and the supernatant stored at +4°C before being directly used for PCR.The non-pathogenic tbpB genes were completed using primers listed in Table 2 system along with the Sequitherm Excel II DNA sequencing kit . Additional sequencing was carried out by cycle sequencing with BigDye Ready Reaction Mix (Applied Biosystems) according to manufacturer's instructions and using an ABI 377 automated DNA sequencer.Amplification and sequencing of tbpB nucleotide sequences were assembled using the Staden sequence analysis package [The  package  and all  package . In the  package .tbpB genes was assessed by calculating p-distances within each tbpB family revealing the proportion (p) of nucleotide sites at which sequences differed. These analyses were conducted using MEGA. The number of fixed differences and shared polymorphisms were obtained using the software DnaSP [tbpB genes are listed in table The level of sequence diversity between re DnaSP . Old andOBH participated in the planning of this study, performed all experimental work, data analysis and drafted the manuscript. MCJM participated in writing the manuscript. BR participated in the planning of this study, coordinated the study and assisted in writing the manuscript.


This paper reports an evaluation of the IL-Phoenix Chemistry/Electrolyte Analyser; the evaluation was carried out in accordance with internationally recognized guidelines. The evaluation was performed in three steps: evaluation in routine conditions; assessment of interferences; and study of practicability. Seven constituents were studied under routine working conditions. Within-run imprecision rangedfrom 0.6% (CV) for chloride to 3.1% (CV) for glucose. Between-run imprecision ranged from 0.9% for sodium to 6.0% (CV) for urea. Sample-related carryover was not significant. The relative inaccuracy was acceptable; drift was negligible; linearity was agreed with the range showed by the supplier. Haemoglobin produced negative interferences with sodium and chloride. Turbidity interfered negatively with sodium, chloride, potassium and total calcium, andpositively with glucose. Bilirubin showed a negative interference with sodium, chloride and creatinine.


Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described.B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae.In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches.The genomic analysis suggests that  Bordetella comprises several human and animal pathogens and nine species are currently described [see Additional file B. pertussis and B. parapertussis cause whooping cough, whereas B. bronchiseptica and B. avium are responsible for respiratory infections in many mammals and birds, respectively [B. pertussis and significant economic losses associated with infections in poultry and cattle. The genomes of these Bordetellae were recently sequenced and analyzed [B. avium revealed that it is quite divergent from the mammalian pathogens. In contrast, the genomes of the human pathogens B. pertussis and B. parapertussis showed that they are independent derivatives of B. bronchiseptica-like ancestors. They both underwent significant gene loss, probably mediated by insertion sequence (IS) elements, during the process of host adaptation, as indicated by their smaller genome sizes compared to that of B. bronchiseptica and the presence of many pseudogenes. The evolution of human-adapted species is characterized by massive genome reduction, lack of horizontal acquisition of genetic material and significant differences in virulence gene expression among species [The genus ectively -3. Despianalyzed ,5. The g species ,6.B. petrii, was isolated from a dechlorinating bioreactor enriched by river sediment [B. petrii strains were repeatedly found in environmental samples, e.g. microbial consortia degrading aromatic compounds [B. petrii strains were isolated from patients suffering from mandibular osteomyelitis and chronic suppurative mastoiditis [B. petrii were also found in cystic fibrosis patients [B. petrii strains were also isolated from marine sponges and a grass root consortium [Achromobacter and Alcaligenes [see Additional file The type strain of the only environmental species of the genus, sediment . B. petrompounds ,9. Recentoiditis ,11. Bactpatients . Moreovensortium ,14. PhylB. petrii exhibits properties of host-associated and environmental bacteria, it is important to perform a genomic analysis of this bacterium in order to understand the genetic basis of its versatility. The B. petrii genome provides insights into the evolutionary history of the Bordetellae, acting also as a spyglass to the Alcaligenes/Achromobacter group for which, despite their biotechnological potential, no sequenced genomes are available yet.Since Bordetella genomes are shown in Table B. petrii has a similar size  and coding-capacity  to that of B. bronchiseptica . The genomes of B. pertussis, B. parapertussis and B. avium are much smaller, showing massive gene loss during host-adaptation. The overall GC content (65.5%) of B. petrii is intermediate between that of B. bronchiseptica (68.08%) and B. avium (61.58%). B. petrii has 125 pseudogenes, less than B. pertussis (358) and B. parapertussis (220), but more than B. bronchiseptica (18) and B. avium (68).The general features of the B. petrii and B. avium show lower similarity and synteny than those of mammalian-adapted Bordetellae, suggesting that they are most distantly related. Reciprocal BLASTP analysis with other sequenced Bordetellae identified coding sequences unique to B. petrii  than with B. avium . Interestingly, B. petrii has 1,825 CDSs that are not present in either B. bronchiseptica or B. avium. A large number of these B. petrii-unique CDSs  are located on mobile elements and encode accessory metabolic functions . The locations of these GIs on the chromosome correlate with atypical GC-content and GC-bias  are related to the clc element of Pseudomonas knackmussii B13, which codes for proteins involved in the biodegradation of chloroaromatics [clc element is mostly confined to the conjugal transfer region and varies substantially (42%–98%) between individual genes within this region [see Additional file clc element and also carries all the chloroaromatic degredation genes found in the clc element. Like the clc element in B13, these four GIs are integrated into the 3'-end of tRNAGly genes, are flanked by a 15–18 bp direct repeat generated by the duplication of the 3'-end of tRNA, and each encodes an integrase highly similar  to that of the clc element (IntB13). GI1, GI2 and GI3 are located in close proximity within a 510 kb region .The most prominent feature that distinguishes slands GI-GI7. Theromatics . The simslands GI-GI7. The habitat . During  habitat . Interes4371 of Ralstonia eutropha A5/Cupriavidus oxalaticus, a 55-kb conjugative transposon that carries genes involved in the degradation of biphenyl or 4-chlorobiphenyl [The remaining GIs  encode a conjugal transfer system similar to the type IV secretion systems associated with plasmids. GI4 is highly similar to Tnbiphenyl . The feaB. pertussis and B. parapertussis carry a large number of IS elements . These IS elements are responsible for the extensive shuffling and deletions in their genomes [B. bronchiseptica and B. avium carry none. B. petrii carries 105 IS elements, most of which belong to the IS3-family. Interestingly, there are six copies of IS1663 which was found in B. pertussis and one truncated copy of IS1001 (Bpet1198) which was found in B. parapertussis. The vast majority (66%) of the B. petrii IS are located within GIs, whereas 34% are randomly dispersed around the chromosome. B. petrii also carries two intact prophages  , IS elements and prophages represent 22% of the B. petrii and the pathogenic Bordetellae. For instance, the glycolytic pathway is incomplete, lacking the determinants for glucokinase and phosphofructokinase. B petrii encodes all the enzymes needed for the TCA cycle and for the synthesis of all essential nucleotides, cofactors, amino acids and fatty acids. However, the in silico analysis of the genome sequence of B. petrii suggests the presence of a set of auxiliary pathways for the use of alternative nutrients, such as gluconate, degraded plant products, cyanate (as N-source), and various aromatic compounds (see below), which may contribute to the ability of B. petrii to thrive in different environments  pathway, which is only found in few bacteria, such as Rhodobacter sphaeroides, but absent in all the other sequenced Bordetellae [kdgA and kdgK genes suggesting that this variant ED pathway may be inducible by this regulator depending on the availability of the external carbon source. In this regard, it is surprising that although a periplasmic glucose dehydrogenase , which converts glucose to gluconate, is encoded in the genome, B. petrii is unable to metabolize glucose [Gluconate is an intermediate in the degradation of sugar acids and sugar derivatives, which are components of more complex molecules, such as plant materials and secondary metabolites. The genome analysis of detellae . In this glucose .B. petrii encodes a protein (Bpet0241) with significant similarity to pectate lyases, extracellular enzymes secreted by phytopathogens, such as Erwinia carotovora, which catalyze the cleavage of pectate [B. petrii uses plant cell-wall constituents as nutrient sources, which is in line with the isolation of a B. petrii strain from a plant root consortium [B. petrii is not only able to cope with the toxicity of environmental cyanate, but like E. coli may use it as a nitrogen source [B. petrii and the other Bordetellae is that it has a facultative anaerobic energy metabolism and can utilize nitrate as the terminal electron acceptor during anaerobic respiration [B. petrii encodes the factors  necessary to carry out complete denitrification from nitrate to N2. Interestingly, only some of the respective genes are present in the pathogenic Bordetellae. B. bronchiseptica appears to encode a functional nitrate reductase, while B. parapertussis has a truncated napD gene probably leading to a non-functional nitrate reductase, and B. pertussis does not encode genes involved in denitrification at all.Interestingly, ein Bpet01 with si pectate . The rea pectate . In agrensortium . The pren source . A strikpiration . Accordiin silico analysis of the B. petrii genome revealed an unusual metabolic capability regarding the degradation of aromatic compounds summarized in Figure The clc element of Pseudomonas knackmussii B13. A second set of chlorocatechol pathway genes (Bpet3747-3752) is located on GI5 and shows high similarity to genes identified in the 1,2,4-trichlorobenzene degrading Pseudomonas strain P51 [A prominent feature is the presence of a surprisingly large number of genes coding for enzymes of the chloroaromatic metabolism. One complete set of chlorocatechol pathway genes (Bpet1533-1538) is localized on GI3 highly related to the rain P51 . A thirdrain P51 -32.Cupriavidus strain PS12 and Pseudomonas strain P51 [On GI5, chlorocatechol genes are preceded by a gene cluster (Bpet3738-3742) encoding a chlorobenzene dioxygenase and chlorobenzene dihydrodiol dehydrogenase, highly homologous to those of the 1,2,4-trichlorobenzene degraders rain P51 ,34. The rain P51 ,30,35.B. petrii also harbors four different central pathways for aromatic metabolism via dihydroxylated central intermediates, the gentisate , the homogentisate (Bpet 4016–4017) and the catechol and protocatechuate branch of the 3-oxoadipate pathway (see below). Such versatility is similar to that observed in bacteria well known for their high metabolic capabilities, such as P. putida KT2440 and Cupriavidus necator [B. avium. necator . Only geProteobacteria, however, all previously sequenced Bordetella strains are devoid of complete pathways and only a catIJCD cluster (Bpet4528-4532) is present as a remnant. In B. petrii, complete pathways are formed by genes of the protocatechuate branch (Bpet0260-0254) located in a genomic region with a quite low GC content, but lacking other typical features of a genomic island . As degradation via CoA derivatives needs less oxygen molecules compared to degradation via dihydroxylated intermediates, this may provide B. petrii with a selective advantage to also thrive in oxygen limited environments. For an overview about the aromatic degradation capabilities of B. petrii see Figure A strategy of aerobic degradation of aromatics alternative to the metabolism via dihydroxylated intermediates is initiated by CoA ligases ,39. B. pB. petrii does not code for the well-characterized toxins of classical Bordetellae . However, it produces a low amount (22–53 nM/OD) of the non-protein tracheal cytotoxin (TCT), a spontaneously released muramylpeptide that disrupts epithelia [B. bronchiseptica.Pathogenic Bordetellae produce many surface associated or secreted virulence factors . B. petrpithelia . This exBordetella virulence [B. bronchiseptica encodes 21 autotransporter proteins, whereas B. petrii encodes only four  [B. bronchiseptica, whereas the other two do not show significant similarity to any database entry. Pathogenic Bordetellae also encode a two-partner secretion system which allows secretion of the colonization factor filamentous hemagglutinin (FhaB) [B. petrii encodes proteins related to FhaB and FhaC (Bpet600-601). B. petrii FhaB has 3039 amino acids and its N-terminus, which is required for export, is well-conserved with respect to pathogenic Bordetellae. However, the overall similarity with FhaB of pathogenic Bordetellae is very low, being more similar to putative secreted proteins of plant pathogens . This suggests that the fha genes found in B. petrii and the pathogenic Bordetellae may not be orthologous.Autotransporter or type V secretion proteins play a major role in irulence . B. bronB. petrii and B. avium lack type I and III protein secretion systems, but encode a type II secretion system (Bpet2855-2866 and BAV0331-0343). Type IV secretion systems are either engaged in protein transport to the extracellular medium or into eukaryotic cells, or they are involved in DNA-transfer. There are three gene clusters in B. petrii, which are associated with GIs and appear to encode complete type IV secretion systems . None of these systems appears to be related to the pertussis toxin exporting type IV system of B. pertussis, being probably involved in conjugal DNA transfer.Other protein secretion systems are differentially distributed among Bordetellae. In contrast to the mammalian pathogens, B. petrii genome carries three fimbrial gene clusters , each encoding a major fimbrial subunit, a fimbrial assembly chaperone, an outer membrane usher protein and a fimbrial adhesin. The chaperone and usher proteins have significant sequence similarities with their counterparts in the pathogenic Bordetellae, with e-values ranging from 6e-23 to 3e-35 for the chaperones and e-values ranging from 3e-71 to 6e-103 for the usher proteins. In contrast, the homology of the major structural proteins and fimbrial adhesins with the proteins of the pathogenic Bordetellae is very low . Similarly to B. avium, B. petrii lacks the genes for the biosynthesis of siderophores. However, it specifies 12 TonB-dependent iron scavenging receptors, which may be important for iron acquisition in soil and plant-associated environments.The B. petrii BvgA (Bpet 4471) is highly homologous to the BvgA proteins of the pathogenic Bordetellae (65% amino acid similarity), the structure of the B. petrii equivalents of BvgS is more complex. First of all, there are two histidine kinases, BvgS1 (Bpet4469) and BvgS2 (Bpet4472), with significant sequence similarities to BvgS. BvgS1 contains a PAS, a transmitter and a receiver domain, whereas BvgS2 contains a transmitter and a receiver domain. In contrast to BvgS, both B. petrii proteins are devoid of a large periplasmic domain. The HPt domain, which in pathogenic Bordetellae is part of the multidomain BvgS protein, in B. petrii is an independent protein (Bpet4470)  and a master virulence regulator (Bvg), but lacks Bordetella toxins. This suggests that pathogenic Bordetellae might have acquired their toxin genes horizontally, while adapting to an obligate pathogenic lifestyle. Alternatively, it is possible that toxin genes have been lost from the B. petrii lineage.Therefore, Bordetella species, B. bronchiseptica, B. pertussis, B. parapertussis, B. avium and B. petrii, represent different states of environmental adaptation. These five organisms have followed different evolutionary paths. Evolution of the B. bronchiseptica-derived host restricted-pathogens B. pertussis and B. parapertussis was dominated by substantial gene decay and loss. This might also hold true for B. avium, which is characterized by a relatively small genome containing 68 pseudogenes. In contrast, the evolution of the environmental isolate B. petrii was dominated by horizontal acquisition of large genomic islands expanding its metabolic capacities. B. bronchiseptica, with a broad host range and the potential to survive in the environment, might represent an intermediate evolutionary state. If the Bordetellae derive from a common environmental ancestor, B. petrii appears to be more closely related to this ancestor than the pathogenic Bordetellae, since it is the only true environmental isolate within the genus, which is also found in association with host organisms. Moreover, B. petrii is the only facultative anaerobic Bordetella species [Achromobacter species are also facultative anaerobes, the common ancestor of the Bordetellae probably had a facultative anaerobic energy metabolism. During adaptation to an obligate host association, the genes for several metabolic functions, e.g. those required for denitrification, may have been deleted from the genomes of pathogenic Bordetellae, leaving just a few remnants in the various species.The five sequenced  species . Since c). The complete genome sequence was finished by using the Staden package (GAP v4.8b1) [For the shotgun phase we produced 103,308 paired-end sequences derived from three pTZ18R genomic libraries  yielding 11.72-fold coverage, using dye terminator chemistry on ABI377 and MegaBACE 1000 automated sequencers. As a scaffold we used 2,688 paired-end sequences pCC1BAC libraries with insert sizes of 17–30 kb and 40–70 kb . Assembly of the paired-end sequences was performed by PHRAP  . AnotherCuration and annotation of the genome was done using the genome annotation system GenDB . A combiBordetella avium 197N , Bordetella pertussis Tohama I , Bordetella parapertussis 12822 , Bordetella bronchiseptica RB50 . Similarity searches were conducted against the genomes on the nucleotide and amino acid sequence level by using BLASTN and BLASTP, respectively [For comparative analyses, the annotated genome sequences of the following bacteria were imported into the genome annotation system GenDB : Bordeteectively . Syntenyectively . For theGenomic regions with atypical GC content were identified using the 'sliding window' technique with a window size of 3,000 bp and a step size of 1,000 bp. For this purpose, the GC content was assumed to follow a Gaussian distribution and regions with at least 2.0 standard deviation differences from the mean were determined.2PO4, 0.2 g KCl, 0.1 g MgCl2 × H2O, 1.5 g Tris/HCl resuspended in 100 ml distilled water), 1.8 μM CaCl2, 36 μM FeSO4 × H2O, 2 mM (NH4)SO4. Aromatic carbon sources were added to final concentrations of 2–5 mM each.Growth experiments were carried out in a minimal medium containing 10% salt solution  was quantified as previously described .The complete, annotated genome sequence has been submitted to EBI under the accession number AM902716.RG and CAG organized and coordinated the work and drafted the manuscript. MS, VMdS, DHP and RK performed the bioinformatic sequence analysis of the genome and participated in manuscript preparation. ML performed molecular genetic experiments to characterize the genomic islands. DB, JB, JVC, TE, LG, SH, ANK, CL, SL, BH, SM, DN, CR, SSB, KS, FJV, and TY annotated the genome sequence. FM, OK and AG annotated the sequence and coordinated annotation, JTE and WEG quantified TCT production, DHP performed growth experiments with various carbon sources, HB and RMA coordinated and performed the genomic sequencing, AP and UBG coordinated sequence analysis and participated in manuscript preparation.Bordetella, Achromobacter and Alcaligenes.Dendrogramm of the genera Click here for fileB. petrii GIs and the clc element of Pseudomonas.Comparison of four Click here for fileAdditional Table 1. List of genes involved in the degradation of aromatic compounds.Click here for fileB. pertussis and B. petrii.Schematic presentation of the Bvg-systems of Click here for fileLegends of the additional figures.Click here for file