RESUMO
Lactobacillus crispatus is a member of the vaginal and gastrointestinal human microbiota. Here we determined the complete genome sequence of the probiotic strain M247 combining Nanopore and Illumina technologies. The M247 genome is organized in one circular chromosome of 2â336â109 bp, with a GC content of 37.04â% and 2303 ORFs, of which 1962 could be annotated. Analysis of the M247 mobilome, which accounts for 14â% of the whole genome, revealed the presence of: (i) Tn7088, a novel 14â105 bp long integrative and mobilizable element (IME) containing 16 ORFs; (ii) ΦM247, a novel 42â510 bp long siphovirus prophage containing 52 ORFs; (iii) three clustered regularly interspaced short palindromic repeats (CRISPRs); and (iv) 226 insertion sequences (ISs) belonging to 14 different families. Tn7088 has a modular organization including a mobilization module encoding FtsK homologous proteins and a relaxase, an integration/excision module coding for an integrase and an excisionase, and an adaptation module coding for a class I bacteriocin and homologous to the listeriolysin S (lls) locus of Listeria monocytogenes. Genome-wide homology search analysis showed the presence of Tn7088-like elements in 12 out of 23 L. crispatus complete public genomes. Mobilization and integration/excision modules are essentially conserved, while the adaptation module is variable since it is the target site for the integration of different ISs. Prophage ΦM247 contains genes for phage structural proteins, DNA replication and packaging, lysogenic and lytic cycles. ΦM247-like prophages are present in seven L. crispatus complete genomes, with sequence variability mainly due to the integration of ISs. PCR and sequencing showed that the Tn7088 IME excises from the M247 chromosome producing a circular form at a concentration of 4.32×10-5 copies per chromosome, and reconstitution of the Tn7088 chromosomal target site occurred at 6.65×10-4 copies per chromosome. The ΦM247 prophage produces an excised form and a reconstituted target site at a level of 3.90×10-5 and 2.48×10-5 copies per chromosome, respectively. This study identified two novel genetic elements in L. crispatus. Tn7088 represents the first example of an IME carrying a biosynthetic gene cluster for a class I bacteriocin in L. crispatus.
Assuntos
Bacteriocinas , Lactobacillus crispatus , Bacteriocinas/genética , Bacteriófagos/genética , Lactobacillus crispatus/genética , Prófagos/genéticaRESUMO
Bacterial SOS response is an inducible system of DNA repair and mutagenesis. Streptococci lack a canonical SOS response, but an SOS-like response was reported in some species. The mef(A)-msr(D)-carrying prophage Ф1207.3 of Streptococcus pyogenes contains a region, spanning orf6 to orf11, showing homology to characterized streptococcal SOS-like cassettes. Genome-wide homology search showed the presence of the whole Φ1207.3 SOS-like cassette in three S. pyogenes prophages, while parts of it were found in other bacterial species. To investigate whether this cassette confers an SOS-mutagenesis phenotype, we constructed Streptococcus pneumoniae R6 isogenic derivative strains: (i) FR172, streptomycin resistant, (ii) FR173, carrying Φ1207.3, and (iii) FR174, carrying a recombinant Φ1207.3, where the SOS-like cassette was deleted. These strains were used in survival and mutation rate assays using a UV-C LED instrument, for which we designed and 3D-printed a customized equipment, constituted of an instrument support and swappable-autoclavable mini-plates and lids. Upon exposure to UV fluences ranging from 0 to 6,400 J/m2 at four different wavelengths, 255, 265, 275, and 285 nm, we found that the presence of Φ1207.3 SOS-like cassette increases bacterial survival up to 34-fold. Mutation rate was determined by measuring rifampicin resistance acquisition upon exposure to UV fluence of 50 J/m2 at the four wavelengths by fluctuation test. The presence of Φ1207.3 SOS-like cassette resulted in a significant increase in the mutation rate (up to 18-fold) at every wavelength. In conclusion, we demonstrated that Φ1207.3 carries a functional SOS-like cassette responsible for an increased survival and increased mutation rate in S. pneumoniae. IMPORTANCE Bacterial mutation rate is generally low, but stress conditions and DNA damage can induce stress response systems, which allow for improved survival and continuous replication. The SOS response is a DNA repair mechanism activated by some bacteria in response to stressful conditions, which leads to a temporary hypermutable phenotype and is usually absent in streptococcal genomes. Here, using a reproducible and controlled UV irradiation system, we demonstrated that the SOS-like gene cassette of prophage Φ1207.3 is functional, responsible for a temporary hypermutable phenotype, and enhances bacterial survival to UV irradiation. Prophage Φ1207.3 also carries erythromycin resistance genes and can lysogenize different pathogenic bacteria, constituting an example of a mobile genetic element which can confer multiple phenotypes to its host.
Assuntos
Taxa de Mutação , Prófagos , Prófagos/genética , Streptococcus pneumoniae , Streptococcus pyogenes/genética , BioensaioRESUMO
Streptococcus pneumoniae is an important human pathogen causing both mild and severe diseases. In this work, we determined the complete genome sequence of the S. pneumoniae clinical isolate BM6001, which is the original host of the ICE Tn5253. The BM6001 genome is organized in one circular chromosome of 2,293,748 base pairs (bp) in length, with an average GC content of 39.54%; the genome harbors a type 19F capsule locus, two tandem copies of pspC, the comC1-comD1 alleles and the type I restriction modification system SpnIII. The BM6001 mobilome accounts for 15.54% (356,521 bp) of the whole genome and includes (i) the ICE Tn5253 composite; (ii) the novel IME Tn7089; (iii) the novel transposon Tn7090; (iv) 3 prophages and 2 satellite prophages; (v) 5 genomic islands (GIs); (vi) 72 insertion sequences (ISs); (vii) 69 RUPs; (viii) 153 BOX elements; and (ix) 31 SPRITEs. All MGEs, except for the GIs, produce excised circular forms and attB site restoration. Tn7089 is 9089 bp long and contains 11 ORFs, of which 6 were annotated and code for three functions: integration/excision, mobilization and adaptation. Tn7090 is 9053 bp in size, flanked by two copies of ISSpn7, and contains seven ORFs organized as a single transcriptional unit, with genes encoding for proteins likely involved in the uptake and binding of Mg2+ cations in the adhesion to host cells and intracellular survival. BM6001 GIs, except for GI-BM6001.4, are variants of the pneumococcal TIGR4 RD5 region of diversity, pathogenicity island PPI1, R6 Cluster 4 and PTS island. Overall, prophages and satellite prophages contain genes predicted to encode proteins involved in DNA replication and lysogeny, in addition to genes encoding phage structural proteins and lytic enzymes carried only by prophages. ΦBM6001.3 has a mosaic structure that shares sequences with prophages IPP69 and MM1 and disrupts the competent comGC/cglC gene after chromosomal integration. Treatment with mitomycin C results in a 10-fold increase in the frequency of ΦBM6001.3 excised forms and comGC/cglC coding sequence restoration but does not restore competence for genetic transformation. In addition, phylogenetic analysis showed that BM6001 clusters in a small lineage with five other historical strains, but it is distantly related to the lineage due to its unique mobilome, suggesting that BM6001 has progressively accumulated many MGEs while losing competence for genetic transformation.
RESUMO
Aim: The aim of the present study was to investigate and correlate the prevalence of Enterococcus faecalis in saliva and in root canals with different pulpal and periapical conditions. Methodology: Sixty-seven patients were divided into five groups based on pulpal and periapical tissue status: healthy vital teeth (HVT, n=7), healthy treated teeth without lesion (HTT, n=9), irreversible pulpitis (IP, n=13), necrosis (N, n=18), and post-treatment apical periodontitis (PTAP, n=20). Saliva, rubber dam, sterility control and pre-treatment root canal samples were collected and microbiologically processed by culture method. The phylogenetic relationship of E. faecalis isolates collected from root canals and saliva were investigated by whole genome sequencing. Fisher's exact test was used to correlate the presence of E. faecalis in root canals or saliva with clinical and/or radiographic findings. Linear/logistic regression analyses were performed to establish the relationship between the presence of E. faecalis in root canals, saliva, and the status of periapical tissues. Results: E. faecalis was found in 18 root canal and saliva samples. E. faecalis root canal isolates were recovered with the highest frequency from post-treatment apical periodontitis. The occurrence of E. faecalis in saliva was strongly associated with its detection in the root canals (P < 0.001). The pretreatment presence of E. faecalis in root canals was associated with significantly higher odds of having periapical lesions (OR=11.03; 95% CI, 1.27-95.70; p < 0.05). Saliva and root canal isolates from the same patient were highly correlated at the phylogenetic level (Jaccard index >0.95). Conclusion: This pilot study confirms the role of E. faecalis in developing peri-radicular lesions in secondary endodontic infections and suggests that saliva could be the main source of infection. Further studies are needed to investigate the exact origin of this bacteria and its true role in the pathogenesis of secondary/persistent endodontic infections.
Assuntos
Enterococcus faecalis , Periodontite Periapical , Humanos , Enterococcus faecalis/genética , Saliva/microbiologia , Filogenia , Projetos Piloto , Periodontite Periapical/microbiologia , Periodontite Periapical/terapia , Fatores de RiscoRESUMO
Streptococcus pyogenes prophage Φ1207.3 (formerly Tn1207.3) carries the mef(A)-msr(D) resistance genes, responsible for type M macrolide resistance. To investigate if Φ1207.3 is a functional bacteriophage, we transferred the element from the original S. pyogenes host in a prophage-free and competence-deficient S. pneumoniae strain. Pneumococcal cultures of the Φ1207.3-carrying lysogen were treated with mitomycin C to assess if Φ1207.3 enters the lytic cycle. Mitomycin C induced a limited phage burst and a growth impairment, resulting in early entrance into the stationary phase. To determine if Φ1207.3 is able to produce mature phage particles, we prepared concentrated supernatants recovered from a mitomycin C-induced pneumococcal culture by sequential centrifugation and ultracentrifugation steps. Negative-staining transmission electron microscopy (TEM) of supernatants revealed the presence of phage particles with an icosahedral, electron-dense capsid and a long, noncontractile tail, typical of a siphovirus. Quantification of Φ1207.3 was performed by quantitative PCR (qPCR) and semiquantitatively by TEM. PCR quantified 3.34 × 104 and 6.06 × 104 excised forms of phage genome per milliliter of supernatant obtained from the untreated and mitomycin C-treated cultures, respectively. By TEM, we estimated 3.02 × 103 and 7.68 × 103 phage particles per milliliter of supernatant. The phage preparations of Φ1207.3 infected and lysogenized pneumococcal recipient strains at a frequency of 7.5 × 10-6 lysogens/recipient but did not show sufficient lytic activity to form plaques. Phage lysogenization efficiently occurred after 30 min of contact of the phages with the recipient cells and required a minimum of 103 phage particles. IMPORTANCE Bacteriophages play an important role in bacterial physiology and genome evolution. The widespread use of genome sequencing revealed that bacterial genomes can contain several different integrated temperate bacteriophages, which can constitute up to 20% of the genome. Most of these bacteriophages are only predicted in silico and are never shown to be functional. In fact, it is often difficult to induce the lytic cycle of temperate bacteriophages. In this work, we show that Φ1207.3, a peculiar bacteriophage originally from Streptococcus pyogenes, which can lysogenize different streptococci and carries the macrolide resistance mef(A)-msr(D) gene pair, is capable of producing mature virions, but only at a low level, while not being able to produce plaques. This temperate phage is probably a partially functional phage, which seems to have lost lytic characteristics to specialize in lysogenization. While we are not used to conceiving phages separately from lysis, this behavior could actually be more frequent than expected.
Assuntos
Bacteriófagos , Bacteriófagos/genética , Antibacterianos/farmacologia , Streptococcus pyogenes , Macrolídeos/farmacologia , Mitomicina/farmacologia , Farmacorresistência Bacteriana/genética , Prófagos/genéticaRESUMO
BACKGROUND: The genomic approach has deeply changed the microbiology perspective, mainly concerning the microbioma identification. In this regard, some microbes colonize the healthy vagina. Vaginitis is a common gynecological ailment and includes bacterial vaginosis (BV), usually caused by local dysbiosis, such as a microbiota imbalance. Lactobacilli are the most prevalent bacteria colonizing the healthy vagina, so guaranteeing local eubiosis. In particular, vaginal colonization by L. crispatus is associated with low susceptibility to BV. Therefore, probiotics, such as life bacteria providing health advantages, are a current strategy in the prevention or treatment of vaginitis, including BV. However, there is a low level of evidence that probiotics after ingestion could really colonize the vagina. In particular, no study evidenced that L. crispatus after ingestion can colonize vagina. Therefore, the current study explored the capacity of Biovaginil® (NTC, Milan, Italy) dietary supplement containing Lactobacillus crispatus NTCVAG04 and vitamin A to colonize the gut and vagina in women with a history of vaginitis/vaginosis. METHODS: Twenty fertile females (mean age 34.0 years) were enrolled in the study. Rectal and vaginal swabs were collected at baseline and after the first and second cycle of Biovaginil®. Each cycle lasted 14 days within two consecutive menstrual periods. RESULTS: Seven women were excluded from the analysis because the samples were technically not evaluable. One woman dropped out because of mild adverse event. At the end of the study, nine women (75%) had positive rectal swab for L. crispatus NTCVAG04, and 8 of them also had positive vaginal swab. CONCLUSIONS: The current study provided the first evidence that L. crispatus NTCVAG04, administered by two Biovaginil® courses, colonized both the gut and vagina. Moreover, the L. crispatus NTCVAG04 strain could be considered the archetype of a new class of oral probiotics that actively colonize the vagina, and that could be called "colpobiotics."
Assuntos
Lactobacillus crispatus , Microbiota , Vaginose Bacteriana , Vulvovaginite , Humanos , Feminino , Adulto , Vagina/microbiologia , Vaginose Bacteriana/tratamento farmacológico , Vaginose Bacteriana/genética , Vaginose Bacteriana/microbiologia , Bactérias , Administração OralRESUMO
Modulation of drug transporter activity at mucosal sites of HIV-1 transmission may be exploited to optimize retention of therapeutic antiretroviral drug concentrations at target submucosal CD4+ T cells. Previously, we showed that darunavir was a substrate for the P-glycoprotein efflux drug transporter in colorectal mucosa. Equivalent studies in the cervicovaginal epithelium have not been reported. Here, we describe the development of a physiologically relevant model to investigate the permeability of antiretroviral drugs across the vaginal epithelium. Barrier properties of the HEC-1A human endometrial epithelial cell line were determined, in a dual chamber model, by measurement of transepithelial electrical resistance, immunofluorescent staining of tight junctions and bi-directional paracellular permeability of mannitol. We then applied this model to investigate the permeability of tenofovir, darunavir and dapivirine. Efflux ratios indicated that the permeability of each drug was transporter-independent in this model. Reduction of pH to physiological levels in the apical compartment increased absorptive transfer of darunavir, an effect that was reversed by inhibition of MRP efflux transport via MK571. Thus, low pH may increase the transfer of darunavir across the epithelial barrier via increased MRP transporter activity. In a previous in vivo study in the macaque model, we demonstrated increased MRP2 expression following intravaginal stimulation with darunavir which may further increase drug uptake. Stimulation with inflammatory modulators had no effect on drug permeability across HEC-1A barrier epithelium but, in the VK2/E6E7 vaginal cell line, increased expression of both efflux and uptake drug transporters which may influence darunavir disposition.
RESUMO
The in vitro stimulation of immune system cells with live or killed bacteria is essential for understanding the host response to pathogens. In the present study, we propose a model combining transcriptomic and cytokine assays on murine splenocytes to describe the immune recall in the days following pneumococcal lung infection. Mice were sacrificed at days 1, 2, 4, and 7 after Streptococcus pneumoniae (TIGR4 serotype 4) intranasal infection and splenocytes were cultured in the presence or absence of the same inactivated bacterial strain to access the transcriptomic and cytokine profiles. The stimulation of splenocytes from infected mice led to a higher number of differentially expressed genes than the infection or stimulation alone, resulting in the enrichment of 40 unique blood transcription modules, including many pathways related to adaptive immunity and cytokines. Together with transcriptomic data, cytokines levels suggested the presence of a recall immune response promoting both innate and adaptive immunity, stronger from the fourth day after infection. Dimensionality reduction and feature selection identified key variables of this recall response and the genes associated with the increase in cytokine concentrations. This model could study the immune responses involved in pneumococcal infection and possibly monitor vaccine immune response and experimental therapies efficacy in future studies.
Assuntos
Infecções Pneumocócicas , Infecções Respiratórias , Animais , Citocinas/metabolismo , Memória Imunológica , Camundongos , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas , Baço , Streptococcus pneumoniae/genética , TranscriptomaRESUMO
Chronic colonization by Pseudomonas aeruginosa is critical in cystic fibrosis (CF) and other chronic lung diseases, contributing to disease progression. Biofilm growth and a propensity to evolve multidrug resistance phenotypes drastically limit the available therapeutic options. In this perspective, there has been growing interest in evaluating combination therapies, especially for drugs that can be administered by nebulization, which allows high drug concentrations to be reached at the site of infections while limiting systemic toxicity. Here, we investigated the potential antibiofilm activity of N-acetylcysteine (NAC) alone and in combination with colistin against a panel of P. aeruginosa strains (most of which are from CF patients) and the transcriptomic response of a P. aeruginosa CF strain to NAC exposure. NAC alone (8,000 mg/L) showed a limited and strain-dependent antibiofilm activity. Nonetheless, a relevant antibiofilm synergism of NAC-colistin combinations (NAC at 8,000 mg/L plus colistin at 2 to 32 mg/L) was observed with all strains. Synergism was also confirmed with the artificial sputum medium model. RNA sequencing of NAC-exposed planktonic cultures revealed that NAC (8,000 mg/L) mainly induced (i) a Zn2+ starvation response (known to induce attenuation of P. aeruginosa virulence), (ii) downregulation of genes of the denitrification apparatus, and (iii) downregulation of flagellar biosynthesis pathway. NAC-mediated inhibition of P. aeruginosa denitrification pathway and flagellum-mediated motility were confirmed experimentally. These findings suggested that NAC-colistin combinations might contribute to the management of biofilm-associated P. aeruginosa lung infections. NAC might also have a role in reducing P. aeruginosa virulence, which could be relevant in the very early stages of lung colonization. IMPORTANCE Pseudomonas aeruginosa biofilm-related chronic lung colonization contributes to cystic fibrosis (CF) disease progression. Colistin is often a last-resort antibiotic for the treatment of such P. aeruginosa infections, and it has been increasingly used in CF, especially by nebulization. N-acetylcysteine (NAC) is a mucolytic agent with antioxidant activity, commonly administered with antibiotics for the treatment of lower respiratory tract infections. Here, we show that NAC potentiated colistin activity against in vitro biofilms models of P. aeruginosa strains, with both drugs tested at the high concentrations achievable after nebulization. In addition, we report the first transcriptomic data on the P. aeruginosa response to NAC exposure.
Assuntos
Fibrose Cística , Infecções por Pseudomonas , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Colistina/farmacologia , Colistina/uso terapêutico , Progressão da Doença , Humanos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/genética , TranscriptomaRESUMO
Streptococcus mitis is a Gram-positive bacterium, member of the oral commensal microbiota, which can occasionally be the etiologic agent of diseases such as infective endocarditis, bacteraemia and septicaemia. The highly recombinogenic and repetitive nature of the S. mitis genome impairs the assembly of a complete genome relying only on short sequencing reads. Oxford Nanopore sequencing can overcome this limitation by generating long reads, enabling the resolution of genomic repeated regions and the assembly of a complete genome sequence. Since the output of a Nanopore sequencing run is strongly influenced by genomic DNA quality and molecular weight, the DNA isolation is the crucial step for an optimal sequencing run. In the present work, we have set up and compared three DNA isolation methods on two S. mitis strains, evaluating their capability of preserving genomic DNA integrity and purity. Sequencing of DNA isolated with a mechanical lysis-based method, despite being cheaper and quicker, did not generate ultra-long reads (maximum read length of 59516 bases) and did not allow the assembly of a circular complete genome. Two methods based on enzymatic lysis of the bacterial cell wall, followed by either (i) a modified CTAB DNA isolation procedure, or (ii) a DNA purification after osmotic lysis of the protoplasts allowed the sequencing of ultra-long reads up to 107294 and 181199 bases in length, respectively. The reconstruction of a circular complete genome was possible sequencing DNAs isolated using the enzymatic lysis-based methods.
Assuntos
DNA Bacteriano/isolamento & purificação , Sequenciamento por Nanoporos/métodos , Streptococcus mitis/genética , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microbiota , Boca/microbiologia , Análise de Sequência de DNA/métodos , Sequenciamento Completo do GenomaRESUMO
The whole-genome sequences of Mycobacterium chimaera strains 850 and 852, which were isolated from two different water samples obtained from a heater-cooler unit at Siena University Hospital (Italy), were determined by combining Nanopore and Illumina technologies. Genomes of both strains 850 and 852 consist of a circular chromosome and five plasmids, with sizes of 6,275,686 bp and 6,453,144 bp, respectively.
RESUMO
BACKGROUND: The aim of this study is to clinically and molecularly evaluate the effect of an interproximal iuxta/subgingival direct composite restoration on periodontal tissue healing. METHODS: Individuals in need of a posterior iuxta/subgingival interproximal restoration were consecutively enrolled. After enrollment, a test (site with tooth decay) and a control site (intact contralateral tooth) were identified. After a periodontal examination (probing depth [PD], clinical attachment level, recession, plaque, and bleeding on probing [BOP]) and a sampling of gingival crevicular fluid, the composite restoration was performed (T0 ). Clinical and molecular assessments were repeated at 3 (T3 ), 6 (T6 ), and 12 (T12 ) months after the restoration. Intragroup pre-post comparisons for quantitative variables were performed either through one-way ANOVA or Kruskal-Wallis test. A multivariate linear regression analysis was then modeled. With α = 0.05, a power of 80% will be reached with the inclusion of 41 individuals. RESULTS: Biometric parameters demonstrated an increased mean PD (ΔPDT0 -T12 = -0.83 mm; P = 0.001) and loss of attachment (AL) (ΔCALT0 -T12 = -0.91 mm; P = 0.005) in the test site at 12 months. Accordingly, in the final multivariate regression model the radiographic distance between the bone crest and the restorative margin at baseline accounted for the dependent variable "attachment loss (AL)" (ΔCALT0 -T12 ) (P <0.05). CONCLUSIONS: Iuxta/subgingival interproximal restorative margins jeopardized clinically and molecularly the periodontal tissue healing at least up to 1 year of follow-up.
Assuntos
Placa Dentária , Periodonto , Citocinas , Índice de Placa Dentária , Líquido do Sulco Gengival , Humanos , Perda da Inserção PeriodontalRESUMO
OBJECTIVES: In streptococci, the type M resistance to macrolides is due to the mef(A)-msr(D) efflux transport system of the ATP-Binding cassette (ABC) superfamily, where it is proposed that mef(A) codes for the transmembrane channel and msr(D) for the two ATP-binding domains. Phage Ï1207.3 of Streptococcus pyogenes, carrying the mef(A)-msr(D) gene pair, is able to transfer the macrolide efflux phenotype to Streptococcus pneumoniae. Deletion of mef(A) in pneumococcal Ï1207.3-carrying strains did not affect erythromycin efflux. In order to identify candidate genes likely involved in complementation of mef(A) deletion, the Mef(A) amino acid sequence was used as probe for database searching. RESULTS: In silico analysis identified 3 putative candidates in the S. pneumoniae R6 genome, namely spr0971, spr1023 and spr1932. Isogenic deletion mutants of each candidate gene were constructed and used in erythromycin sensitivity assays to investigate their contribution to mef(A) complementation. Since no change in erythromycin sensitivity was observed compared to the parental strain, we produced double and triple mutants to assess the potential synergic activity of the selected genes. Also these mutants did not complement the mef(A) function.
Assuntos
Macrolídeos , Streptococcus pneumoniae , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Streptococcus pneumoniae/genéticaRESUMO
BACKGROUND: Tn5253, a composite Integrative Conjugative Element (ICE) of Streptococcus pneumoniae carrying tet(M) and cat resistance determinants, was found to (i) integrate at specific 83-bp integration site (attB), (ii) produce circular forms joined by a 84-bp sequence (attTn), and (iii) restore the chromosomal integration site. The purpose of this study is to functionally characterize the attB in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, and to investigate the presence of Tn5253 attB site into bacterial genomes. RESULTS: Analysis of representative Tn5253-carryng transconjugants obtained in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, namely Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes, and Enterococcus faecalis showed that: (i) Tn5253 integrates in rbgA of S. pneumoniae and in orthologous rbgA genes of other bacterial species, (ii) integration occurs always downstream of a 11-bp sequence conserved among streptococcal and enterococcal hosts, (iii) length of the attB site corresponds to length of the duplication after Tn5253 integration, (iv) attB duplication restores rbgA CDS, (v) Tn5253 produced circular forms containing the attTn site at a concentration ranging between 2.0 × 10-5 to 1.2 × 10-2 copies per chromosome depending on bacterial species and strain, (vi) reconstitution of attB sites occurred at 3.7 × 10-5 to 1.7 × 10-2 copies per chromosome. A database search of complete microbial genomes using Tn5253 attB as a probe showed that (i) thirteen attB variants were present in the 85 complete pneumococcal genomes, (ii) in 75 pneumococcal genomes (88.3 %), the attB site was 83 or 84 nucleotides in length, while in 10 (11.7 %) it was 41 nucleotides, (iii) in other 19 bacterial species attB was located in orthologous rbgA genes and its size ranged between 17 and 84 nucleotides, (iv) the 11-bp sequence, which correspond to the last 11 nucleotides of attB sites, is conserved among the different bacterial species and can be considered the core of the Tn5253 integration site. CONCLUSIONS: A functional characterization of the Tn5253 attB integration site combined with genome analysis contributed to elucidating the potential of Tn5253 horizontal gene transfer among different bacterial species.
RESUMO
The complete genome sequence of Streptococcus pneumoniae strain Rx1, a Hex mismatch repair-deficient standard transformation recipient, was obtained by combining Nanopore and Illumina sequencing technologies. The genome consists of a 2.03-Mb circular chromosome, with 2,054 open reading frames and a GC content of 39.72%.
RESUMO
DNA sequencing of whole bacterial genomes has revealed that the entire set of mobile genes (mobilome) represents as much as 25% of the bacterial genome. Despite the huge availability of sequence data, the functional analysis of the mobile genetic elements (MGEs) is rarely reported. Therefore, established laboratory protocols are needed to investigate the biology of this important part of the bacterial genome. Conjugation is a mechanism of horizontal gene transfer which allows the exchange of MGEs among strains of the same or different bacterial species. In streptococci and enterococci, integrative and conjugative elements (ICEs) represent a large part of the mobilome. Here, we describe an efficient and easy-to-perform plate mating protocol for in vitro conjugative transfer of ICEs in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis. Conjugative transfer is carried out on solid media and selection of transconjugants is performed with a multilayer plating. This protocol allows the transfer of large genetic elements with a size up to 81 kb, and a transfer frequency up to 6.7 × 10-3 transconjugants/donor cells.
RESUMO
The complete genome sequence of Lactobacillus crispatus type strain ATCC 33820 was obtained by combining Nanopore and Illumina sequencing technologies. The genome consists of a 2.2-Mb circular chromosome with 2,194 open reading frames and an average GC content of 37.0%.
RESUMO
Salmonella Typhimurium (STm) represents the most prevalent cause of invasive non-typhoidal Salmonella (iNTS) disease, and currently no licensed vaccine is available. In this work we characterized the long-term anti-bacterial immunity elicited by a STm vaccine based on Generalized Modules of Membrane Antigens (GMMA) delivering O:4,5 antigen, using a murine model of systemic infection. Subcutaneous immunization of mice with STmGMMA/Alhydrogel elicited rapid, high, and persistent antigen-specific serum IgG and IgM responses. The serum was bactericidal in vitro. O:4,5-specific IgG were also detected in fecal samples after immunization and positively correlated with IgG observed in intestinal washes. Long-lived plasma cells and O:4,5-specific memory B cells were detected in spleen and bone marrow. After systemic STm challenge, a significant reduction of bacterial load in blood, spleen, and liver, as well as a reduction of circulating neutrophils and G-CSF glycoprotein was observed in STmGMMA/Alhydrogel immunized mice compared to untreated animals. Taken together, these data support the development of a GMMA-based vaccine for prevention of iNTS disease.
RESUMO
Ebolavirus Disease (EVD) is a severe haemorrhagic fever that occurs in epidemic outbreaks, with a high fatality rate and no specific therapies available. rVSVΔG-ZEBOV-GP (Ervebo®), a live-attenuated recombinant vesicular stomatitis virus vector expressing the glycoprotein G of Zaire Ebolavirus, is the first vaccine approved for prevention of EVD. Both innate and adaptive responses are deemed to be involved in vaccine-induced protection, yet the mechanisms are not fully elucidated. A global transcriptomic approach was used to profile the blood host-response in 51 healthy volunteers enrolled in a phase 1/2 clinical trial. Signatures of the host responses were investigated assessing the enrichment in differentially expressed genes (DEGs) of specific "blood transcription modules" (BTM). Comparison of gene-expression levels showed that vaccination produces a peak of 5469 DEGs at day one, representing 38.6% of the expressed genes. Out of 346 BTMs, 144 were significantly affected by vaccination. Innate immunity pathways were induced from day 1 to day 14. At days 2 and 3, neutrophil modules were downregulated and complement-related modules upregulated. T-cell and cell-cycle associated modules were upregulated at days 7 and 14, while at day 28, no modules remained activated. At day 14, a direct correlation was observed between ZEBOV glycoprotein-specific antibody titres and activation of seven BTMs, including two related to B-cell activation and B cell receptor signalling. Transcriptomic analysis identified an rVSVΔG-ZEBOV-GP-induced signature and demonstrated a direct correlation of blood transcriptomic changes with ZEBOV glycoprotein-specific antibody titres.