Phylogenomics of Leptospira santarosai, a prevalent pathogenic species in the Americas.

BACKGROUND: Leptospirosis is a complex zoonotic disease mostly caused by a group of eight pathogenic species (L. interrogans, L. borgpetersenii, L. kirschneri, L. mayottensis, L. noguchii, L. santarosai, L. weilii, L. alexanderi), with a wide spectrum of animal reservoirs and patient outcomes. Leptospira interrogans is considered as the leading causative agent of leptospirosis worldwide and it is the most studied species. However, the genomic features and phylogeography of other Leptospira pathogenic species remain to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the genome diversity of the main pathogenic Leptospira species based on a collection of 914 genomes from strains isolated around the world. Genome analyses revealed species-specific genome size and GC content, and an open pangenome in the pathogenic species, except for L. mayottensis. Taking advantage of a new set of genomes of L. santarosai strains isolated from patients in Costa Rica, we took a closer look at this species. L. santarosai strains are largely distributed in America, including the Caribbean islands, with over 96% of the available genomes originating from this continent. Phylogenetic analysis showed high genetic diversity within L. santarosai, and the clonal groups identified by cgMLST were strongly associated with geographical areas. Serotype identification based on serogrouping and/or analysis of the O-antigen biosynthesis gene loci further confirmed the great diversity of strains within the species. CONCLUSIONS/SIGNIFICANCE: In conclusion, we report a comprehensive genome analysis of pathogenic Leptospira species with a focus on L. santarosai. Our study sheds new light onto the genomic diversity, evolutionary history, and epidemiology of leptospirosis in America and globally. Our findings also expand our knowledge of the genes driving O-antigen diversity. In addition, our work provides a framework for understanding the virulence and spread of L. santarosai and for improving its surveillance in both humans and animals.

The chemical structure of the O-antigen and enterobacterial common antigen of Pectobacterium brasiliense NCPPB 4609TS.

Pectobacterium brasiliense is a widely distributed phytopathogenic bacterium that causes diseases such as soft rot and blackleg, leading to significant yield losses in potatoes as well as other vegetables and ornamental plants. Lipopolysaccharide (LPS) is an important virulence factor that plays an essential role in colonisation of plant tissues and overcoming the host defence mechanisms. The O-polysaccharide from the LPS of P. brasiliense strain NCPPB 4609TS (=CFBP 6617TS = LMG 21371TS = IFB5390) was structurally characterised using spectroscopic techniques and chemical methods. The analyses revealed that the polysaccharide repeating unit consists of Gal, GlcN and an unusual 3-amino-3,6-dideoxyglucose decorated with (R)-3-hydroxybutyric acid according to the structure shown below: In addition, another polysaccharide was isolated from bacterial cells, analysis of which led to the identification of an enterobacterial common antigen, containing N-acetyl-d-glucosamine, N-acetyl-d-mannosaminouronic acid, and 4-acetamido-4,6-dideoxy-d-galactose.

Bm Delta-pgm, a vaccine for the control of Brucella melitensis with cross-species protective properties.

Brucellosis remains one of the most worldwide distributed zoonosis inflicting serious economical and human health problems in many areas of the world. The disease is caused by different species of the genus Brucella that have different tropisms towards different mammals being the most relevant for human health Brucella abortus, Brucella melitensis and Brucella suis that infect cows, goats/sheep, and swine respectively. For B. melitensis, considered the species with more zoonotic potential and highly aggressive for animals, only one vaccine is available to date in the market: Rev 1. This attenuated strain has the disadvantage that is has a very high residual virulence for animals and humans and, for this reason, it is applied by ocular instillation which is technically challenging in many productive settings. For this reason, the search for new vaccines for caprine and ovine brucellosis is an active topic of research. We describe here the construction of a novel highly attenuated vaccine strain (Bm Delta-pgm) that confers excellent levels of protection against B. melitensis in the mouse model of infection. This strain is a clean deletion of the phosphoglucomutase (pgm) gene that codes for a protein that catalyzes the conversion of glucose-6-P to glucose-1-P, which is used as a precursor for the biosynthesis of many polysaccharides, including the O-antigen of the lipopolysaccharide and cyclic beta glucans. Our results indicate that vaccination with Bm Delta-pgm induces a robust memory cellular immune response but no antibody production against the O-antigen. Cross protection experiments show that this new vaccine protects against B. abortus and B. suis raising the possibility that Bm Delta-pgm could be used as a universal vaccine for the most important Brucella species.

Salmonella Typhimurium O-antigen and VisP play an important role in swarming and osmotic stress response during intracellular conditions.

Salmonella Typhimurium is a pathogen of clinical relevance and a model of study in host-pathogen interactions. The virulence and stress-related periplasmic protein VisP is important during S. Typhimurium pathogenesis. It supports bacteria invading host cells, surviving inside macrophages, swimming, and succeeding in murine colitis model, O-antigen assembly, and responding to cationic antimicrobial peptides. This study aimed to investigate the role of the O-antigen molecular ruler WzzST and the periplasmic protein VisP in swarming motility and osmotic stress response. Lambda red mutagenesis was performed to generate single and double mutants, followed by swarming motility, qRT-PCR, Western blot, and growth curves. Here we demonstrate that the deletion of visP affects swarming under osmotic stress and changes the expression levels of genes responsible for chemotaxis, flagella assembly, and general stress response. The deletion of the gene encoding for the O-antigen co-polymerase wzzST increases swarming motility but not under osmotic stress. A second mutation in O-antigen co-polymerase wzzST in a ΔvisP background affected gene expression levels. The ΔvisP growth was affected by sodium and magnesium levels on N-minimum media. These data indicate that WzzST has a role in swarming the motility of S. Typhimurium, as the VisP is involved in chemotaxis and osmotic stress, specifically in response to MgCl2 and NaCl.

Bivalent oral cholera vaccination induces a memory B cell response to the V. cholerae O1-polysaccharide antigen in Haitian adults.

The bivalent killed whole-cell oral cholera vaccine (BivWC) is being increasingly used to prevent cholera. The presence of O-antigen-specific memory B cells (MBC) has been associated with protective immunity against cholera, yet MBC responses have not been evaluated after BivWC vaccination. To address this knowledge gap, we measured V. cholerae O1-antigen MBC responses following BivWC vaccination. Adults in St. Marc, Haiti, received 2 doses of the BivWC vaccine, Shanchol, two weeks apart. Participants were invited to return at days 7, 21, 44, 90, 180 and 360 after the initial vaccination. Serum antibody and MBC responses were assessed at each time-point before and following vaccination. We observed that vaccination with BivWC resulted in significant O-antigen specific MBC responses to both Ogawa and Inaba serotypes that were detected by day 21 and remained significantly elevated over baseline for up to 12 months following vaccination. The BivWC oral cholera vaccine induces durable MBC responses to the V. cholerae O1-antigen. This suggests that long-term protection observed following vaccination with BivWC could be mediated or maintained by MBC responses.

Structural studies of O-specific polysaccharide(s) and biological activity toward plants of the lipopolysaccharide from Azospirillum brasilense SR8.

Lipopolysaccharide (LPS) was extracted from dry bacterial cells of plant-growth-promoting bacterium Azospirillum brasilense SR8 (IBPPM 5). The O-specific polysaccharide (OPS) was obtained by mild acid hydrolysis of the lipopolysaccharide and studied by sugar analysis, 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC and HMBC experiments, computational NMR-based structure analysis, and Smith degradation. The OPS was shown to contain two types of repeating units of the following structure: Both OPS structures are present in A. brasilense 54, from which structure 1 has been reported earlier (Fedonenko et al., 2011), whereas to our knowledge structure 2 has not been hitherto found in bacterial saccharides. Treatment of wheat seedling roots with LPS of A. brasilense SR8 increased the number of root hair deformations as compared to seedlings grown without LPS, but had no effect on adsorption of the bacteria to the root surface. A. brasilense SR8 was able to utilize LPS of several structurally related Azospirillum strains.

Outer-Membrane-Vesicle-Associated O Antigen, a Crucial Component for Protecting Against Bordetella parapertussis Infection.

Bordetella parapertussis is a respiratory-disease pathogen producing symptomatology similar to that of pertussis but of underestimated incidence and with no specific vaccine existing. We recently designed a vaccine candidate from B. parapertussis outer-membrane vesicles (OMVs) that proved to be safe and protective in a murine-infection model. Based on protection recently reported for the B. parapertussis O antigen in aqueous solution, we assessed here whether the B. parapertussis O-antigen-containing lipopolysaccharide (BppLPS-O+) embedded in the membranes, as present in B. parapertussis-derived OMVs (OMVs(Bpp-LPS-O+)), was the component responsible for that previously observed protection by OMVs. By performing a comparative study with OMVs from a human strain with undetectable O antigen (OMVs(Bpp-LPS-O-)), we demonstrated that the OMVs(Bpp-LPS-O+), but not the OMVs(Bpp-LPS-O-), protected mice against sublethal B. parapertussis infections. Indeed, the B. parapertussis loads were significantly reduced in the lungs of OMVs(Bpp-LPS-O+) -vaccinated animals, with the CFUs recovered being decreased by 4 log units below those detected in the non-immunized animals or in the animals treated with the OMVs(Bpp-LPS-O-), (p < 0.001). We detected that the OMVs(Bpp-LPS-O+) induced IgG antibodies against B. parapertussis whole-cell lysates, which immunocomponents recognized, among others, the O antigen and accordingly conferred protection against B. parapertussis infection, as observed in in-vivo-passive-transfer experiments. Of interest was that the OMVs(Bpp-LPS-O+) -generated sera had opsonophagocytic and bactericidal capabilities that were not detected with the OMVs(Bpp-LPS-O-)-induced sera, suggesting that those activities were involved in the clearance of B. parapertussis. Though stimulation of cultured spleen cells from immunized mice with formulations containing the O antigen resulted in gamma interferon (IFN-γ) and interleukin-17 production, spleen cells from OMVs(Bpp-LPS-O+) -immunized mice did not significantly contribute to the observed protection against B. parapertussis infection. The protective capability of the B. parapertussis O antigen was also detected in formulations containing both the OMVs derived from B. pertussis and purified BppLPS-O+. This combined formulation protected mice against B. pertussis along with B. parapertussis.

Synthesis of the tetrasaccharide related to the repeating unit of the O-antigen from Azospirillum brasilense Jm125A2 in the form of its 2-aminoethyl glycoside.

Total chemical synthesis of the linear tetrasaccharide repeating unit ß-D-Glc-(1 → 2)-α-L-Rha-(1 → 3)-α-L-Rha-(1 → 2)-α-L-Rha-CH2CH2NH2 of the O-antigen from Azospirillum brasilense Jm125A2 is accomplished through rational protecting group manipulations of commercially available monosaccharides and stereoselective glycosylations. The target tetrasaccharide in the form of its 2-aminoethyl glycoside is obtained in ∼24% yield over 10 steps following a linear strategy. The structure is particularly suitable for further glycoconjugate formation through the terminal free amine without hampering the reducing end stereochemistry.

New envelope stress factors involved in σE activation and conditional lethality of rpoE mutations in Salmonella enterica.

Salmonella enterica serovar Typhimurium (S. typhimurium) can cause food- and water-borne illness with diverse clinical manifestations. One key factor for S. typhimurium pathogenesis is the alternative sigma factor σE, which is encoded by the rpoE gene and controls the transcription of genes required for outer-membrane integrity in response to alterations in the bacterial envelope. The canonical pathway for σE activation involves proteolysis of the antisigma factor RseA, which is triggered by unfolded outer-membrane porins (OMPs) and lipopolysaccharides (LPS) that have accumulated in the periplasm. This study reports new stress factors that are able to activate σE expression. We demonstrate that UVA radiation induces σE activity in a pathway that is dependent on the stringent response regulator ppGpp. Survival assays revealed that rpoE has a role in the defence against lethal UVA doses that is mediated by functions that are dependent on and independent of the alternative sigma factor RpoS. We also report that the envelope stress generated by phage infection requires a functional rpoE gene for optimal bacterial tolerance and that it is able to induce σE activity in an RseA-dependent fashion. σE activity is also induced by hypo-osmotic shock in the absence of osmoregulated periplasmic glucans (OPGs). It is known that the rpoE gene is not essential in S. typhimurium. However, we report here two cases of the conditional lethality of rpoE mutations in this micro-organism. We demonstrate that rpoE mutations are not tolerated in the absence of OPGs (at low to moderate osmolarity) or LPS O-antigen. The latter case resembles that of the prototypic Escherichia coli strain K12, which neither synthesizes a complete LPS nor tolerates null rpoE mutations.

Novel Role of VisP and the Wzz System during O-Antigen Assembly in Salmonella enterica Serovar Typhimurium Pathogenesis.

Salmonella enterica serovars are associated with diarrhea and gastroenteritis and are a helpful model for understanding host-pathogen mechanisms. Salmonella enterica serovar Typhimurium regulates the distribution of O antigen (OAg) and presents a trimodal distribution based on Wzy polymerase and the WzzST (long-chain-length OAg [L-OAg]) and WzzfepE (very-long-chain-length OAg [VL-OAg]) copolymerases; however, several mechanisms regulating this process remain unclear. Here, we report that LPS modifications modulate the infectious process and that OAg chain length determination plays an essential role during infection. An increase in VL-OAg is dependent on Wzy polymerase, which is promoted by a growth condition resembling the environment of Salmonella-containing vacuoles (SCVs). The virulence- and stress-related periplasmic protein (VisP) participates in OAg synthesis, as a ΔvisP mutant presents a semirough OAg phenotype. The ΔvisP mutant has greatly decreased motility and J774 macrophage survival in a colitis model of infection. Interestingly, the phenotype is restored after mutation of the wzzST or wzzfepE gene in a ΔvisP background. Loss of both the visP and wzzST genes promotes an imbalance in flagellin secretion. L-OAg may function as a shield against host immune systems in the beginning of an infectious process, and VL-OAg protects bacteria during SCV maturation and facilitates intramacrophage replication. Taken together, these data highlight the roles of OAg length in generating phenotypes during S Typhimurium pathogenesis and show the periplasmic protein VisP as a novel protein in the OAg biosynthesis pathway.

Design of Two Multiplex PCR Assays for Serotyping Shigella flexneri.

Shigella flexneri is a major health problem in developing countries. There are 19 serotypes recognized based on O-antigen structure and its typing is important for epidemiological purposes. However, the diversity of serotypes and the difficulties presented by phenotypic serotyping, for example, unavailable antisera for less common antigens, require the implementation of molecular techniques. In this study, we developed two multiplex PCR assays targeting the O-antigen synthesis genes and the O-antigen modification genes, for the rapid identification of S. flexneri serotypes 1/7, 2, 4, 5, and 6 (PCR A) and serotype 7 and group antigenic factors (3,4; 6; 7,8; E1037) (PCR B). A total of 73 S. flexneri strains representing 18 serotypes, except serotype 1d, were used in the study. Specific amplification patterns were obtained for each of the different serotypes. All strains tested had concordant results with phenotypic and genotypic serotyping; therefore, its implementation in the microbiology clinical laboratory will significantly improve S. flexneri serotyping.

Pro-inflammatory Analysis of Macrophages in Contact with Titanium Particles and Porphyromonas gingivalis.

During insertion of titanium dental implants, particles may shear from the implant to the periimplant region causing osteolysis, and their association with bacteria can exacerbate the inflammatory reaction. However, the association of a high invasive bacterium from the oral cavity, Porphyromonas gingivalis (Pg), and titanium particles remains unknown. This study evaluated pro-inflammatory reaction of human macrophages in contact with micro and nanoparticles of titanium associated with Porphyromonas gingivalis lipopolysaccharide (PgLPS). THP-1 cell were used and treated for 12, 24 and 48 h following 6 groups: Control(C), PgLPS (L); Microparticles (M); Nanoparticles (N); PgLPS and microparticles (LM); PgLPS and nanoparticles (LN). The following assays were carried out: i) cell viability using MTS, ii) cell morphology by SEM and iii) expression of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) by qRT-PCR and ELISA. For statistics two-way ANOVA followed by Tukey's test was used (p<0.05). After treatment, cells presented similar viability and morphology demonstrating that the treatments were not able to induce cell death. Gene expression was significantly higher for TNF-α and IL1-ß after 12 h, and for IL-6 after 24 h in the N and LN groups. Cytokine production over time was an ascending curve for TNF-α with the peak at 48 h and IL1-ß and IL-6 had a straight line among the time points, although cells from N group presented a significant production of IL-6 at 48 h. In conclusion, these results suggest that titanium nanoparticles stimulate stronger pro-inflammatory response in macrophages, independent of their association with LPS from P.gingivalis.

A recombinant O-polysaccharide-protein conjugate approach to develop highly specific monoclonal antibodies to Shiga toxin-producing Escherichia coli O157 and O145 serogroups.

Shiga toxin-producing Escherichia coli (STEC) is the major etiologic agent of hemolytic-uremic syndrome (HUS). The high rate of HUS emphasizes the urgency for the implementation of primary prevention strategies to reduce its public health impact. Argentina shows the highest rate of HUS worldwide, being E. coli O157 the predominant STEC-associated HUS serogroup (>70%), followed by E. coli O145 (>9%). To specifically detect these serogroups we aimed at developing highly specific monoclonal antibodies (mAbs) against the O-polysaccharide (O-PS) section of the lipopolysaccharide (LPS) of the dominant STEC-associated HUS serogroups in Argentina. The development of hybridomas secreting mAbs against O157 or O145 was carried out through a combined immunization strategy, involving adjuvated-bacterial immunizations followed by immunizations with recombinant O-PS-protein conjugates. We selected hybridoma clones that specifically recognized the engineered O-PS-protein conjugates of O157 or O145 serogroups. Indirect ELISA of heat-killed bacteria showed specific binding to O157 or O145 serogroups, respectively, while no cross-reactivity with other epidemiological important STEC strains, Brucella abortus, Salmonella group N or Yersinia enterocolitica O9 was observed. Western blot analysis showed specific recognition of the sought O-PS section of the LPS by all mAbs. Finally, the ability of the developed mAbs to bind the surface of whole bacteria cells was confirmed by flow cytometry, confocal microscopy and agglutination assays, indicating that these mAbs present an exceptional degree of specificity and relative affinity in the detection and identification of E. coli O157 and O145 serogroups. These mAbs may be of significant value for clinical diagnosis and food quality control applications. Thus, engineered O-PS specific moieties contained in the recombinant glycoconjugates used for combined immunization and hybridoma selection are an invaluable resource for the development of highly specific mAbs.

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis.

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis. It was found that this homolog, named AfuABpp , is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O-antigen, a molecule that has been found to shield surface antigens on B. parapertussis, showed no influence on antibody recognition of AfuABpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.

Phylogeographic, genomic, and meropenem susceptibility analysis of Burkholderia ubonensis.

The bacterium Burkholderia ubonensis is commonly co-isolated from environmental specimens harbouring the melioidosis pathogen, Burkholderia pseudomallei. B. ubonensis has been reported in northern Australia and Thailand but not North America, suggesting similar geographic distribution to B. pseudomallei. Unlike most other Burkholderia cepacia complex (Bcc) species, B. ubonensis is considered non-pathogenic, although its virulence potential has not been tested. Antibiotic resistance in B. ubonensis, particularly towards drugs used to treat the most severe B. pseudomallei infections, has also been poorly characterised. This study examined the population biology of B. ubonensis, and includes the first reported isolates from the Caribbean. Phylogenomic analysis of 264 B. ubonensis genomes identified distinct clades that corresponded with geographic origin, similar to B. pseudomallei. A small proportion (4%) of strains lacked the 920kb chromosome III replicon, with discordance of presence/absence amongst genetically highly related strains, demonstrating that the third chromosome of B. ubonensis, like other Bcc species, probably encodes for a nonessential pC3 megaplasmid. Multilocus sequence typing using the B. pseudomallei scheme revealed that one-third of strains lack the "housekeeping" narK locus. In comparison, all strains could be genotyped using the Bcc scheme. Several strains possessed high-level meropenem resistance (≥32 µg/mL), a concern due to potential transmission of this phenotype to B. pseudomallei. In silico analysis uncovered a high degree of heterogeneity among the lipopolysaccharide O-antigen cluster loci, with at least 35 different variants identified. Finally, we show that Asian B. ubonensis isolate RF23-BP41 is avirulent in the BALB/c mouse model via a subcutaneous route of infection. Our results provide several new insights into the biology of this understudied species.

Pro-inflammatory Analysis of Macrophages in Contact with Titanium Particles and Porphyromonas gingivalis

Abstract During insertion of titanium dental implants, particles may shear from the implant to the periimplant region causing osteolysis, and their association with bacteria can exacerbate the inflammatory reaction. However, the association of a high invasive bacterium from the oral cavity, Porphyromonas gingivalis (Pg), and titanium particles remains unknown. This study evaluated pro-inflammatory reaction of human macrophages in contact with micro and nanoparticles of titanium associated with Porphyromonas gingivalis lipopolysaccharide (PgLPS). THP-1 cell were used and treated for 12, 24 and 48 h following 6 groups: Control(C), PgLPS (L); Microparticles (M); Nanoparticles (N); PgLPS and microparticles (LM); PgLPS and nanoparticles (LN). The following assays were carried out: i) cell viability using MTS, ii) cell morphology by SEM and iii) expression of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) by qRT-PCR and ELISA. For statistics two-way ANOVA followed by Tukey's test was used (p<0.05). After treatment, cells presented similar viability and morphology demonstrating that the treatments were not able to induce cell death. Gene expression was significantly higher for TNF-α and IL1-β after 12 h, and for IL-6 after 24 h in the N and LN groups. Cytokine production over time was an ascending curve for TNF-α with the peak at 48 h and IL1-β and IL-6 had a straight line among the time points, although cells from N group presented a significant production of IL-6 at 48 h. In conclusion, these results suggest that titanium nanoparticles stimulate stronger pro-inflammatory response in macrophages, independent of their association with LPS from P.gingivalis.

Resumo Durante a inserção de implantes dentários partículas de titânio podem ser liberadas na região peri-implantar levando ao processo de osteólise e a associação com a bactéria pode exacerbar ainda mais a reação inflamatória. Entretanto, a associação de uma bactéria altamente invasiva da cavidade oral, Porphyromonas gingivalis (Pg) e partículas de titânio ainda não foi investigada. Este estudo avaliou a reação pró-inflamatória de macrófagos humanos em contato com micro e nanopartículas de titânio associada a lipopolissacarídeo P. gingivalis (PgLPS). As células THP-1 foram utilizadas e tratadas durante 12, 24 e 48 h nos 6 seguintes grupos: Controle (C), PgLPS (L); micropartículas (M); nanopartículas (N); PgLPS e micropartículas (LM); PgLPS e nanopartículas (LN). Em seguida foram realizados os seguintes ensaios: i) a viabilidade celular utilizando MTS, ii) a morfologia celular por MEV e iii) expressão do fator de necrose tumoral alfa (TNF-α), interleucina-1 beta (IL-1β) e interleucina 6 (IL-6) por qRT-PCR e ELISA. Como estatística foi realizado o teste ANOVA two-way seguido pelo teste de Tukey (p<0,05). Após o tratamento, as células apresentaram viabilidade e morfologia semelhantes, demonstrando que os tratamentos não foram capazes de induzir a morte celular. A expressão de genes foi significativamente mais elevada para o TNF-α e IL1-β após 12h, e para a IL-6 após 24 horas em N e grupos de LN. A produção de citocinas em relação ao tempo representou uma curva ascendente para o TNF-α com o pico em 48 h, enquanto que para IL1-β e IL-6 se apresentou como uma linha reta com relação ao tempo, exceto pelo grupo N que foi significativo para IL-6 em 48 h . Conclui-se, a partir destes resultados, que as nanopartículas de titânio produziram o maior estímulo na resposta pró-inflamatória nos macrófagos, independente da sua associação com LPS de P. gingivalis.

Virulence factors, antibiotic resistance phenotypes and O-serogroups of Escherichia coli strains isolated from community-acquired urinary tract infection patients in Mexico.

BACKGROUND/PURPOSE: Uropathogenic Escherichia coli (UPEC) strains isolated from patients with community-acquired urinary tract infections (UTIs) were assessed to determine the prevalence of virulence genes, antibiotic resistance, and the O-serogroup of the strains. METHODS: Consenting patients with community-acquired UTI were enrolled at Unidad Médica Familiar Number 64 (Instituto Mexicano del Seguro Social, Estado de Mexico, Mexico) and 321 urine samples were collected. Polymerase chain reaction (PCR) was used to assess 24 virulence genes and 14 O-serogroups. The Kirby-Bauer method was used to evaluate the antibiotic susceptibility of the isolated strains to 12 commonly used antibiotics. RESULTS: A total of 194 strains were identified as E. coli using standard biochemical tests, followed by PCR amplification of 16S ribosomal RNA gene. Only 58.2% of the strains belonged to the assessed 14 O-serogroups. The serogroups O25, O15, O8, and O75 were present in 20.6%, 17%, 6.1%, and 4.6% of strains, respectively. The most frequently occurring virulence genes among UPEC strains included kpsMT (92.2% strains), usp (87.1%), irp2 (79.3%), iha (64.9%), fim (61.3%), set (36%), astA (33.5%), pap (24.7%), and papGII (21.1%). In addition, 97% of the strains were multi-drug resistant (coresistance to 3-11 antibiotics). CONCLUSION: The isolated UPEC strains predominantly belonged to three serogroups (O25, O15, and O8), harboured numerous virulence genes, and are multiresistant to antibiotics. The findings of this study could be used to orient UTI treatment strategies and in epidemiological studies in Mexico.

The Porphyromonas gingivalis O antigen is required for inhibition of apoptosis in gingival epithelial cells following bacterial infection.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection induces apoptosis inhibition in gingival epithelial cells; however, it is not fully understood which bacterial effectors are involved in this process. The aim of this study is to evaluate whether the P. gingivalis lipopolysaccharide (LPS), specifically the O-antigen region, affects adherence, invasion, viability and apoptosis of gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells (OKF6/TERT2 line) were infected by different freshly prepared P. gingivalis clinical isolates, obtained from subjects with chronic periodontitis (CP3 and CP4) and healthy individuals (H1 and H3). Periodontitis and healthy isolates show differences in O-antigen production, as healthy isolates lack the O-antigen region. In addition, cells were infected by a site-specific mutant lacking the O-antigen portion. After 24 h postinfection, cell proliferation, viability and apoptosis were evaluated by Trypan blue, MTS and annexin V assays, respectively. Bacterial invasion, adhesion and proliferation were measured by gentamicin/metronidazole protection assays. Finally, toll-like receptor (TLR)2 and TLR4 mRNA expression was evaluated by quantitative reverse transcription-polymerase chain reaction. Statistical analysis was performed using ANOVA, Tukey's or Dunnett's tests (p < 0.05). RESULTS: At 24 h postinfection, strains lacking the O-antigen region (healthy isolates and O-antigen ligase-deficient strain) were unable to increase proliferation and viability, or decrease apoptosis as compared with strains producing intact LPS (periodontitis isolates and reference strain). However, the presence of the O-antigen neither contributed to changes in the ability of the bacteria to adhere to or invade cells, nor to intracellular survival. The presence of O-antigen also increased the expression of TLR4 (nearly sixfold), which correlated with inhibition of apoptosis. CONCLUSION: The O-antigen region of P. gingivalis LPS is required to increase gingival epithelial cell viability upon infection by bacteria and this increase is attributable to a reduction in apoptosis. Moreover, although bacterial internalization is required, the effects observed are not due to alterations in P. gingivalis adherence, invasion or intracellular survival. Interestingly, inhibition of apoptosis correlates with increased TLR4 expression, suggesting a role for TLR4 in this process.

Structure of the polysaccharides from the lipopolysaccharide of Azospirillum brasilense Jm125A2.

Two polysaccharides were obtained by mild acid degradation of the lipopolysaccharide of associative nitrogen-fixing bacteria Azospirillum brasilense Jm125A2 isolated from the rhizosphere of a pearl millet. The following structures of the polysaccharides were established by sugar and methylation analyses, Smith degradation, and (1)H and (13)C NMR spectroscopy: [Formula: see text] Structure 1 has been reported earlier for a polysaccharide from A. brasilense S17 (Fedonenko YP, Konnova ON, Zdorovenko EL, Konnova SA, Zatonsky GV, Shaskov AS, Ignatov VV, Knirel YA. Carbohydr Res 2008;343:810-6), whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides.

Adherence and virulence genes of Escherichia coli from children diarrhoea in the Brazilian Amazon.

The bacterial pathogen most commonly associated with endemic forms of childhood diarrhoea is Escherichia coli . Studies of epidemiological characteristics of HEp-2 cell-adherent E. coli in diarrhoeal disease are required, particularly in developing countries. The aim of this study was evaluate the presence and significance of adherent Escherichia coli from diarrhoeal disease in children. The prevalence of LA, AA, and DA adherence patterns were determined in HEp-2 cells, the presence of virulence genes and the presence of the O serogroups in samples obtained from 470 children with acute diarrhoea and 407 controls in Porto Velho, Rondônia, Brazil. E. coli isolates were identified by PCR specific for groups of adherent E. coli . Out of 1,156 isolates obtained, 128 (11.0%) were positive for eae genes corresponding to EPEC, however only 38 (29.6%) of these amplified bfpA gene . EAEC were isolated from 164 (14.1%) samples; of those 41(25%), 32 (19%) and 16 (9.7%) amplified eagg , aggA or aafA genes, respectively and aggA was significantly associated with diarrhoea ( P = 0.00006). DAEC identified by their adhesion pattern and there were few isolates. In conclusion, EAEC was the main cause of diarrhoea in children, especially when the aggA gene was present, followed by EPEC and with a negligible presence of DAEC.