Detection of type III secretion genes as a general indicator of bacterial virulence

Type III secretion systems of Gram-negative bacteria are specific export machineries for virulence factors which allow their translocation to eukaryotic cells. Since they correlate with bacterial pathogenicity, their presence is used as a general indicator of bacterial virulence. By comparing the genetic relationship of the major type III secretion systems we found the family of genes encoding the inner-membrane channel proteins represented by the Yersinia enterocolitica lcrD (synonym yscV) and its homologous genes from other species an ideal component for establishing a general detection approach for type III secretion systems. Based on the genes of the lcrD family we developed gene probes for Gram-negative human, animal and plant pathogens. The probes comprise lcrD from Y. enterocolitica, sepA from enteropathogenic Escherichia coli, invA from Salmonella typhimurium, mxiA from Shigella sonnei, as well as hrcV from Erwinia amylovora. In addition we included as a control probe the flhA gene from E. coli K-12 to validate our approach. FlhA is part of the flagellar export apparatus which shows a high degree of similarity with type III secretions systems, but is not involved in pathogenicity. The probes were evaluated by screening a series of pathogenic as well as non-pathogenic bacteria. The probes detected type III secretion in pathogens where such systems were either known or were expected to be present, whereas no positive hybridization signals could be found in non-pathogenic Gram-negative bacteria. Gram-positive bacteria were devoid of known type III secretion systems. No interference due to the genetic similarity between the type III secretion system and the flagellar export apparatus was observed. However, potential type III secretion systems could be detected in bacteria where no such systems have been described yet. The presented approach provides therefore a useful tool for the assessment of the virulence potential of bacterial isolates of human, animal and plant origin. Moreover, it is a powerful means for a first safety assessment of poorly characterized strains intended to be used in biotechnological applications.

Effect of photoactivated disinfection with a light-emitting diode on bacterial species and biofilms associated with periodontitis and peri-implantitis

BACKGROUND To determine the effect of photoactivated disinfection (PAD) using toluidine blue and a light-emitting diode (LED) in the red spectrum (wave length at 625-635 nm) on species associated with periodontitis and peri-implantitis and bacteria within a periodontopathic biofilm. METHODS Sixteen single microbial species including 2 Porphyromonas gingivalis and 2 Aggregatibacter actinomycetemcomitans and a multispecies mixture consisting of 12 species suspended in saline without and with 25% human serum were exposed to PAD. Moreover, single-species biofilms consisting of 2 P. gingivalis and 2 A. actinomycetemcomitans strains and a multi-species biofilm on 24-well-plates, grown on titanium discs and in artificial periodontal pockets were exposed to PAD with and without pretreatment with 0.25% hydrogen peroxide. Changes in the viability were determined by counting the colony forming units (cfu). RESULTS PAD reduced the cfu counts in saline by 1.42 log₁₀ after LED application for 30s and by 1.99 log₁₀ after LED application for 60s compared with negative controls (each p<0.001). Serum did not inhibit the efficacy of PAD. PAD reduced statistically significantly (p<0.05) the cfu counts of the P. gingivalis biofilms. The viability of the A. actinomycetemcomitans biofilms and the multi-species biofilms was statistically significantly decreased when PAD was applied after a pretreatment with 0.25% hydrogen peroxide. The biofilm formed in artificial pockets was more sensitive to PAD with and without pretreatment with hydrogen peroxide compared with those formed on titanium discs. CONCLUSIONS PAD using a LED was effective against periodontopathic bacterial species and reduced viability in biofilms but was not able to completely destroy complex biofilms. The use of PAD following pretreatment with hydrogen peroxide resulted in an additional increase in the antimicrobial activity which may represent a new alternative to treat periodontal and peri-implant infections thus warranting further testing in clinical studies.

Gingival fluid cytokine expression and subgingival bacterial counts during pregnancy and postpartum: a case series

OBJECTIVES The aim of this study was to assess gingival fluid (GCF) cytokine messenger RNA (mRNA) levels, subgingival bacteria, and clinical periodontal conditions during a normal pregnancy to postpartum. MATERIALS AND METHODS Subgingival bacterial samples were analyzed with the checkerboard DNA-DNA hybridization method. GCF samples were assessed with real-time PCR including five proinflammatory cytokines and secretory leukocyte protease inhibitor. RESULTS Nineteen pregnant women with a mean age of 32 years (S.D. ± 4 years, range 26-42) participated in the study. Full-mouth bleeding scores (BOP) decreased from an average of 41.2% (S.D. ± 18.6%) at the 12th week of pregnancy to 26.6% (S.D. ± 14.4%) at the 4-6 weeks postpartum (p < 0.001). Between week 12 and 4-6 weeks postpartum, the mean probing pocket depth changed from 2.4 mm (S.D. ± 0.4) to 2.3 mm (S.D. ± 0.3) (p = 0.34). Higher counts of Eubacterium saburreum, Parvimonas micra, Selenomonas noxia, and Staphylococcus aureus were found at week 12 of pregnancy than at the 4-6 weeks postpartum examinations (p < 0.001). During and after pregnancy, statistically significant correlations between BOP scores and bacterial counts were observed. BOP scores and GCF levels of selected cytokines were not related to each other and no differences in GCF levels of the cytokines were observed between samples from the 12th week of pregnancy to 4-6 weeks postpartum. Decreasing postpartum counts of Porphyromonas endodontalis and Pseudomonas aeruginosa were associated with decreasing levels of Il-8 and Il-1β. CONCLUSIONS BOP decreased after pregnancy without any active periodontal therapy. Associations between bacterial counts and cytokine levels varied greatly in pregnant women with gingivitis and a normal pregnancy outcome. Postpartum associations between GCF cytokines and bacterial counts were more consistent. CLINICAL RELEVANCE Combined assessments of gingival fluid cytokines and subgingival bacteria may provide important information on host response.

The Bacterial Amyloid Curli Is Associated with Urinary Source Bloodstream Infection

Urinary tract infections are the most common cause of E. coli bloodstream infections (BSI) but the mechanism of bloodstream invasion is poorly understood. Some clinical isolates have been observed to shield themselves with extracellular amyloid fibers called curli at physiologic temperature. We hypothesize that curli fiber assembly at 37 °C promotes bacteremic progression by urinary E. coli strains. Curli expression by cultured E. coli isolates from bacteriuric patients in the presence and absence of bacteremia were compared using Western blotting following amyloid fiber disruption with hexafluoroisopropanol. At 37 °C, urinary isolates from bacteremic patients were more likely to express curli than those from non-bacteremic patients [16/22 (73%) vs. 7/21 (33%); p = 0.01]. No significant difference in curli expression was observed at 30 °C [86% (19/22) vs. 76% (16/21); p = 0.5]. Isolates were clonally diverse between patients, indicating that this phenotype is distributed across multiple lineages. Most same-patient urine and blood isolates were highly related, consistent with direct invasion of urinary bacteria into the bloodstream. 37 °C curli expression was associated with bacteremic progression of urinary E. coli isolates in this population. These findings suggest new future diagnostic and virulence-targeting therapeutic approaches

Temporal and Spatial Coexistence of Archaeal and Bacterial amoA Genes and Gene Transcripts in Lake Lucerne

Despite their crucial role in the nitrogen cycle, freshwater ecosystems are relatively rarely studied for active ammonia oxidizers (AO). This study of Lake Lucerne determined the abundance of both amoA genes and gene transcripts of ammonia-oxidizing archaea (AOA) and bacteria (AOB) over a period of 16 months, shedding more light on the role of both AO in a deep, alpine lake environment. At the surface, at 42 m water depth, and in the water layer immediately above the sediment, AOA generally outnumbered AOB. However, in the surface water during summer stratification, when both AO were low in abundance, AOB were more numerous than AOA. Temporal distribution patterns of AOA and AOB were comparable. Higher abundances of amoA gene transcripts were observed at the onset and end of summer stratification. In summer, archaeal amoA genes and transcripts correlated negatively with temperature and conductivity. Concentrations of ammonium and oxygen did not vary enough to explain the amoA gene and transcript dynamics. The observed herbivorous zooplankton may have caused a hidden flux of mineralized ammonium and a change in abundance of genes and transcripts. At the surface, AO might have been repressed during summer stratification due to nutrient limitation caused by active phytoplankton.

Bacterial Meningitis After Sinus Surgery in Five Adult Horses.

OBJECTIVE To report meningoencephalitis as a complication after paranasal sinus surgery in 5 horses. STUDY DESIGN Case series. ANIMALS Adult horses (n = 5). METHODS Medical records (2005-2010) of 5 horses that developed neurologic signs after sinus surgery were reviewed to identify potential risk factors, cause(s), or common pathways for infection. RESULTS Underlying diseases were primary (n = 1) and secondary sinusitis (4) because of apical dental infection (1), sinus cyst (2), or masses in the ethmoturbinate region (2). Horses were treated by conventional surgical approaches and aftercare including repeated sinus lavage. Four horses had undulating pyrexia postoperatively despite antimicrobial therapy. All horses developed neurologic signs, eventually unresponsive to treatment. Suppurative meningoencephalitis was diagnosed macro- and/or microscopically on necropsy in all horses. CONCLUSION Meningitis is a rare but fatal complication after sinus surgery in horses.

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

BACKGROUND: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown. PRINCIPAL FINDINGS: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2. SIGNIFICANCE: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.

Adenine nucleotide-dependent regulation of assembly of bacterial tubulin-like FtsZ by a hypermorph of bacterial actin-like FtsA.

Cytokinesis in bacteria depends upon the contractile Z ring, which is composed of dynamic polymers of the tubulin homolog FtsZ as well as other membrane-associated proteins such as FtsA, a homolog of actin that is required for membrane attachment of the Z ring and its subsequent constriction. Here we show that a previously characterized hypermorphic mutant FtsA (FtsA*) partially disassembled FtsZ polymers in vitro. This effect was strictly dependent on ATP or ADP binding to FtsA* and occurred at substoichiometric levels relative to FtsZ, similar to cellular levels. Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical concentration for FtsZ assembly but was able to disassemble preformed FtsZ polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination of the inhibited FtsZ polymers revealed a transition from long, straight polymers and polymer bundles to mainly short, curved protofilaments. These results indicate that a bacterial actin, when activated by adenine nucleotides, can modify the length distribution of bacterial tubulin polymers, analogous to the effects of actin-depolymerizing factor/cofilin on F-actin.

The effect of body mass index on therapeutic response to bacterial vaginosis in pregnancy.

Our objective was to determine the effect of body mass index (BMI) on response to bacterial vaginosis (BV) treatment. A secondary analysis was conducted of two multicenter trials of therapy for BV and TRICHOMONAS VAGINALIS. Gravida were screened for BV between 8 and 22 weeks and randomized between 16 and 23 weeks to metronidazole or placebo. Of 1497 gravida with asymptomatic BV and preconceptional BMI, 738 were randomized to metronidazole; BMI was divided into categories: < 25, 25 to 29.9, and > or = 30. Rates of BV persistence at follow-up were compared using the Mantel-Haenszel chi square. Multiple logistic regression was used to evaluate the effect of BMI on BV persistence at follow-up, adjusting for potential confounders. No association was identified between BMI and BV rate at follow-up ( P = 0.21). BMI was associated with maternal age, smoking, marital status, and black race. Compared with women with BMI of < 25, adjusted odds ratio (OR) of BV at follow-up were BMI 25 to 29.9: OR, 0.66, 95% CI 0.43 to 1.02; BMI > or = 30: OR, 0.83, 95% CI 0.54 to 1.26. We concluded that the persistence of BV after treatment was not related to BMI.

The effect of body mass index on therapeutic response to bacterial vaginosis in pregnancy.

Our objective was to determine the effect of body mass index (BMI) on response to bacterial vaginosis (BV) treatment. A secondary analysis was conducted of two multicenter trials of therapy for BV and TRICHOMONAS VAGINALIS. Gravida were screened for BV between 8 and 22 weeks and randomized between 16 and 23 weeks to metronidazole or placebo. Of 1497 gravida with asymptomatic BV and preconceptional BMI, 738 were randomized to metronidazole; BMI was divided into categories: < 25, 25 to 29.9, and > or = 30. Rates of BV persistence at follow-up were compared using the Mantel-Haenszel chi square. Multiple logistic regression was used to evaluate the effect of BMI on BV persistence at follow-up, adjusting for potential confounders. No association was identified between BMI and BV rate at follow-up ( P = 0.21). BMI was associated with maternal age, smoking, marital status, and black race. Compared with women with BMI of < 25, adjusted odds ratio (OR) of BV at follow-up were BMI 25 to 29.9: OR, 0.66, 95% CI 0.43 to 1.02; BMI > or = 30: OR, 0.83, 95% CI 0.54 to 1.26. We concluded that the persistence of BV after treatment was not related to BMI.

Functional taxonomy of bacterial hyperstructures.

The levels of organization that exist in bacteria extend from macromolecules to populations. Evidence that there is also a level of organization intermediate between the macromolecule and the bacterial cell is accumulating. This is the level of hyperstructures. Here, we review a variety of spatially extended structures, complexes, and assemblies that might be termed hyperstructures. These include ribosomal or "nucleolar" hyperstructures; transertion hyperstructures; putative phosphotransferase system and glycolytic hyperstructures; chemosignaling and flagellar hyperstructures; DNA repair hyperstructures; cytoskeletal hyperstructures based on EF-Tu, FtsZ, and MreB; and cell cycle hyperstructures responsible for DNA replication, sequestration of newly replicated origins, segregation, compaction, and division. We propose principles for classifying these hyperstructures and finally illustrate how thinking in terms of hyperstructures may lead to a different vision of the bacterial cell.

Down-Regulation of Annexin A1 in the Urothelium Decreases Cell Survival After Bacterial Toxin Exposure

PURPOSE: We examined the role of annexins in bladder urothelium. We characterized expression and distribution in normal bladders, biopsies from patients with bladder pain syndrome, cultured human urothelium and urothelial TEU-2 cells. MATERIALS AND METHODS: Annexin expression in bladder layers was analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunofluorescence. We assessed cell survival after exposure to the pore forming bacterial toxin streptolysin O by microscopy and alamarBlue® assay. Bladder dome biopsies were obtained from 8 asymptomatic controls and 28 patients with symptoms of bladder pain syndrome. RESULTS: Annexin A1, A2, A5 and A6 were differentially distributed in bladder layers. Annexin A6 was abundant in detrusor smooth muscle and low in urothelium, while annexin A1 was the highest in urothelium. Annexin A2 was localized to the lateral membrane of umbrella cells but excluded from tight junctions. TEU-2 cell differentiation caused up-regulation of annexin A1 and A2 and down-regulation of annexin A6 mRNA. Mature urothelium dedifferentiation during culture caused the opposite effect, decreasing annexin A1 and increasing annexin A6. Annexin A2 influenced TEU-2 cell epithelial permeability. siRNA mediated knockdown of annexin A1 in TEU-2 cells caused significantly decreased cell survival after streptolysin O exposure. Annexin A1 was significantly reduced in biopsies from patients with bladder pain syndrome. CONCLUSIONS: Several annexins are expressed in human bladder and TEU-2 cells, in which levels are regulated during urothelial differentiation. Annexin A1 down-regulation in patients with bladder pain syndrome might decrease cell survival and contribute to compromised urothelial function.