Antibacterial activity of a complex bacteriocin secreted by Staphylococcus epidermidis against Porphyromonas gingivalis.

OBJECTIVE: To characterize the inhibitory activity of a novel bacteriocin produced by Staphylococcus epidermidis against this periodontal pathogen. DESIGN: The bacteriocin activity was evaluated by the agar diffusion method over a lawn of P. gingivalis ATCC 33277. The bacteriocin was purified by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) and Matrix Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry (MALDI-TOF-MS). In addition, the bacteriocin host specificity, production on different media cultures and susceptibility to enzymes, pH, and heat treatment were determined. RESULTS: The bacteriocin BAC 14990 was selective to P. gingivalis, suggesting a narrow activity range. The production during the growth curve indicated that S. epidermidis had a continued production of this antimicrobial, showing the highest concentration in the stationary phase. The purification of BAC 14990 showed that bacteriocin had a molecular mass of 5795 Da. BAC 14990 was partially resistant to the treatment with proteinase K and papain, however, was fully susceptible to amylase treatment indicating the presence of sugar residues in the protein, suggesting a conjugated type of bacteriocin. Also, this diffusible inhibitory substance was heat and pH treatment resistant. CONCLUSIONS: The results indicate the isolation of a new staphylococcal complex bacteriocin that is able to eliminate a Gram-negative bacterium. These results could contribute to the development of treatments directed against pathogens in mixed communities, as is the case with oral diseases.

Synthesis, characterisation, crystal structure and antimicrobial evaluation of novel 6-alkoxyergosta-4,6,8(14),22-tetraen-3-one derived from natural ergosta-5,7,22-trien-3ß-ol.

In this study, we report a facile transformation starting from 5α-hydroxyergosta-7,22-dien-3,6-dione (1) to afford two novel compounds: 6-methoxyergosta-4,6,8(14),22-tetraen-3-one (2) and 6-ethoxyergosta-4,6,8(14),22-tetraen-3-one (3) using alcoholic acid catalysis. Their structures were elucidated using NMR experiments, FT-IR, MS and X-ray analysis. These compounds were evaluated for antibacterial activity using the disk and broth diffusion test. In those tests, compound 3 was found to be the most significant antibacterial agent. In general, compounds 1-3 showed inhibition zone in the range of 7.00-12.3 mm for S. aureus and S. mutans, meanwhile for Gram-negative bacteria E. coli and Pseudomonas sp. was found to be in the range of 7.00-8.00 mm. For the most active, compound 3, MIC was significantly lower than that reported for ergosterol, in a value of 160 µg/mL. Overall, these compounds were more active than their natural precursor.

Antibacterial Effect of Luma apiculata (DC.) Burret Extracts in Clinically Important Bacteria.

Nosocomial infections caused by bacteria are one of the main public health problems. Moreover, the resistance to antibiotics by these bacteria makes it necessary to find new treatments to fight them. Objective. To evaluate the antibacterial activity of Luma apiculata (DC.) Burret extracts on bacteria of clinical importance. Materials and Methods. In this study, extracts were obtained at room temperature by successive extraction of L. apiculata leaves, flowers, and branches and treated separately with solvents of ascending polarity (i.e., hexane, methylene dichloride, ethyl acetate, ethanol, methanol, and water) to extract the compounds depending on their polarity. Then, the extract's antibacterial activity was tested against Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Enterococcus sp, Acinetobacter baumanii, Pseudomonas aeruginosa, and Escherichia coli. Results. The hexane extract of L. apiculata leaves resulted to be active against all bacteria tested. Among them, S. aureus showed to be the more susceptible, showing a minimum inhibitory concentration (MIC) of 120 µg/ml. In addition, a growth curve was performed, and colonies were counted. A decrease in bacterial growth was observed when the hexane extract of L. apiculata leaves was added. Besides, the hexane extracts of L. apiculata flowers resulted to be active against all Gram-positive tested bacteria. However, at higher concentrations, this extract resulted inactive for the Gram-negative bacteria tested. The hexane extract of L. apiculata branches resulted to be inactive in all cases. The extracts obtained treating separately leaves, flowers, or branches with solvents of major polarity than the hexane in a successive extraction of ascending polarity methodology resulted also to be inactive as an antimicrobial against all bacteria tested. Discussion/Conclusion. The hexane extract of L. apiculata leaves showed the lower MIC against S. aureus when compared with extracts obtained from other parts of the plant. The growth curve and the colonies count suggest a bacteriostatic activity of the L. apiculata leaves extract against Staphylococcus aureus.

Prevalence and risk factors for HPV infection in normal oral mucosa of chilean dentistry students / Prevalencia y factores de riesgo de infección por virus papiloma humano en mucosa oral normal de estudiantes de odontología chilenos

Abstract: aim: to determine the prevalence and risk factors for HPV infection in normal oral mucosa of Chilean dentistry students. Materials and methods: A cross-sectional study was performed. The study group was comprised of 103 individuals between 18 and 33 years old. A self-administered survey of cancer family history, sexual habits, smoking and alcohol drinking was applied. Oral mucosal samples were taken using a sterile swab. Subsequently, all samples were analyzed by polymerase chain reaction technique (PCR). Results: Results were negative for HPV detection in all analyses. Of the study population, 58 percent had a family history of cancer, 40.9 percent had had more than 3 sexual partners, 76.3 percent had sexual intercourse before the age of 19, 66.3 percent had engaged in oral sex, 69.9 percent drank alcohol and 20.6 percent were smokers. Conclusions: The group studied is exposed to various risk factors for HPV infection, so it is necessary to educate about the relationship between them and the spread of the virus. Despite the presence of risk factors, the detected prevalence of HPV was 0 percent.

Resumen: se realizó un estudio de corte transversal. El grupo de estudio fue constituido por 103 individuos entre 18 y 33 años. Se aplicó una encuesta autoadministrada sobre antecedentes familiares de cáncer, hábitos sexuales y consumo de tabaco y alcohol. Se tomaron muestras de la mucosa oral utilizando un hisopo estéril. Posteriormente, todas las muestras fueron analizadas mediante técnica de reacción en cadena de la polimerasa (PCR). Resultados: Los resultados fueron negativos para la detección de VPH en todos los análisis. De la población estudiada el 58 por ciento presentó antecedentes familiares de cáncer, el 40,9 por ciento ha tenido más de 3 parejas sexuales, el 76,3 por ciento se inició sexualmente antes de los 19 años, el 66,3 por ciento ha practicado sexo oral, un 69,9 por ciento es bebedor de alcohol y el 20,6 por ciento es fumador. Conclusiones: El grupo analizado está expuesto a diversos factores de riesgo de infección VPH, por lo que es necesario educar acerca de la relación entre estos y el contagio con el virus. A pesar de la presencia de factores de riesgo en los encuestados, la prevalencia detectada de VPH fue de 0 por ciento.

Synthesis of new antibacterial composite coating for titanium based on highly ordered nanoporous silica and silver nanoparticles.

Infection is the most common factor that leads to dental titanium implant failure. Antibacterial implant surfaces based on nano-scale modifications of the titanium appear as an attractive strategy for control of peri-implantitis. In the present work, the preparation and antibacterial properties of a novel composite coating for titanium based on nanoporous silica and silver nanoparticles are presented. Starch-capped silver nanoparticles (AgNPs) were synthesized and then incorporated into sol-gel based solution system. The AgNP-doped nanoporous silica coatings were prepared on titanium surface using a combined sol-gel and evaporation-induced self-assembly (EISA) method. The coating nanostructure was characterized by XRD, SEM-EDX, and HR-TEM. Antibacterial activity was evaluated against Aggregatibacter actinomycetemcomitans, a representative pathogen of dental peri-implantitis. Colony-forming units (CFUs) were counted within the biofilm and at the planktonic state. Biofilm development was quantified using crystal violet staining and viability of adherent bacteria was confirmed with the Live/Dead fluorescence assay. Silica-based composite coating containing AgNPs (AgNP/NSC) was prepared on titanium surface by direct incorporation of AgNP suspension into the sol-gel system. The self-assembly technique enabled the spontaneous formation of a highly ordered nanoporosity in the coating structure, which is a desired property for osseointegration aspects of titanium implant surface. AgNP/NSC coating produces a strong antibacterial effect on titanium surface by not only killing the adherent bacteria but also reducing the extent of biofilm formation. Biofilm survival is reduced by more than 70% on the AgNP/NSC-modified titanium surface, compared to the control. This antibacterial effect was verified for up to 7 days of incubation. The long-term antibacterial activity exhibited by the nanostructured AgNP/NSC-titanium surface against A. actinomycetemcomitans suggests that this type of nano-scale surface modification is a promissory strategy to control infections associated with dental implant rehabilitation.

Detección Temprana Mediante PCR de Patógenos Periodontales en Relación a Implantes Oseointegrados / Early Detection of Periodontal Pathogens by PCR in Relation to Osseointegrated Implants

El objetivo de este trabajo fue valuar la presencia temprana en sitios dentarios e implantarios de cuatro bacterias periodontopatógenas (A. actinomycetemcomitans, P. gingivalis, T. forsythensis y T. denticola) luego de dos semanas de la cirugía de segunda fase, además se propone comparar la presencia de estas 4 bacterias en sitios subgingivales dentarios e implantarios. Se estudiaron mediante reacción en cadena de polimerasa muestras de placa subgingival de implantes y de dientes vecinos a ellos, dos semanas luego de la cirugía de segunda fase. Dieciséis implantes y trece dientes en diez pacientes fueron seleccionados. Luego de dos semanas se encontró presencia de bacterias periodontopatógenas en sitios tanto periodontales como peri-implantarios, no se encontró relación entre diente e implante para P. gingivalis, T. forsythensis y T. denticola. Se encontró una relación significativa para A. actinomycetemcomitans (P<0,005). La bacteria detectada con mayor frecuencia fue P. gingivalis y la menos encontrada fue T. denticola. La dependencia para A. actinomycetemcomitans estuvo relacionada a pacientes con historia de periodontitis. Dentro de los límites de este estudio, los resultados muestran la presencia temprana de los cuatro patógenos periodontales alrededor de los sitios implantarios, y una relación estadísticamente significativa (P<0,005) entre implante y diente para A. actinomycetemcomitans.

The aim of this study was to assess the early colonization of four periodontopathogenic bacteria (A. actinomycetemcomitans, P. gingivalis, T. forsythensis and T. denticola) on titanium implants immediately after two weeks following second stage surgery; to compare the presence of these four periodontopathogenic bacteria at subgingival implant and adjacent tooth sites. Subgingival plaque samples from implant and neighboring teeth were studied by PCR after two weeks following second stage surgery. Sixteen implants and thirteen teeth, in ten patients were selected. At two weeks, pathogenic bacteria presence was found in both peri-implant and periodontal sites, there was no relation found between tooth and implant for P. gingivalis, T. forsythensis and T. denticola. A significant relation was found for A. actinomycetemcomitans (P<0.005). The more frequently detected bacteria were P. gingivalis, and the less was T. denticola. The dependence for A. actinomycetemcomitans was related to patients with a history of periodontitis. Within the limits of this study, the findings showed the early presence of the four periodontopathogenic bacteria around implant sites and a statistically significant (P<0.005) relation between implants and teeth sites for A. actinomycetemcomitans.

Isolation of a novel Aggregatibacter actinomycetemcomitans serotype b bacteriophage capable of lysing bacteria within a biofilm.

A bacteriophage specific for Aggregatibacter actinomycetemcomitans serotype b, able to kill the bacterium within a biofilm, was isolated. Random mutagenesis of this phage rendered a bacteriophage able to kill 99% of the bacteria within a biofilm. This is the first report of a biocontrol experiment against A. actinomycetemcomitans.

Isolation of a novel bacteriophage specific for the periodontal pathogen Fusobacterium nucleatum.

Fusobacterium nucleatum is a periodontal pathogen that has been directly associated with the development and progression of periodontal disease, a widespread pathology that affects the support tissues of the tooth. We isolated a new bacteriophage (FnpΦ02) that specifically infects this bacterium. Transmission electron microscopy showed that the virion is composed of an icosahedral head and a segmented tail. The size of the phage genome was estimated to be approximately 59 kbp of double-stranded DNA. The morphological features and the genetic characteristics suggest that FnpΦ02 is part of the Siphoviridae family. Using one-step growth and adsorption experiments, the latent period, burst size, and adsorption rate were estimated to be 15 h, 100 infectious units per cell, and 7.5 × 10⁻¹° ml min⁻¹, respectively. A small fragment of phage DNA was cloned and sequenced, showing 93% nucleotide identity with the phage PA6 of Propionibacterium acnes and amino acid identity with fragments of two proteins (Gp3 and Gp4) of this phage. To our knowledge, FnpΦ02 is the first phage described to infect Fusobacterium nucleatum and provides the base for future exploration of phages in the control of periodontal disease.

Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit.

WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in DeltawbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the DeltawbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.

The outer core lipopolysaccharide of Salmonella enterica serovar Typhi is required for bacterial entry into epithelial cells.

Salmonella enterica serovar Typhi causes typhoid fever in humans. Central to the pathogenicity of serovar Typhi is its capacity to invade intestinal epithelial cells. The role of lipopolysaccharide (LPS) in the invasion process of serovar Typhi is unclear. In this work, we constructed a series of mutants with defined deletions in genes for the synthesis and polymerization of the O antigen (wbaP, wzy, and wzz) and the assembly of the outer core (waaK, waaJ, waaI, waaB, and waaG). The abilities of each mutant to associate with and enter HEp-2 cells and the importance of the O antigen in serum resistance of serovar Typhi were investigated. We demonstrate here that the presence and proper chain length distribution of the O-antigen polysaccharide are essential for serum resistance but not for invasion of epithelial cells. In contrast, the outer core oligosaccharide structure is required for serovar Typhi internalization in HEp-2 cells. We also show that the outer core terminal glucose residue (Glc II) is necessary for efficient entry of serovar Typhi into epithelial cells. The Glc I residue, when it becomes terminal due to a polar insertion in the waaB gene affecting the assembly of the remaining outer core residues, can partially substitute for Glc II to mediate bacterial entry into epithelial cells. Therefore, we conclude that a terminal glucose in the LPS core is a critical residue for bacterial recognition and internalization by epithelial cells.

RpoS and RpoN are involved in the growth-dependent regulation of rfaH transcription and O antigen expression in Salmonella enterica serovar Typhi.

We reported earlier that the production of O antigen lipopolysaccharide (LPS) by Salmonella enterica serovar Typhi (Salmonella typhi) increases at the onset of stationary phase and correlates with a growth-regulated expression of the rfaH gene under the control of the alternative sigma factor RpoN (Microbiology 148 (2002) 3789). In this study, we demonstrate that RpoS also modulates rfaH promoter activity as revealed by the absence of growth-dependent regulation of an rfaH-lacZ transcriptional fusion and O antigen production in a S. typhi rpoS mutant. Introduction of a constitutively expressed rpoN gene into the rpoS mutant restored increased production of O antigen during stationary phase, suggesting that constitutive production of RpoN could overcome the RpoS defect. Similar results were observed when an rpoS rpoN double mutant was transformed with the intact rpoN gene. Thus, we conclude that both RpoS and RpoN control the rfaH promoter activity and concomitantly, the production of O-specific LPS in S. typhi.

O-antigen expression in Salmonella enterica serovar Typhi is regulated by nitrogen availability through RpoN-mediated transcriptional control of the rfaH gene.

The authors previously reported increased expression of the Salmonella enterica serovar Typhi (S. typhi) rfaH gene when the bacterial cells reach stationary phase. In this study, using a lacZ fusion to the rfaH promoter region, they demonstrate that growth-dependent regulation of rfaH expression occurs at the level of transcription initiation. It was also observed that production of the lipopolysaccharide (LPS) O-antigen by S. typhi Ty2 correlated with the differential expression of rfaH during bacterial growth. This was probably due to the increased cellular levels of RfaH, since expression of the distal gene in the O-antigen gene cluster of S. typhi Ty2, wbaP, was also increased during stationary growth, as demonstrated by RT-PCR analysis. Examination of the sequences upstream of the rfaH coding region revealed homologies to potential binding sites for the RcsB/RcsA dimer of the RcsC/YopJ/RcsB phosphorelay regulatory system and for the RpoN alternative sigma factor. The expression of the rfaH gene in rpoN and rcsB mutants of S. typhi Ty2 was measured. The results indicate that inactivation of rpoN, but not of rcsB, suppresses the growth-phase-dependent induction of rfaH expression. Furthermore, production of beta-galactosidase mediated by the rfaH-lacZ fusion increased approximately fourfold when bacteria were grown in a nitrogen-limited medium. Nitrogen limitation was also shown to increase the expression of the O-antigen by the wild-type S. typhi Ty2, as demonstrated by a similar electrophoretic profile to that observed during the stationary phase of growth in rich media. It is therefore concluded that the relationship between LPS production and nitrogen limitation parallels the pattern of rfaH regulation under the control of RpoN and is consistent with the idea that RpoN modulates LPS formation via its effect on rfaH gene expression during bacterial growth.