BHI Medium Research Articles

Regulation and RNA-binding properties of Hfq-like RNA chaperones in Bacillus anthracis

BackgroundSmall RNAs (sRNAs) are important modulators of gene expression in bacteria. Regulation by sRNAs is yet to be established in Bacillus anthracis. Here, regulation and RNA-binding properties of Hfq-like RNA chaperones in B. anthracis are investigated. MethodsTranscript levels were measured by RT-PCR. Proteins were cloned, purified, and their ability to bind sRNA was seen by EMSA. Regulators of Hfq1 were identified by in silico analysis and validated by EMSA. Conserved sRNAs were identified by homology search and their ability to bind Hfq1 was seen by EMSA. Residues crucial for sRNA binding were identified by mutational studies. Resultshfq1 and hfq3 showed expression during exponential phase on BHI medium, while hfq2 showed no expression. Hfq1 and Hfq3 formed hexamer and sRNA–Hfq complex, not Hfq2. In silico prediction and EMSA confirmed AbrB binding to the promoter of Hfq1. Homology search identified 7 sRNAs in B. anthracis. The sRNA CsfG showed binding to Hfq1 via residues Y24, F29, Q30, R32, K56, and H57. ConclusionsHfq1 and Hfq3 can function as RNA chaperones in B. anthracis. The transition phase repressor AbrB might be responsible for the growth-dependent expression of Hfq1. The sporulation-specific sRNA CsfG binds to Hfq1 via its distal surface and requires an intact hexameric structure for forming CsfG-Hfq1 complex. General significanceThis is the first report demonstrating the regulation and functional properties of Hfq-like RNA chaperones in B. anthracis and paves the path towards establishing sRNA-based regulation in B. anthracis.

Low Xylitol Concentration induces Ultrastructural Cellular Changes in Streptococcus mutans and Streptococcus sobrinus

ABSTRACT Background Xylitol is a sugar alcohol frequently used in the prevention of dental caries. However, the mechanism of action of xylitol on mutans streptococci is still unclear. This study evaluated the ultrastructural cellular changes in Streptococcus mutans and Streptococcus sobrinus after xylitol treatment. Materials and methods S. mutans ATCC 25175 and S. sobrinus ATCC 33478 were grown separately in BHI medium plus 0.2% sucrose at 37°C under microaerophilic conditions overnight. Then, planktonic cells were harvested by centrifugation, resuspended in a fresh medium and incubated with either 0.2% glucose or 0.2% xylitol solutions for 2 hours. To prepare bacterial cells for the evaluation of morphology of cellular walls and polysaccharide capsules, cultures were subsequently fixed with 2.5% glutaraldehyde, stained with ruthenium red, post-fixed with 1% osmium tetroxide, dehydrated in acetone, embedded in Epon LX112 and analyzed by a transmission electron microscopy. Results Bacterial cells incubated with glucose showed welldefined cellular walls and well-stained cytoplasmic membrane. Unlike, xylitol yielded diffuse cellular wall and irregular polysaccharide capsules in both microorganisms. Conclusion Low xylitol concentrations also can induce cellular damage to both S. mutans and S. sobrinus. Further studies are needed to provide more evidence on the anticariogenic effect of xylitol. How to cite this article Pereira AFF, Silva TC, Machado MAAM, Buzalaf MAR. Low Xylitol Concentration induces Ultrastructural Cellular Changes in Streptococcus mutans and Streptococcus sobrinus. Int J Experiment Dent Sci 2015;4(1):1-3.

Dentifrice pH but not consistency may affect fluoride uptake in plaque

ObjectiveTest the ability of acidic fluoridated solutions to enhance fluoride (F) bound on bacteria (1) and the effect of dentifrice consistency on plaque fluid F uptake (2). Methods(1) Streptococcus mutans isolates were grown in BHI medium (37°C/18h). Bacteria were washed either with EDTA or CaCl2 both at 1mM to remove or add calcium, respectively. Pellets were incubated with 12mM NaF at pH 4.5 or 7 for 1min and F was quantified in the lysates and supernatants with the electrode, after HMDS-facilitated diffusion. (2) A randomized, double-blind, crossover clinical trial was performed in three phases with nineteen adults (20–35 years) that used one of the dentifrices: commercial toothpaste (1100ppm F, pH7.0 and conventional viscosity (Sorriso Fresh®)); experimental liquid dentifrice (ELD) (1100ppm F, pH7.0 and low viscosity [1.1% carboxymethylcellulose (CMC)]) and ELD (1100ppm F and high viscosity pH7.0 (2.2% CMC)). F concentration in plaque fluid was analyzed using an inverted F electrode. Results(1) Significantly higher F amounts were detected in the lysates of bacteria incubated with NaF solution at pH4.5 compared to the supernatant, which was not seen at pH7.0, being this effect calcium-dependent. (2) Significantly higher F concentrations in plaque fluid were found 1h after toothbrushing compared to 12h, but no significant differences were seen among the toothpastes. ConclusionsF at low pH binds more efficiently to S. mutans than at neutral pH and dentifrice viscosity does not interfere in plaque fluid fluoride incorporation. Clinical significancepH of the dentifrice but not consistency may be important to F uptake in plaque.

Differential gene expression profiling of Actinobacillus pleuropneumoniae during induction of primary alveolar macrophage apoptosis in piglets

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the causative agent of porcine pleuropneumonia, a disease that causes serious problems for the swine industry. Successful infection by this bacterium requires breaking the first line of defence in the lungs, the primary alveolar macrophages (PAMs). Therefore, exploring A. pleuropneumoniae–PAM interactions will provide vital groundwork for the scientific control of this infectious disease, which has been little studied up to now. In this work, PAMs were isolated from piglets and co-incubated with A. pleuropneumoniae serovar 5b strain L20 in vitro, and their interaction, PAM cell death, and differential gene expression of A. pleuropneumoniae in response to PAM cell death were observed and analysed using confocal microscopy, electron microscopy, RT-PCR, Western blot, flow cytometry and the use of a gene expression profile chip. A. pleuropneumoniae quickly adhered to and invaded PAMs, inducing apoptosis, which was confirmed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The highest percentage of apoptosis in cells was confirmed using flow cytometry when the cells were infected at a multiplicity of infection (MOI) of 10 and incubated for 5 h, with higher expression of activated caspase-3 as measured by Western blot. Using microarray gene chips with 2868 probes containing nearly all of the genomic sequence of A. pleuropneumoniae serotype 5b strain L20, a total of 185 bacterial genes were found to be differentially expressed (including 92 up-regulated and 93 down-regulated genes) and involved in the process of apoptosis, as compared with the expression of control bacteria cultured without PAMs in BHI medium (mean expression ratios >1.5-fold, p < 0.05). The up-regulated genes are involved in energy metabolism, gene transcription and translation, virulence related gene such as LPS, Trimeric Autotransporter Adhesin, RTX and similar genes. The down-regulated genes are involved in amino acid, cofactor, and vitamin metabolism, and also include ABC transporters. These data demonstrate that A. pleuropneumoniae induces apoptosis of PAMs and undergoes complex changes in gene transcription, including expression changes in known and potential virulence factors. Some potentially novel virulence targets have been identified, suggesting new strategies for the development of vaccines and medicines for both preventive and clinical use.

Effect of a Previous Acid Adaptation of Zygosaccharomyces bailii on its Growth Kinetic in Acidic Media

The growth response of Zygosaccharomyces bailii acid adapted cells was assessed in acidified media. Yeast cells were first pre-cultured in nutrient broth adjusted with hydrochloric, citric and malic acid to pH 4; 4.5; 5; 5.5; 6 and 6.5. Moreover, they were also grown in two controls consisting of nutrient broth and nutrient broth supplemented with 1% of glucose both adjusted at pH 7. The variation of pH before and after the growth along with yeast concentration was measured. The cells pre-cultured in controls conditions and in the three conditions at pH 5 were then each inoculated in six BHI medium consisting of BHI adjusted with hydrochloric, citric and malic acid at pH 5.5 and 3.5. The growth was monitored by spectrophotometry and the yeast concentration after incubation was obtained by microscopy using a Thoma cell chamber. DMFit 2.1 was used to plot the growth curves and to estimate the growth parameters. All the pre-cultures and cultures were made at 37°C during 24 hours. During the pre-cultures, an important decrease of pH was noted in nutrient broth supplemented with glucose, moving from 7 to 3.81. In all the other pre-cultures, just a little variation was observed ranging from -0.57 to 0.50. Growth was observed in all the conditions, except at pH4. By growing the cells coming from the selected pre-cultures conditions in the different acidic BHI media, it appears that acid adaptation enhance the growth at pH 5.5 no matter the acid contains in the medium and the acid to which the cells were adapted. However, this acid adaptation was not sufficient to initiate growth at pH 3.5 after 24 hours of incubation at 37°C. Growth rate was significantly affected by the pH of the pre-culture medium and the acid present in the culture medium. Pre-culture with glucose supplementation was the only parameter studied affecting the latency.

A selective medium for the isolation of Corynebacterium species in oral cavities

Corynebacterium matruchotii is a microbial inhabitant in the oral cavity of humans and is associated with the formation of dental calculi. C. matruchotii forms highly specific morphological units, which are referred to as corn-cobs. Although other Corynebacterium species have frequently been isolated from the oral cavity of humans, their distribution has not been reported as extensively. The aim of the present study was to develop a selective medium to isolate the genus Corynebacterium and examine the distribution Corynebacterium species in the oral cavity of humans. The growth recoveries of representative Corynebacterium species on the selective medium were sufficient. Moreover, the growth of other representative oral bacteria was markedly inhibited on the selective medium. The proportion of Corynebacterium species in saliva samples collected from 20 subjects was examined. PCR primers were designed for the oral Corynebacterium species. C. matruchotii and Corynebacterium durum accounted for 0.3% and 1.5% of the total cultivable bacteria number on the BHI medium from saliva samples, respectively. The selective medium could distinguish C. matruchotii from C. durum by each colony color using differences in acid production from galactose. The selective medium, designated OCM, was useful for isolating oral Corynebacterium species.

Microbial biodiversity in cheese consortia and comparative Listeria growth on surfaces of uncooked pressed cheeses

The study set out to determine how changes in the microbial diversity of a complex antilisterial consortium from the surface of St-Nectaire cheese modify its antilisterial activities. On the basis of the microbial composition of a natural complex consortium named TR15 (Truefood consortium 15), three new consortia of different species and strain compositions were defined: TR15-SC (58 isolates from TR15 collection), TR15-M (pools of isolates from selective counting media) and TR15-BHI (pools of isolates from BHI medium). Their antilisterial activities on the surfaces of uncooked pressed cheese made with pasteurised milk were compared with the activity of complex consortium TR15 and a control cheese inoculated only with starter culture (Streptococcus thermophilus, Lactobacillus delbrueckii). The natural consortium TR15 was the most inhibitory, followed by reconstituted consortium TR15-BHI. The dynamics of the cheese rind microbial flora were monitored by counting on media and by isolate identification using 16S rDNA sequencing and direct 16S rDNA Single Strand Conformation Polymorphism analysis. The combination of these methods showed that rind with natural consortium TR15 had greater microbial diversity and different microbial dynamics than cheese rinds with reconstituted consortia. Cheese rind with the natural consortium showed higher citrate consumption and the highest concentrations of lactic and acetic acids, connected with high levels of lactic acid bacteria such as Carnobacterium maltaromaticum, Vagococcus fluvialis, Enterococcus gilvus, Leuconostoc mesenteroides, Brochothrix thermosphacta and Lactococcus lactis, ripening bacteria such as Arthrobacter nicotianae/arilaitensis, and Gram negative bacteria (Pseudomonas psychrophila and Enterobacter spp.). The highest L. monocytogenes count was on rind with TR15-M and was positively associated with the highest pH value, high succinic and citric acid contents, and the highest levels of Marinilactibacillus psychrotolerans and Gram positive catalase positive bacteria represented by Staphylococcus vitulinus, Brevibacterium linens, Microbacterium gubbeenense and Brachybacterium tyrofermentans.The results show that the species composition of consortium is more important than the number of species. It is likely that inhibition mechanisms differ from one consortium to another; investigating gene expression will be an effective way to elucidate microbial interactions in cheese.

Dental Care Clothing: An Investigation the Presence of Bacteria Contamination by Public Health Professionals in Southern Brazil

This study aimed to identify microorganisms present in contaminated dental lab coats and determine the risks of microbial contamination to professionals and patients. The study included 10 dentists of the city of Foz do Iguacu, state of Parana, Brazil, who signed an informed consent form according to the ethics committee on human research. Microbiological samples were collected from coats using the rolling swab technique, passing a single swab moist in BHI medium in the collar, cuffs and pockets. A questionnaire with open and closed questions was used. Statistical data were tabulated and described in a descriptive analysis through the bioestatic software 5.0. Regarding coat sanitization, 80% of respondents adopted this procedure every five days of use. Diagnosed microorganisms were: Klebsiella sp., Staphylococcus saprophyticus, Enterobacter sp., Staphylococcus epidermidis, Staphylococcus aureus. Staphylococcus aureus (50%) and Staphylococcus epidermidis (40%) were the most predominant bacteria found, especially in coats analyzed at the end of the workday, which may cause diseases such as: folliculitis, furuncle, endocarditis, meningitis, osteomyelitis, arthritis, urinary tract infections and others. Based on results, it was found that the lab coats of dentists are contaminated by microorganisms considered of clinical importance, contributing to a possible spread of diseasecausing microorganisms among dentists and patients.

Inhibitory effect of enterocin KP in combination with sublethal factors on Escherichia coli O157:H7 or Salmonella Typhimurium in BHI broth and UHT milk

The effects of physical and chemical sublethal treatments on the antibacterial activity of enterocin KP produced by Enterococcus faecalis KP against Escherichia coli O157:H7 and Salmonella Typhimurium were investigated. Enterocin KP was not active against intact cells of E. coli O157:H7 or S. Typhimurium. However, the use of enterocin KP together with ethylenediaminetetraacetic acid (50 mM), sodium tripolyphosphate (50 mM), sublethal heating (60 °C for 10 min), cold shock (-20 °C for 2 h), or acid stress (mixture of 40% lactic acid, 16% propionic acid, 16% acetic acid) in BHI medium decreased the cell number of E. coli O157:H7 by 7.27, 6.28, 3.39, 3.06, 4.20 log and S. Typhimurium by 7.21, 6.20, 3.64, 3.38, 3.98 log cfu/mL, respectively. The combination of enterocin KP with ethylenediaminetetraacetic acid decreased the cell number of E. coli O157:H7 in UHT milk to undetectable level, enterocin KP plus sodium tripolyphosphate or enterocin KP plus sublethal heating caused a reduction by 6.07 and 5.68 log cycles. The results of this study showed that enterocin KP could be applied as a biopreservative to inhibit E. coli O157:H7 and S. Typhimurium in combination with physical and food grade chemical hurdles.

Optimal specimen collection and transport methods for the detection of avian influenza virus and Newcastle disease virus

BackgroundActive and passive surveillance for avian influenza virus (AIV) and Newcastle disease virus (NDV) is widespread in commercial poultry worldwide, therefore optimization of sample collection and transport would be valuable to achieve the best sensitivity and specificity possible, and to develop the most accurate and efficient testing programs. A H7N2 low pathogenicity (LP) AIV strain was selected and used as an indicator virus because it is present in lower concentrations in swabbings and thus requires greater sensitivity for detection compared to highly pathogenic (HP) AIV. For similar reasons a mesogenic strain of NDV was selected. Using oro-pharyngeal and cloacal swabs collected from chickens experimentally exposed to the viruses we evaluated the effects of numerous aspects of sample collection and transport: 1) swab construction material (flocked nylon, non-flocked Dacron, or urethane foam), 2) transport media (brain heart infusion broth [BHI] or phosphate buffered saline [PBS]), 3) media volume (2 ml or 3.5 ml), 4) transporting the swab wet in the vial or removing the swab prior to transport, or transporting the swab dry with no media, and 5) single swabs versus pooling 5 or 11 swabs per vial.ResultsUsing real-time RT-PCR (rRT-PCR), virus isolation (VI) and commercial antigen detection immunoassays for AIV we observed statistically significant differences and consistent trends with some elements of sample collection and transport; media, dry transport and swab construction. Conversely, the number of swabs pooled (1, 5 or 11) and whether the swab was removed prior to transport did not impact virus detection. Similarly, with NDV detection by both VI and rRT-PCR was not affected by the numbers of swabs collected in a single vial (1, 5 or 11).ConclusionsWe observed that flocked and foam swabs were superior to non-flocked swabs, BHI media was better than PBS, and transporting swabs wet was better for virus recovery and detection than transporting them dry. There was no observable difference in detection whether the swab was removed prior to transport or left in the vial. Also, with both AIV and NDV, there was no observed difference in virus detection between pools of 1, 5 or 11 swabs.

Phenotypic Detection of Virulence Traits and Antibiotic Susceptibility of Endodontic &lt;i&gt;Enterococcus &lt;/i&gt;&lt;i&gt;f&lt;/i&gt;&lt;i&gt;aecalis&lt;/i&gt; Isolates

Enterococcus faecalis is a Gram-positive member of human gastrointestinal flora that is in recent years emerging as an important cause of endodontic infections. In this study, we have investigated the occurrence of virulence determinants and antibiotic susceptibility pattern of E. faecalis isolates, originating from root canals of apical periodontitis. Among 52 E. faecalis isolates, 32 (61.5%) isolates produced hemolysin on blood agar while all (100%) isolates showed hemolysin production on BHI-GA ((BHI medium supplemented with 1% glucose and 0.03% L-arginine), 18 (34.6%) produced gelatinase, 38 (73%) produced caseinase, no hemagglutination was observed in E. faecalis isolates, whereas all 52 (100%) resistant to serum and formed biofilm. Antibiotic susceptibility results showed that all (100%) E. faecalis isolates were susceptible to amoxicillin, amoxicillin/clavulanate, and vancomycin. Whereas, 32 (61.5%) E. faecalis isolates were resistant to chloramphenicol, 30 (57.6%) isolates were resistant to ciprofloxacin, 39 (75%) isolates were resistant to erythromycin, and 34 (65.3%) isolates were resistant to tetracycline. Multi-drug resistance was observed in 16 (30.7%) isolates of E. faecalis to chloramphenicol, ciprofloxacin, erythromycin and tetracycline antibiotics. These findings demonstrate the presence of putative virulence determinants in E. faecalis isolates originating from root canal and suggest amoxicillin, amoxicillin/clavulanate, and vancomycin as more effective than other antibiotics tested.

Antimicrobial activity of the synthetic peptide Lys-a1 against oral streptococci

The peptide LYS-[TRP6]-Hy-A1 (Lys-a1) is a synthetic derivative of the peptide Hy-A1, initially isolated from the frog species Hypsiboas albopunctatus. According to previous research, it is a molecule with broad antimicrobial activity. The objective of this study was to evaluate the antimicrobial activity of the synthetic peptide Lys-a1 (KIFGAIWPLALGALKNLIK-NH2) on the planktonic and biofilm growth of oral bacteria. The methods used to evaluate antimicrobial activity include the following: determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in microtiter plates for growth in suspension and quantification of biomass by crystal violet staining and counting of colony forming units for biofilm growth. The microorganisms Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis, Streptococcus salivarius, Streptococcus mutans and Streptococcus sobrinus were grown in Brain Heart Infusion broth at 37°C under atmospheric pressure with 10% CO2. The peptide was solubilized in 0.1% acetic acid (v/v) at various concentrations (500–1.9μgmL−1). Chlorhexidine gluconate 0.12% was used as the positive control, and BHI culture medium was used as the negative control. The tested peptide demonstrated a remarkable antimicrobial effect, inhibiting the planktonic and biofilm growth of all strains tested, even at low concentrations. Thus, the peptide Lys-a1 is an important source for potential antimicrobial agents, especially for the control and prevention of microbial biofilms, which is one of the most important factors in cariogenic processes.

Use of Rich BHI Medium Instead of Synthetic TMH Medium for Gene Regulation Study in Yersinia pestis

ObjectiveThis study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis. MethodsThe transcriptional regulation of rovA by PhoP or via temperature upshift, and that of pla by CRP were investigated when Y. pestis was cultured in BHI. After cultivation under 26 °C, and with temperature shifting from 26 to 37 °C, the wild-type (WT) strain or its phoP or crp null mutant (ΔphoP or Δcrp, respectively) was subject to RNA isolation, and then the promoter activity of rovA or pla in the above strains was detected by the primer extension assay. The rovA promoter-proximal region was cloned into the pRW50 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and ΔphoP to measure the promoter activity of rovA in these two strains with the β-Galactosidase enzyme assay system. ResultsWhen Y. pestis was cultured in BHI, the transcription of rovA was inhibited by PhoP and upon temperature upshift while that of pla was stimulated by CRP. ConclusionThe rich BHI medium without the need for modification to be introduced into the relevant stimulating conditions (which are essential to triggering relevant gene regulatory cascades), can be used in lieu of synthetic TMH media to cultivate Y. pestis for gene regulation studies.

Pollution Impacts on Bacterioplankton Diversity in a Tropical Urban Coastal Lagoon System

Despite a great number of published studies addressing estuarine, freshwater and marine bacterial diversity, few have examined urban coastal lagoons in tropical habitats. There is an increasing interest in monitoring opportunistic pathogens as well as indigenous microbial community members in these water bodies by current molecular and microbiological approaches. In this work, bacterial isolates were obtained through selective plate dilution methods to evaluate antibiotic resistances. In addition, 16S rRNA gene libraries were prepared from environmental waters and mixed cultures grown in BHI medium inoculated with Jacarepaguá lagoon waters. Denaturing gradient gel electrophoresis (DGGE) analyses showed distinct community profiles between environmental communities from each studied site and their cultured counterparts. A total of 497 bacterial sequences were analyzed by MOTHUR, yielding 245 operational taxonomic units (OTUs) grouped at 97% similarity. CCA diagrams showcased how several environmental variables affect the distribution of 18 bacterial orders throughout the three distinct habitats. UniFrac metrics and Venn diagrams revealed that bacterial communities retrieved through each experimental approach were significantly different and that only one OTU, closely related to Vibrio cholerae, was shared between them. Potentially pathogenic bacteria were isolated from most sampled environments, fifty percent of which showed antibiotic resistance.

Ex vivo antimicrobial efficacy of the EndoVac® system plus photodynamic therapy associated with calcium hydroxide against intracanal Enterococcus faecalis

To evaluate the ex vivo efficacy of the EndoVac system and photodynamic treatment (PDT) as adjuncts to chemomechanical debridement associated with calcium hydroxide (CaOH2 ) in reducing the levels of intracanal Enterococcus faecalis. One hundred and twenty-five sterile premolar teeth were conventionally accessed, prepared and then contaminated with E. faecalis (ATCC 29212) for 30 days. Teeth were randomly divided into 4 groups: Control (chemomechanical debridement with conventional irrigation); Endovac (chemomechanical debridement with EndoVac system); PDT (chemomechanical debridement with conventional irrigation and PDT) and Endovac+PDT (chemomechanical debridement with EndoVac and PDT). The irrigants used in all groups were 5.25% sodium hypochlorite and 17% EDTA. After treatment, an intracanal dressing (CaOH2 ) was applied in all canals for 7 days. Samples were obtained before (T1) and after the therapeutic procedures (T2) and, after intracanal medication (T3), plated onto BHI media and incubated (37 °C, 48 h) to determine the colony-forming units (CFU mL(-1) ). The overall mean cell counts (CFU mL(-1) ) of E. faecalis were high at the initial contamination (T1). A significant reduction (P < 0.05) of E. faecalis mean counts was observed in all groups from baseline (T1) to both post-therapy samplings (T2 and T3); no differences amongst the groups were detected. No significant change in bacterial counts from T2 to T3 was detected. The adjunctive use of the EndoVac system and the photodynamic treatment, in combination or not, was as effective as the conventional chemomechanical debridement associated with CaOH2 on reducing the counts of intracanal E. faecalis.

Toksisitas Produk Ekstrasellular (ECP) Streptococcus agalactiae pada Ikan Nila (Oreochromis niloticus)

This research aimed to know the toxicity of extracellular products (ECP) of Streptococcus agalactiae was tastedin cultured Nile tilapia (Oreochromis niloticus). Streptococcus agalactiae had two haemolytic types: β-haemolyticand non-haemolytic type. Toxicity test of ECP to know the virulancy factor of S. agalactiae was still limited. It wasfound that after tested on 15 fish weighing 15 g through intraperitoneal injection 0,1 ml/fish, both bacteria causedchanges in swimming pattern, palatability, external and internal anatomy macroscopically and microscopically.Extracellular products of S. agalactiae non-haemolytic type (BHIA and BHI 24 h) and β-haemolytic type (BHI 72 h)caused mortality 12 hours after injection and the mortality continued till day 7 th of culture. Whirling happened 96hours after injection with ECP S. agalactiae β-haemolytic type (BHIA 72 h incubation) whereas injection with ECP(BHI 24 h) on 72 h after injection and continued untill day 7 th. Behavior disease signs caused by S. agalactiaeoccured on eyes. There were opacity, purulens, eye shrink, lateral and bilateral exopthalmia and haemorrhage oninfected-fish. Silver staining of sodium dodecyl sulphate-polyacrylamide gels to S. agalactiae revealed thatpredominant 51.8-69.6 kDa bands were present in BHIA ECP fraction. The 69.6 kDa was absent from the BHI ECP.Total protein on non-haemolytic S. agalactiae ECP are 28.18 ppm on BHIA medium and 13.64 ppm on BHI medium.Whereas β-haemolytic S. agalactiae ECP are 2.73 ppm on BHIA medium and 8.18 ppm on BHI medium. Concentrationof protein in ECP was one of factor that caused non-haemolytic S. agalactiae more virulent than β-haemolytic type.The conclusion from the research that ECP was virulent factor on β-haemolytic and non-haemolytic S. agalactiaein fish which caused changes in behavior disease signs.

An assessment of bactericidal effect of two different types of lasers on enterococcus faecalis: An in vitro study

Aim and Objective: To evaluate the bactericidal efficacy of the Nd:YAG laser and the Diode laser at different parameters to that of calcium hydroxide, and to know the effect of increase in lasing time on degree of disinfection. Materials and Methods: Fifty human single-rooted teeth were taken and were kept in 3% NaOCl for 15 mins and access cavities were prepared, the apical third of these roots was gradually enlarged until reaching the ISO 40- K file. Later the teeth were autoclaved and mounted on 2.5 ml eppendorf tubes filled with BHI medium which was autoclaved again. The teeth were inoculated with a standard strain of Enterococcus faecalis and incubated for one week. They were randomly divided into five groups and treated as follows, G-I: - Negative control. G-II:- 980 nm diode laser irradiation for five seconds and five cycles at 2W power, 10 Hz frequency and pulsed mode. G-III:- 980 nm diode laser irradiation for 10 seconds and five cycles at 2W power, 10 Hz frequency and pulsed mode. G-IV:- Nd:YAG laser irradiation for five seconds and five cycles at 2 W power, 15 Hz frequency and pulsed mode. G-V:- Nd:YAG laser irradiation for 10 seconds and five cycles at 2 W power, 15 Hz frequency and pulsed mode. The dentin dust was collected from superficial and deep dentin of coronal and apical halves of all the samples and CFU/ ml were evaluated after three days. Results: The results were calculated and statistical analysis was done using Kruskal Wallis ANOVA test by ranks and Mann Whitney's U test. Comparisons were done between the superficial and deep dentin of different groups and between the groups. Conclusion: Under the parameters of the samples tested Nd:YAG laser provided the best results for disinfection compared to Diode laser. However, both the Nd:YAG and Diode lasers provided clinically acceptable disinfection.

Construction ofEdwardsiella tardaghosts from eel and their optimization of preparation conditions

Edwardsiellosis caused by Edwardsiella tarda is one of the most serious diseases in the eel culture industry.Bacterial ghosts,which have native surface antigenic structures and can induce humoral and cellular immune response,have become candidate of vaccine development.To explore the feasibility of E.tarda ghost vaccine in eel,E.tarda ghosts were generated by the thermal controlled expression of the PhiX174 lysis gene E in present study.At the same time the morphology,lysis kinetics,lysis efficiency and preparation conditions were also studied.Holes were observed in Escherichia coli and E.tarda ghosts by scanning electron microscopy and the bacteria become withered due to the loss of bacterial content.The lysis kinetics in E.tarda were also compared with those in E.coli.Generation of ghosts in the transformants of E.coli and E.tarda carrying plasmid pBVlysisE was performed successfully by increasing the incubation temperature up to 42℃.Compared to E.coli,in which lysis was observed within 30 min and was completed 3 h after induction of E gene expression,onset of E.tarda lysis occurred 1 h after temperature elevation and the lysis process was completed 5 h after induction.At the end of the lysis process,the efficiency of ghost induction in non-lyophilized E.tarda was 99.99%,as the results of 5 replicate experiments showed.However,no bacterial growth was detected in lyophilized E.tarda and there is no obvious difference in bacterial morphology before and after lyophilization.The lysis kinetics and lysis efficiency had no difference between beginning of induction at OD600=0.4 and 0.6.Among LB,BHI and NB medium,LB medium is the best for preparation of E.tarda because in this medium the ghosts are more integrated and lysed completely.In the study we successfully constructed E.tarda ghosts and optimized their preparation conditions,which will provide the foundation for development of vaccine against edwardsiellosis in eel.

Hyicin 3682, a bioactive peptide produced by Staphylococcus hyicus 3682 with potential applications for food preservation

Bacteriocins are peptides produced by bacteria and having inhibitory activity against other bacteria. Many of these substances may be useful as antibacterial agents for practical applications. In this study, 21 Staphylococcus spp. isolated from pigs, dogs and bovine milk in different states of Brazil were investigated for staphylococcin production. Hyicin 3682, a bacteriocin produced by one such strain, inhibited almost all strains tested, including Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. PCR experiments showed that hyicin 3682 is lantibiotic-related, but not identical, to both epidermin and Bsa. The maximum production of hyicin 3682 (6,400 AU/ml) was observed after 24 h of growth in BHI medium at 37 °C. Hyicin 3682 proved to be a cationic, small antimicrobial peptide with a molecular mass of 2,139 Da. It exhibited resistance to low pH and to heating at 65 °C, and partial sensitivity to proteolytic enzymes. Taken together, these results suggest that hyicin 3682, the first bacteriocin characterized in Staphylococcus hyicus, has potential biotechnological applications as a food preservative. Moreover, hyicin 3682 was able to inhibit its producer strain, suggesting that an effective immune system for specific protection against hyicin 3682 is not found in its producer strain, a characteristic not described thus far for other staphylococcins.

Structure and Mechanism of Iron Translocation by a Dps Protein from Microbacterium arborescens

Dps (DNA protection during starvation) enzymes are a major class of dodecameric proteins that bacteria use to detoxify their cytosol through the uptake of reactive iron species. In the stationary growth phase of bacteria, Dps enzymes are primarily used to protect DNA by biocrystallization. To characterize the wild type Dps protein from Microbacterium arborescens that displays additional catalytic functions (amide hydrolysis and synthesis), we determined the crystal structure to a resolution of 2.05 Å at low iron content. The structure shows a single iron at the ferroxidase center coordinated by an oxo atom, one water molecule, and three ligating residues. An iron-enriched protein structure was obtained at 2 Å and shows the stepwise uptake of two hexahydrated iron atoms moving along channels at the 3-fold axis before a restriction site inside the channels requires removal of the hydration sphere. Supporting biochemical data provide insight into the regulation of this acylamino acid hydrolase. Moreover, the peroxidase activity of the protein was determined. The influence of iron and siderophores on the expression of acylamino acid hydrolase was monitored during several stages of cell growth. Altogether our data provide an interesting view of an unusual Dps-like enzyme evolutionarily located apart from the large Dps sequence clusters.