Tryptic Soy Broth Research Articles

Evaluation of nitrogen fixers for drought tolerance and growth promotion in Tomato

Drought stress is one of the most fatal environmental phenomena that affect plant growth and productivity worldwide. In the present study, an attempt was made to screen and characterize nitrogen fixing bacteria for drought tolerance by inoculation in trypticase soy broth with different concentrations of PEG 6000. The strains were further evaluated for induction of drought tolerance and growth promotion in tomato. It was found that inoculation improved the plant physiological parameters of tomato seedlings. Furthermore, plants inoculated with nitrogen fixers viz., Microbacterium, Cellulosimicrobium and Paenibacillus showed better growth in terms of plant height, number of leaves, stem girth, root length, fresh and dry weight of plants and yield compared to uninoculated control plants under normal and reduced irrigation. The study demonstrates nitrogen fixers with plant growth promoting traits and drought tolerance can be a useful approach for the development of bioinoculants for drought stress management in crops.

Antibacterial efficacy of bioceramic root canal sealers against planktonic Enterococcus faecalis after different contact and setting time: An in vitro study

Introduction: Bioceramic root canal sealers are the newest generation of root canal sealers. Τhere are contradictory results in the literature about their antimicrobial activity. The aim of the study was to evaluate the antimicrobial efficacy of four root canal sealers against Enterococcus faecalis.Materials and Methods: Four root canal sealers were used in order to examine antibacterial efficacy: TotalFill, BioRoot Root Canal Sealer, mineral trioxide aggregate (MTA) Fillapex, and AH Plus. The bacterial suspension was placed on a freshly mixed sealer, one or 3 days set sealer in vertically held microtiter plates. After incubation of 2, 5, 20, and 60 min in 100% humidity at 37 C, Trypticase Soy Broth was added to each well and mixed. Then, bacterial suspension from each well was transferred, serially diluted, and placed on Mitis salivarius agar plates. After incubation, colony-forming units were counted. All experiments were performed in triplicate. The outcomes of antimicrobial properties of tested materials were analyzed by one-way analysis of variance.Results: All bioceramic root canal sealers showed significantly better efficacy than the control group and epoxy resin sealer (P < 0.05). TotalFill presented the highest efficacy comparable with BioRoot RCS (P > 0.05), followed with MTA Fillapex (P < 0.05). Also, freshly mixed sealers showed comparable efficacy as sealers set for 3 days (P > 0.05), but better than sealers set for 1 day (P < 0.05). All sealers exhibited the highest efficacy after 20 min of contact time, independently of materials that were freshly mixed or set for 1 or 3 days.Conclusion: Bioceramic sealers have greater antimicrobial activity than commercially used epoxy resin sealer. These sealers exhibited the strongest efficacy at 20 min of contact time, independently of their setting condition.

Influence of Free Chlorine and Contact Time on the Reduction of Salmonella Cross-Contamination of Tomatoes in a Model Flume System

The process of washing tomatoes in dump (flume) tanks has been identified as a potential source of cross-contamination. This study's objective was to assess the potential for Salmonella enterica cross-contamination at various inoculation levels in the presence of free chlorine (HOCl) and organic matter. Uninoculated tomatoes were introduced into a laboratory-based model flume containing tomatoes inoculated with a cocktail of five rifampin-resistant S. enterica serovars at 104, 106, or 108 CFU per tomato in water containing 0 or 25 mg/L HOCl and 0 or 300 mg/L chemical oxygen demand (COD). Uninoculated tomatoes exposed to the inoculated tomatoes were removed from the water after 5, 30, 60, and 120 s and placed in bags containing tryptic soy broth supplemented with rifampin and 0.1% sodium thiosulfate. Following incubation, enrichment cultures were plated on tryptic soy agar supplemented with rifampin and xylose lysine deoxycholate agar to determine the presence of Salmonella. HOCl and pH were measured before and after each trial. The HOCl in water containing 300 mg/L COD significantly declined (P ≤ 0.05) by the end of each 120-s trial, most likely due to the increased demand for the oxidant. Higher inoculum levels and lower HOCl concentrations were significant factors (P ≤ 0.05) that contributed to increased cross-contamination. At 25 mg/L HOCl, no Salmonella was recovered under all conditions from uninoculated tomatoes exposed to tomatoes inoculated at 104 CFU per tomato. When the inoculum was increased to 106 and 108 CFU per tomato, cross-contamination was observed, independent of COD levels. The results from this study indicate that the currently required sanitizer concentration (e.g., 100 or 150 mg/L) for flume water may be higher than necessary and warrants reevaluation.

Physical and Chemical Fermentation Conditions Affect the Growth and Metabolite Production of Endophytic Fungi Athelia rolfsii

Background: The effort to explore antimicrobial agents isolated from an endophytic fungus of Coleus amboinicus resulted in the finding of a potential aromatic compound having methoxy, hydroxyl, and methyl groups produced by Athelia rolfsii. This compound exhibited antibacterial activities with IC50 values of &lt;2 μg/mL against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Streptococcus mutans, and Pseudomonas aeruginosa. This study was aimed to determine the effect of varying fermentation conditions (types of media, carbon sources, nitrogen sources, temperature, and pH) on biomass, total metabolites, and bioactive compound production. Materials and Methods: The fungal cultures were subjected to various treatment conditions and incubated for 12 days at 25°C 160 rpm followed by analyzing the biomass, metabolite, and bioactive compound production. Results and Conclusion: The study found that although the maximum total metabolite production was achieved in the Tryptic Soy Broth medium, both biomass and bioactive compound production accumulated at the highest amount in Potato Dextrose Broth and Sabouraud Dextrose Broth media. Adding 1% of different types of carbon sources did not significantly enhance biomass, total metabolite, and bioactive compound production. Three types of organic nitrogen sources used in this study did not significantly affect biomass and total metabolite production, but adding peptone produced the highest amount of bioactive compound. Supplementing inorganic source of nitrogen to the culture medium decreased the production. While pH 5.5 was found to be the optimum condition for total metabolite production, pH 6–7 resulted in higher productivity of the bioactive compound. The total metabolite production was best produced at 25°C, whereas higher temperatures were needed to get optimum bioactive and biomass production. This study found that the total metabolite production was 7.8 times higher when the culture was grown in PDB medium supplemented with 1% sucrose and 1% peptone and incubated at 27°C at pH 6.5; whereas a 15% increase was observed in the bioactive compound production.

Effect of High Hydrostatic Pressure Treatment Combined with Alkaline Electrolyzed Water on the Injury and Growth Characteristics of Bacterial Spores.

The characteristics of growth and injury of Bacillus subtilis spores whose heat resistance was reduced by the treatment of high hydrostatic pressure processing (HPP) combined with/without alkaline electrolyzed water (AlEW) were investigated. The delay in the lag phase of growth curve observed when used in combination with AlEW, especially at 50 MPa or less, and was prolonged by about 4 to 6 hours. However, the effects of temperature and time during treatment on the lag phase were not significantly different between solo-HPP and in combination with AlEW. The injury characteristics of the treated bacterial spores were evaluated by using antibiotics (penicillin G, rifampicin and chloramphenicol) supplemented trypticase soy broth. In the chloramphenicol supplemented TSB, although the lag phase of bacterial spores treated by HPP with AlEW was not prolonged as compared with the normal-TSB, the decrease in growth rate during logarithmic phase and increase in maximum growth amount were observed. This result could suggest due to a damage by combined treatment of HPP with AlEW such as the inhibition of protein synthesis. Furthermore, the combined treatment with AlEW increased the catalase activity by 1.45, 1.63 and 4.25 times at 30, 80 and 100 MPa, respectively, as compared with solo-HPP, therefore the combined treatment could cause high oxidative stress on bacterial spores.

Identification and Antimicrobial Activity of Actinobacteria Isolated from Rhizosphere of the Local Endemic Plants

The search for novel antimicrobial compounds from the environment has been gaining momentum with the increase in resistance of pathogens. In the present study, the antimicrobial potential of 11 local isolates of Actinobacteria which were isolated from rhizosphere soils of the three local endemic plants in Turkey was evaluated against different pathogenic bacteria and Candida albicans. Antimicrobial activity was investigated by the disk diffusion agar method using the organic extracts obtained from the isolates grown in the modified Bennett's medium and Tryptic Soy Broth (TSB). Eight isolates were revealed to show remarkable antimicrobial activity against to pathogens. Study of the production of bioactive metabolites in two different culture media indicated that the higher antimicrobial activity was observed in modified Bennett's medium when compared to TSB. Also, partial 16S rDNA sequence analyses revealed that 10 of these local isolates belong to member of Streptomyces and one of them to member Promicromonospora genera.

Sterility Test of Syringes As A Pharmaceutical Preparation That Obtained From Pasar Pramuka

Syringes are one of the pharmaceutical preparations that are in high demand. In healthcare institutions, syringes are used to aid in patient care and examination. Pharmaceutical preparations, such as various syringes, are widely available in the Pasar Pramuka. Syringes must be free from microbes and used syringes should not be reused. Microbiological sterility tests can be performed on a syringe to determine whether it is sterile or not. The purpose of the study is to test the sterility of syringes obtained from the Pasar Pramuka. A random sample of a syringe is being used in the study as an experimental method. The syringes were isolated and incubated for 14 days in Fluid Thioglycollate Medium (FTM) and Trypticase Soy Broth (TSB) at 30-35 oC and 20-25 oC, respectively, with frequent observations. If FTM and TSB media were turbid, then isolated into selective media based on their microbe as controls, namely Clostridium sporogenes ATCC 19404, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, Escherichia coli ATCC 8739, Bacillus subtilis ATCC 6633, Salmonella typhi. The results showed that the B syringe was turbid on FTM media and did not contain pathogenic microbes after being identified on selective media, as in controls. The A and C syringes on TSB media were turbid, and after identification on selective media, Candida albicans and Aspergillus brasiliensis were found. In conclusion, the A, B, and C syringes are not steril.

Laktobasillerden elde edilen hücresiz süzüntülerin metisiline dirençli Staphylococcus aureus, vankomisine dirençli Enterococcus ve karbapeneme dirençli Klebsiella suşlarını inhibisyonu

Aim: Antibiotic resistance is a major health problem. Recently, probiotics are used in the field of alternative/supportive medicine. Thus, in this study, we aimed to evaluate the in vitro inhibitory effects of four different cell-free supernatants (CFSs) of lactobacilli species (L. fermentum, L. plantarum, L. acidophilus and L. rhamnosus) on clinically isolated Methicilin-resistant Staphylococcus aureus (MRSA) strains, Vancomycin-resistant Enterococcus (VRE) strains, and Carbapenem-resistant Klebsiella (CRK) strains. Materials and Methods: Lactobacillus strains were grown in de Man Rogosa Sharpe broth; after filtration, CFSs were diluted to obtain 25, 50, and 100% concentrations. Pathogen bacteria were grown in tryptic soy broth with and without CFSs in a micro-plate. The bacterial growths were measured using spectrophotometric method after four hours of incubation at 37°C. One-way ANOVA followed by Dunnett’s multiple comparisons and Kruskal-Wallis test were used for statistical analyses. Results: All tested CFSs at all concentrations were found to inhibit growth of MRSA, VRE, CRK strains; the results were found statistically significant (p&amp;lt;0.0001). At 50% concentrations, all CFSs were found to be most effective on MRSA growth. The CFSs of L.fermentum, L.acidophilus and L.plantarum were found to be most inhibitory at 50% concentration on VRE growth. 50% diluted CFSs of L.fermentum and L.plantarum were found to be effective on growth of CRK. All results were found statistically significant (p&amp;lt;0.0001). Conclusion: In our study, our results support that CFSs of lactobacilli strains inhibit growth of multi-drug resistant bacteria. Their inhibitory effects were dependent on microorganisms and CFS concentrations.

Establishing in vitro and in vivo Co-culture Models of Staphylococcus epidermidis and Enterococcus faecalis to Evaluate the Effect of Topical Fluoroquinolone on Ocular Microbes.

Purpose: To establish in vitro and in vivo ocular co-culture models of Staphylococcus epidermidis and Enterococcus faecalis and to study how various concentrations of moxifloxacin affect the survival of these two endophthalmitis-causing bacteria.Methods: Standard strains of S. epidermidis and E. faecalis were used. Color detection agar plates were employed to distinguish their colonies. To establish the in vitro and in vivo co-culture models, S. epidermidis and E. faecalis were co-cultivated at different ratios for various periods. For the in vivo model, various volumes and concentrations of either a mono-culture or co-culture were inoculated into the lower conjunctival sac of rabbits. Finally, the newly developed in vitro and in vivo co-culture models were subjected to the moxifloxacin treatment to access its effect on S. epidermidis and E. faecalis.Results: When S. epidermidis and E. faecalis were cultured separately in tryptic soy broth, their growth peaked and plateaued at approximately 16 and 6 h, respectively. When they were co-cultured, the growth peak of S. epidermidis got delayed, whereas the growth peak of E. faecalis did not change. The number of E. faecalis was significantly higher in the co-culture than that in the mono-culture. Treatment with moxifloxacin in the in vitro co-culture model rapidly decreased the number of S. epidermidis cells at doses ≥ 0.125 μg/ml. In contrast, the number of E. faecalis did not change significantly up to 16 μg/ml moxifloxacin. In in vivo co-culture (at 1:1), the S. epidermidis count decreased in a pattern similar to that seen in in vivo mono-culture and was barely detectable at 24 h after inoculation. In contrast, the of E. faecalis count increased up to 16 h and then decreased. When moxifloxacin was applied (zero, one, or two times) to this model, the S. epidermidis count decreased in proportion to the number of treatments. In contrast, the E. faecalis count increased with moxifloxacin treatment.Conclusions: The in vitro and in vivo co-culture models of S. epidermidis and E. faecalis were established to determine the influence of moxifloxacin eye drops on these bacteria. The results clearly show that the moxifloxacin eye drops can make E. faecalis dominant on the ocular surface.

Fast Identification and Quantification of Uropathogenic E. coli through Cluster Analysis.

Rapid diagnostic tools to detect, identify, and enumerate bacteria are key to maintaining effective antibiotic stewardship and avoiding the unnecessary prescription of broad-spectrum agents. In this study, a 15 min agglutination assay is developed that relies on the use of mannose-functionalized polymeric microspheres in combination with cluster analysis. This allows for the identification and enumeration of laboratory (BW25113), clinical isolate (NCTC 12241), and uropathogenic Escherichia coli strains (NCTC 9001, NCTC 13958, J96, and CFT073) at clinically relevant concentrations in tryptic soy broth (103-108 CFU/mL) and in urine (105-108 CFU/mL). This fast, simple, and efficient assay offers a step forward toward efficient point-of-care diagnostics for common urinary tract infections.

Inhibitory activity of aqueous extracts of pomegranate peel products and juice powder against Salmonella enterica

Pomegranate peel contains high amounts of polyphenols that could potentially be used as natural preservatives. This study evaluated the inhibitory effect of aqueous extracts of three commercial powdered peels, a whole peel, and a juice powder, against strains of Salmonella Tennessee and S. Enteritidis. Cells of the two Salmonella strains (4.88–4.96 log CFU/ml) were treated in tryptic soy broth containing 9 or 23% of the extracts prepared by two different methods for 5, 10, or 24 h at 25 °C, and reduction in Salmonella population compared to the untreated control was determined. The extracts from the three powdered peels were significantly more effective (P ≤ 0.05) in reducing the Salmonella population (1.67–2.03 Log CFU/ml) than the extracts from the whole peel (1.05 Log CFU/ml) and juice powder (0.94 Log CFU/ml). The higher dose of the extracts resulted in a greater reduction in the Salmonella population (3.02 Log CFU/ml) than the lower dose (1.47 Log CFU/ml). The level of Salmonella population reduction correlated positively with the total polyphenolic content (R2 0.66–0.99) and titratable acidity (R2 0.72–0.98) in the treatment system. The study suggests that pomegranate peels have the potential to be used as preservatives in foods.

1276. Assessment of Anti-biofilm Activity of Staphylococcus aureus Bacteriophages Against Clinical Isolates from Patients with Left Ventricular Assist Device Infections

Abstract Background Staphylococcus aureus is a common cause of biofilm-mediated left ventricular device (LVAD) infections which are difficult to resolve with antibiotics alone and are associated with substantial morbidity and mortality. Recently, bacteriophages (phage) therapy has been used to resolve LVAD infections in a few cases. Our goal was to assess in vitro susceptibility and anti-biofilm activity of two S. aureus bacteriophages against clinical isolates from patients with S. aureus LVAD infections in order to develop a S. aureus phage cocktail for clinical use. Methods Two bacteriophages, OMS1 and OMS2, from the Israeli Phage Bank, were assessed for lytic activity against 15 S.aureus isolates (9 methicillin resistant, MRSA and 6 methicillin susceptible, MSSA) via agar overlay method and plaque forming units (PFU)/mL were enumerated. We then formed bacterial biofilms after overnight incubation at 120 rpm in a 96-well plates in duplicate; experiments were repeated thrice. Wells were then treated with tryptic soy broth (TSB, control) or TSB containing phage at 109 PFU/mL for 24 hours. After washing, biofilms were stained with crystal violet and biomass quantified via optical density at 570mm. Results All bacterial isolates were susceptible to both phages via agar overlay to varying degrees as determined by phage titers obtained via serial dilutions (Figure 1a, 1b). OMS1 led to significant reduction in biofilm of 7/9 MRSA and 3/6 MSSA isolates and OMS2 reduced biofilms in 9/9 MRSA and 4/6 MSSA isolates (Figure 1c). Phage titers and Biofilm Biomass Figure 1a) Phage titers in PFU/ml obtained from each bacterial isolate via agar overlay method. 1b) Representative agar plate demonstrating lytic plaques from OMS1 and OMS2 on a Staphylococcus aureus LVAD isolate. 1c) Biofilm biomass of Staphylococcus aureus isolates alone (SA) or with individual phage (SA+OMSA1 and SA=OMS2) assessed by optical density readings at 570 nm; error bars represent standard error of the mean. Conclusion We demonstrated in vitro lytic and anti-biofilm activity of 2 S. aureus phages against clinical S. aureus isolates from patients with LVAD infection. Our data suggests that phage susceptibility measured with agar overlay does not always correlate with phage susceptibility of S. aureus biofilms, suggesting that more than one method should be used to assess in vitro activity. We plan to assess for synergistic activity with the phage combination. Disclosures Ryan K. Shields, PharmD, MS, Shionogi (Consultant, Research Grant or Support) Ran Nir-Paz, MD, BiomX (Consultant)Technophage (Scientific Research Study Investigator, Advisor or Review Panel member) Saima Aslam, MD, MS, BioMx (Consultant)Cystic Fibrosis Foundation (Grant/Research Support)Gilead (Consultant)Johnson and Johnson (Consultant)Merck (Consultant)

Divalent metal release and antimicrobial effects of layered double hydroxides

Resistance to antimicrobial agents is responsible for major social and economic losses. The World Health Organization estimated 700,000 global deaths a year due to antimicrobial resistance. The use of layered double hydroxides (LDHs) as antibacterial materials could present a way to reduce the risk of bacterial infections and antibacterial resistance, by partial release of metallic ions in aqueous dispersion. The partial dissolution of different synthetic LDHs MII-AlIII (M = Zn, Cu, Ni, Co, Mg) was studied in Lysogeny Broth (LB) and Tryptic Soy Broth (TSB), growth media of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacteria, respectively. The influence of several parameters (crystallinity, MII:AlIII ratio, type of intercalated anion {CO32−, Cl−, NO3−, ClO4−} and nature of MII cations) was investigated. In the absence of any post-synthetic hydrothermal treatment, ZnII-AlIII LDH showed a release of ZnII ions 6 times enhanced. Upon increasing ZnII:AlIII molar ratio to 3: 1 and exchanging carbonate anions with other anions having lower intercalating affinities, a similar effect was observed. The dissolution properties of MII-AlIII LDHs were correlated with the thermodynamic stability of their MII(OH)2 hydroxide counterparts with the exception of CuII-based LDH, which showed an amplified release of CuII due to its irregular structure presenting defects. Finally, the antibacterial activity was only noted for ZnII and CuII-based LDHs. The antimicrobial effect of the studied LDHs was linked in the first place to the nature of divalent metal itself, and to the amount of released MII ions into the culture media in the second place. This effect was more easily identified in ZnII-AlIII LDHs whose minimum inhibitory concentration was decreased significantly from 12 to 0.375 mg.mL−1 when higher amounts of ZnII ions were released.

1249. Metabolomic Profile of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA) in Latin-American MRSA Isolates

BackgroundVancomycin (VAN) is a first-line therapeutic option in severe infections caused by MRSA in Latin-America (LA). Development of reduced susceptibility to VAN has been associated with multiple changes in genes encoding pathways for cell wall metabolism and envelope stress responses. Nevertheless, a detailed and coherent mechanistic model to explain the phenotype remains elusive. To gain further insights into the hVISA phenotype, we sought to explore the metabolomic profile of hVISA isolates from LA. MethodsThe undirected profile of intracellular S. aureus metabolites was analysed in four clinical isolates (two hVISA and two VSSA [Vancomycin susceptible S. aureus]) belonging to the Chilean/Cordobes clone-ST5 (the predominant hVISA lineage in LA), and two reference strains Mu3 and N315. The metabolites were obtained in mid-exponential growth phase in trypticase soy broth in five independent replicates. The metabolites were determined by reverse phase liquid chromatography and hydrophilic interaction. The metabolic profile was determined by variable importance in the projection score (VIP > 1). The differences between hVISA and VSSA were maximized by orthogonal partial least squares discriminant analysis (OPLS-DA) and the affected metabolic pathways were identified with MetaboAnalyst.ResultsAmong the differences identified in the metabolic profiles of hVISA respect to VSSA, 69 metabolites were relevant. Of these, 47 were fatty acids (including glycerol), 7 amino acids and 6 nucleosides (Table 1). These changes mainly impact the biosynthesis of amino acids derived from pyruvate since tyrosine, valine and leucine, had a reduction of 34%, 57% and 41% in hVISA compared to VSSA, respectively, which suggests alterations of the acid cycle tricarboxylic (TCA). Additionally, a reduction in purine and pyrimidine metabolism in hVISA was identified with reduction of nucleosides and dinucleotides derived from the pentose phosphate pathway.Table 1. Metabolites with higher VIP scores in the comparison of the hVISA and VSSA profile ConclusionWe were able to observe metabolic alterations in TCA, pentose phosphate pathway and purine intermediates in hVISA-ST5 isolates. Our results support that gluconeogenesis and biosynthesis of carbohydrates and nucleic acids are the main pathways involved in the reduced susceptibility to VAN as reported in VISA isolates.Disclosures Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support) Lorena Diaz, PhD , Nothing to disclose

Antibacterial Activities of Methanol and Aqueous Extracts of Salvadora persica against Streptococcus mutans Biofilms: An In Vitro Study.

The use of herbal products in oral hygiene care has a long history, and their use is popular today. A tree stick, named Salvadora persica (S. persica), is commonly used to remove dental plaque and clean teeth in many countries. In addition, extracts of S. persica can be used as a mouthwash, as they demonstrate antimicrobial properties. This study aimed to investigate the antibacterial effect of S. persica methanol and aqueous extracts against Streptococcus mutans (S. mutans) biofilm. A S. mutans biofilm formation assay was conducted using different concentrations of S. persica methanol or water extracts in tryptic soy broth (TSB) supplemented with 1% sucrose. The biofilm was stained with crystal violet dye, and the absorbance was assessed to examine biofilm formation. One-way analysis of variance (ANOVA) and Tukey tests were used to analyze the results. The S. persica methanol extract displayed a significant inhibition (p ≤ 0.001) against the S. mutans biofilm. The 10 mg/mL concentration of the S. persica methanol extract was determined as the minimum biofilm inhibitory concentration (MBIC). The used methanol concentration, mixed with TSB supplemented with 1% sucrose and without the S. persica extract, did not inhibit the S. mutans biofilm. The S. persica aqueous extract did not demonstrate any biofilm inhibition at any concentration (p ≥ 0.05). The findings of this study suggest the potential of using S. persica methanol extract as a mouthwash or adjunctive to oral hygiene tools.

Control Efficacy of Bacillus velezensis AFB2-2 against Potato Late Blight Caused by Phytophthora infestans in Organic Potato Cultivation.

Although late blight is an important disease in ecofriendly potato cultivation in Korea, it is highly dependent on the use of eco-friendly agricultural materials and the development of biological control technology is low. It is a necessary to develop an effective biocontrol agent to inactivate late blight in the field. AFB2-2 strain is a gram-positive with peritrichous flagella. It can utilize 20 types of carbon sources, like L-arabinose, and D-trehalose at 35°C. The optimal growth temperature of the strain is 37°C. It can survive at 20–50°C in tryptic soy broth. The maximum salt concentration tolerated by AFB2-2 strain is 7.5% NaCl. AFB2-2 strain inhibited the mycelial growth of seven plant pathogens by an average inhibitory zone of 10.2 mm or more. Among the concentrations of AFB2-2, 107 cfu/ml showed the highest control value of 85.7% in the greenhouse. Among the three concentrations of AFB2-2, the disease incidence and severity of potato late blight at 107 cfu/ml was lowest at 0.07 and 6.7, respectively. The nucleotide sequences of AFB2-2 strain were searched in the NCBI GenBank; Bacillus siamensis strain KCTC 13613, Bacillus velezensis strain CR-502, and Bacillus amyloliquefaciens strain DSM7 were found to have a genetic similarity of 99.7%, 99.7%, and 99.5%, respectively. The AFB2-2 strain was found to harbor the biosynthetic genes for bacillomycin D, iturin, and surfactin. Obtained data recommended that the B. velezensis AFB2-2 strain could be considered as a promising biocontrol agent for P. infestans in the field.

Characterization of \u03b2-galactosidase and \u03b1-galactosidase activities from the halophilic bacterium Gracilibacillus dipsosauri

PurposeGracilibacillus dipsosauri strain DD1 is a salt-tolerant Gram-positive bacterium that can hydrolyze the synthetic substrates o-nitrophenyl-β-d-galactopyranoside (β-ONP-galactose) and p-nitrophenyl-α-d-galactopyranoside (α-PNP-galactose). The goals of this project were to characterize the enzymes responsible for these activities and to identify the genes encoding them.MethodsG. dipsosauri strain DD1 was grown in tryptic soy broth containing various carbohydrates at 37 °C with aeration. Enzyme activities in cell extracts and whole cells were measured colorimetrically by hydrolysis of synthetic substrates containing nitrophenyl moieties. Two enzymes with β-galactosidase activity and one with α-galactosidase activity were partially purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel-filtration chromatography from G. dipsosauri. Coomassie Blue-stained bands corresponding to each activity were excised from nondenaturing polyacrylamide gels and subjected to peptide sequencing after trypsin digestion and HPLC/MS analysis.ResultFormation of β-galactosidase and α-galactosidase activities was repressed by d-glucose and not induced by lactose or d-melibiose. β-Galactosidase I had hydrolytic and transgalactosylation activity with lactose as the substrate but β-galactosidase II showed no activity towards lactose. The α-galactosidase had hydrolytic and transgalactosylation activity with d-melibiose but not with d-raffinose. β-Galactosidase I had a lower Km with β-ONP-galactose as the substrate (0.693 mmol l−1) than β-galactosidase II (1.662 mmol l−1), was active at more alkaline pH, and was inhibited by the product d-galactose. β-Galactosidase II was active at more acidic pH, was partially inhibited by ammonium salts, and showed higher activity with α-PNP-arabinose as a substrate. The α-galactosidase had a low Km with α-PNP-galactose as the substrate (0.338 mmol l−1), a pH optimum of about 7, and was inhibited by chloride-containing salts. β-Galactosidase I activity was found to be due to the protein A0A317L6F0 (encoded by gene DLJ74_04930), β-galactosidase II activity to the protein A0A317KZG3 (encoded by gene DLJ74_12640), and the α-galactosidase activity to the protein A0A317KU47 (encoded by gene DLJ74_17745).ConclusionsG. dipsosauri forms three intracellular enzymes with different physiological properties which are responsible for the hydrolysis of β-ONP-galactose and α-PNP-galactose. BLAST analysis indicated that similar β-galactosidases may be formed by G. ureilyticus, G. orientalis, and G. kekensis and similar α-galactosidases by these bacteria and G. halophilus.

A Simple and Affordable Biofilm Assay for the Undergraduate Microbiology Classroom

Through experience and surveying undergraduate microbiology curriculum, we found that most schools discuss biofilms in lecture classes but not laboratory courses. Biofilms are a concern for both industry and the medical field and should be studied in student laboratories. To study this at an institution, there would need to be an affordable method to assay them. As a sample specimen, Pseudomonas aeruginosa was used as it proliferates into biofilms when it is starved for nutrients, which can be easily simulated in a laboratory environment. Known assays for studying biofilms are expensive, and most departments do not have the materials. In Trypticase Soy Broth (TSB), P. aeruginosa can also be induced to form a biofilm, but the technique is not seen very often because it has not been sufficiently standardized for undergraduate microbiology education laboratories. To account for the absence of specialized reagents, we incubated bacteria for longer periods of time as a means to diminish nutrients or starve the specimen. Using a two-tailed t-test, we were able to show that glass tubes inoculated with P. aeruginosa in TSB for 48 hours were forming distinct biofilms on the glass surface, which is appropriate for undergraduate microbiology laboratory studies.

Effect of a Tea Polyphenol on Different Levels of Exposure of Nicotine and Tobacco Extract on Streptococcus mutans Biofilm Formation.

Objective: The purpose of this study was to compare the effects of different levels of nicotine and tobacco extract exposure on Streptococcus mutans biofilm formation and the inhibitory effect of the polyphenol epigallocatechin-3 gallate (EGCG) found in green tea. This study addressed the results of biofilm assays with EGCG and varying relative concentrations of nicotine and tobacco extract consistent with primary, secondary and tertiary levels of smoking exposure. Primary smoking exposure to nicotine has been demonstrated to significantly increase biofilm formation, while EGCG has been demonstrated to reduce S. mutans biofilm formation.Methods: S. mutans was treated with varying levels of nicotine or cigarette smoke condensate (CSC) concentrations (0–32 mg/ml and 0–2 mg/ml, respectively) in Tryptic Soy broth supplemented with 1% sucrose for different lengths of time simulating primary, secondary and tertiary smoking exposure with and without 0.25 mg/ml EGCG. The amount of total growth and biofilm formed was determined using a spectrophotometric crystal violet dye staining assay.Results: For both nicotine and CSC, primary exposure displayed overall significantly less growth compared to secondary exposure. For nicotine, secondary exposure demonstrated significantly greater growth than tertiary exposure levels. Overall, significantly greater total bacterial growth and biofilm formation in the presence of nicotine and CSC was observed in the absence of EGCG than in the presence of EGCG. However, biofilm growth was not significantly different among different concentrations of CSC.Conclusion: The results of this study help illustrate that nicotine-induced S. mutans biofilm formation is reduced by the presence of EGCG. This provides further evidence of the potential beneficial properties of polyphenols.

Strain Variability in Growth and Thermal Inactivation Characteristics of Listeria monocytogenes Strains after Acid Adaptation

Given the importance of strain variability to predictive microbiology and risk assessment, this study aimed to quantify the magnitude of strain variability in growth and thermal inactivation kinetics behaviors after acid adaptation. Thirty-three Listeria monocytogenes strains were exposed to acid-adapted tryptic soy broth supplemented with yeast extract (TSBYE; pH 5.5) and non-acid-adapted TSBYE (pH 7.0) for 20 h. Next, the growth parameters of these adapted and nonadapted strains that grew in nonbuffered TSBYE at 25°C were estimated. The tested strains were inactivated at 60°C in nonbuffered broth to obtain the heat resistance parameters. The results revealed that strain variability was present in the growth and thermal inactivation characteristics. The maximum specific growth rate ranged from 0.21 to 0.44 h-1 and from 0.20 to 0.45 h-1 after acid and nonacid adaptation, respectively. The lag times were from 0.69 to 2.56 h and from 0.24 to 3.36 h for acid-adapted and non-acid-adapted cells, respectively. The apparent D-values at 60°C of the pathogen ranged between 0.56 and 3.93 min and between 0.52 and 3.63 min for the presence and absence of acid adaptation condition, respectively. Acid adaptation significantly (P < 0.05) increased the magnitude of strain variability in the thermal inactivation characteristics of L. monocytogenes, with the coefficient of variation increasing to 0.17, whereas acid adaptation did not significantly (P ≥ 0.05) influence the variabilities in the growth parameters of the tested strains. Furthermore, the subsequent growth behaviors of all strains did not exhibit significant (P > 0.05) changes after exposure to acidic broth. However, the thermal resistance of most (25 of 33) of the tested strains increased (P < 0.05) after growing in acid-adapted broth. The relevant data generated in the present study can be used to describe the strain variability in predictive microbiology and to deeply understand the behavioral responses of different strains to acid adaptation.