Tryptic Soy Broth Research Articles

Aphicidal activity of Bacillus thuringiensis strain AH‐2 against cotton aphid (Aphis gossypii)

AbstractBacillus thuringiensis (Bt) is the most commercialized entomopathogenic species. However, the low efficacy against notorious sap‐sucking Hemipteran pests and the high costs of production, reduce the applicability of Bt biopesticides. In this context, B. thuringiensis AH‐2 (Bt‐2) isolated from soil was found to release parasporal inclusions and aphicidal activity. Bt‐2 showed optimal growth at pH 8 and 30°C in tryptic soy broth (TSB) and maintained normal growth when cultured in cost‐effective test media, pink brown (PB), supplemented with either glucose (PBG) or sucrose (PBS). To study aphicidal activity against cotton aphid (Aphis gossypii), Bt‐2 and a commercial B. thuringiensis subspecies kurstaki (Btk) were used. Using third nymph (3 N) instars, treatment with Bt‐2 caused 70.2, 78.1 and 88.4% mortality at 10, 25 and 50% culture concentrations, respectively. Similarly, Btk resulted in 79.9, 80.4 and 92.9% mortality at 10, 25 and 50%, respectively. When cultured in TSB, PBG and PBS media, Bt‐2 culture (50%) resulted in 85.9, 82.9 and 82.2% 3 N mortality, respectively. Equally, Btk caused a mortality of 79.9, 78.9 and 78.9% in TSB, PBG and PBS, respectively. Bt‐2 exerted a notably higher aphicidal efficacy at 24 h after treatment compared to Btk, especially at low concentration. Moreover, both Bt strains significantly suppressed adult fecundity compared to control. At 50% culture concentration, Bt‐2 and Btk suppressed adult fecundity by 98.3 and 91.1%, respectively. Our result demonstrates a strong aphicidal efficacy of Bt‐2, and its potential production using cost‐effective PB medium.

Short communication: A competitive exclusion study reveals the emergence of Bacillus subtilis as a predominant constitutive microorganism of a whey reverse osmosis membrane biofilm matrix

Microbial attachment and colonization on separation membranes lead to biofilm formation. Some isolates within the biofilm microflora acquire greater resistance to the chemical cleaning protocols on prolonged use of membranes. It is thus likely that the constitutive microflora might compete with each other and result in certain species emerging as predominant, especially within older biofilms. To understand the microbial interactions within biofilms, the emergence of predominance was studied in the current investigation. An 18-mo-old reverse osmosis membrane was procured from a whey processing plant. The membrane pieces (2.54 × 2.54 cm2) were neutralized by dipping in Letheen broth. The resuscitation step was done in tryptic soy broth (TSB) at 37°C, followed by plating on tryptic soy agar (TSA) to recover the constitutive microflora. Distinct colonies of isolates were further identified using MALDI-TOF as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, Bacillus subtilis (rpoB sequencing), and 1 unidentified species each of Exiguobacterium and Bacillus. Further, the competitive exclusion study helped establish the emergence of predominance using a co-culturing technique. Fifteen combinations (of 2 isolates each) were prepared from the isolates. Pure cultures of the respective isolates were spiked in a ratio of 1:1 in TSB and incubated at 37°C for 24 h, followed by plating on TSA. The enumerated colonies were distinguished based on colony morphology, Gram staining, and MALDI-TOF to identify the type of the isolate. Plate counts of B. subtilis emerged as predominant with mean log counts of 7.22 ± 0.22 cfu/mL. The predominance of B. subtilis was also validated using the process of natural selection in a multispecies growth environment. In this instance, the TSB culture with overnight-incubated membrane piece (with mixed-species biofilm) at 37°C for 12 h was inoculated in fresh TSB and incubated for the second cycle. Overall, 5 such sequential broth-culture incubation cycles were carried out, followed by pour plating on TSA plates, at the end of each cycle. The isolates obtained were identified using a similar methodology as mentioned above. The fifth subsequent transfer depicted the presence of only 1 B. subtilis isolate on plating, thereby validating its predominance under the conditions of the experiment.

Quantitative assay of Indole Acetic Acid-producing bacteria isolated from several lakes in East Java, Indonesia

Abstract. Ramadhani SI, Prabaningtyas S, Witjoro A, Saptawati TR, Rodiansyah A. 2020. Quantitative assay of Indole Acetic Acid-producing bacteria isolated from several lakes in East Java, Indonesia. Biodiversitas 21: 5448-5454. Biofuel is an alternative to fossil fuels that are environmentally friendly with low emissions. Biofuel from biomass microalgae, especially Chlorella vulgaris, has an essential role in biofuel production. Increasing biomass microalgae was done by co-culture between microalgae and bacteria. This research aims to determine the potential of bacterial isolates to produce the IAA hormone and identify the highest isolate with the ability to synthesis IAA from four lakes in East Java. This research was conducted by culturing bacterial isolates in the Tryptic Soy Broth (TSB) to add tryptophan media in various periods of incubation. The absorbance was measured with UV-Vis spectrophotometry at a wavelength of 530 nm for determining IAA-production from bacterial isolates. The results showed that the "12" code bacterial isolate from Ranu Grati produced the highest IAA hormone concentration, with an average of 30.23 ppm. The morphological characterization of the highest IAA-producing bacteria showed that isolate included the Enterobacteriaceae group and phenotypic characterization include Enterobacter cloacae complex (ECC).

Chemical, Electrochemical, and Surface Study on Microbial Attack of CoCrMo Dental Alloy by Streptococcus mutans

In this investigation, the objective of our research was to evaluate the influence of the corrosion behavior of CoCrMo dental alloy in the presence of the oral bacteria Streptococcus mutans (Streptococcus mutans) and its growth byproducts. The corrosion resistance of the dental alloy CoCrMo followed a comparative study in the sterilized Fusayama’s artificial saliva (FAS) containing Trypticase soy broth (TSB) (medium 1) and Streptococcus mutans mixed with (TSB) and (FAS) (medium 2). To perform this study, electrochemical corrosion tests including open circuit potential (OCP), potentiodynamic polarization (PDP), and impedance spectroscopy (EIS) measurements and inductively coupled plasma atomic emission spectrometry (ICP-AES) were used. The formed films biomass was observed by an optical microscope, after 216 h (9 days) of exposition to medium containing Streptococcus mutans; then, it was verified by SEM–EDS analysis. The results of the electrochemical corrosion showed a significant shift of the corrosion potential and an increase in the corrosion current density (icorr) and the corrosion resistance (Rp) after 24 h of immersion of the alloys in the medium containing Streptococcus mutans. This led to alter the formation of the passive film which reveals the high reactivity of CoCrMo in the presence of Streptococcus mutans, whereas the surface analysis confirmed the biofilm formation once the contact of Streptococcus mutans to CoCrMo alloy surfaces was insured.

Proteome analysis of virulent Aeromonas hydrophila reveals the upregulation of iron acquisition systems in the presence of a xenosiderophore.

The Gram-negative bacterium, Aeromonas hydrophila, has been responsible for extensive losses in the catfish industry for over a decade. Due to this impact, there are ongoing efforts to understand the basic mechanisms that contribute to virulent A. hydrophila (vAh) outbreaks. Recent challenge models demonstrated that vAh cultured in the presence of the iron chelating agent deferoxamine mesylate (DFO) were more virulent to channel catfish (Ictalurus punctatus). Interestingly, differential gene expression of select iron acquisition genes was unremarkable between DFO and non-DFO cultures, posing the question: why the increased virulence? The current work sought to evaluate growth characteristics and protein expression of vAh after the addition of DFO. A comparative proteome analysis revealed differentially expressed proteins among tryptic soy broth (TSB) and TSB+DFO treatments. Upregulated proteins identified among the TSB+DFO treatment were enriched for gene ontology groups including iron ion transport, siderophore transport and siderophore uptake transport, all iron acquisition pathways. Protein-protein interactions were also evaluated among the differentially expressed proteins and predicted that many of the upregulated iron acquisition proteins likely form functional physiological networks. The proteome analysis of the vAh reveals valuable information about the basic biological processes likely leading to increased virulence during iron restriction in this organism.

10.4314/cajeb.v3i1.37959

Environmental factors may greatly influence the expression of cell surface components of bacterial pathogens. Few studies have described the effect of growth conditions on the cell surface hydrophobicity of bacterial isolates of certain Gramnegative and Gram-positive bacteria. The present study describes the effects of CUltivation in four common liquid growth media on the cell surface hydrophobicity of non-clinical Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus using the microbial adhesion to hydrocarbon (MATH) test which assesses the partition of bacterial cells into a hydrocarbon phase. It was observed that growth in all the test media yielded cells with varying surface hydrophobicity increasing in the order: nutrient broth<tryptic soy broth<brain heart infusion broth<MacConkey broth, irrespective of the test strain. The changes were however, more pronounced in the Gram-positive strain. It was also observed that the surface of Staphylococcus aureus was more hydrophobic than that of Escherichia coli , irrespective of the cultivation media. The outcome of this work further point to the fact that environmental changes can influence cell surface hydrophobicity of bacteria which in turn can affect their adhesion to certain kinds of host targets through hydrophobic interactions. Key words : Hydrophobicity; Escherichia coli; Staphylococcus aureus; Growth media

Synergistic efficacy of alkyltrimethylammonium bromide, chlorhexidine digluconate on diverse bacterial strains causing red-heat and purple-stain deteriorations of leather.

This investigation assessed the synergistic effectiveness of alkyltrimethylammonium bromide (ATMB) and chlorhexidine digluconate (CDG) on selected microbes causing red-heat and purple stain degradations on salt-cured hides and leather products. This biological phenomenon ultimately deteriorates the finished leather quality and grounds considerable economic losses for leather industry. In tryptic soy broth, combination of ATMB and/CDG at the concentration of 900/90ppm for predefined time interval of 1, 3, and 6min caused in 0.73, 2.45, and 2.95 log CFU/mL reductions of bacterial cocktail population cell survival, respectively. Whereas in saturated brine solution (SBS), the bacterial cocktail treated with 600/60 and 900/90ppm of ATMB/CDG for 18h resulted ~ 74% and 98% reduction, respectively, in comparison to their respective controls. Furthermore, lessening of individual bacterial cultivations by combined ATMB and CDG in SBS was also elucidated.

Effects of Irregularities in the Microstructure of Surgical Instruments on Microbial Adherence and Challenges for Processing

Background: A surgical instrument can have areas that pose different challenges to cleaning, hindering the removal of dirt. This can directly impact the effectiveness of its processing while potentially promoting cross contamination. Moreover, structural changes (ie, cracks or fissures) on the instrument surface, although rarely addressed, can act as reservoirs for microorganisms, contributing to organic matter retention, microbial growth, and biofilm formation. Our aim was to determine the effect of irregularities in the microstructure of surgical instruments on microbial adherence. Methods: We analyzed 18 fragments of 3 distinct areas of new crile forceps: the ratchet, shank, and jaw. Of these fragments, 15 were artificially contaminated by immersion in tryptic soy broth containing 10 6 CFU/mL of Escherichia coli (ATCC 25922), for 4, 6, 8, and 12 hours of incubation at 37C with agitation at 100 rpm. The other 3 fragments were used as controls. All fragments were subjected to scanning electron microscopy to evaluate the adhesion of the microorganism. Results: An irregular surface was found in 3 of 6 shank fragments (50%) (Fig. 1) and in all the jaw and ratchet fragments, grooves, and cracks. Initially, there was less adherence of E. coli to the smooth shank surface after contamination, but the concentration of the microorganism increased progressively over time in relation to that in the jaw or ratchet at the same time, and a higher concentration occurred in the cracks and grooves. Conclusions: Structural damage was observed in all fragments, especially in the ratchet and jaw areas, favoring microbial accumulation. Microorganisms housed in the cracks and grooves were better protected from removal by scrubbing with a brush (being unlikely to reach them), making these areas a microbial reservoir and source of contamination. Prolonged contact of the instrument with the contaminating microorganism allowed for greater adherence, even on the smooth areas. The results support the relevance of the early onset of cleaning, considering that even microscopic changes on the surface of the instrument may represent an additional challenge to its effective processing.Funding: NoneDisclosures: None

Heterojunction formation between copper(II) oxide nanoparticles and single-walled carbon nanotubes to enhance antibacterial performance.

We delineate the excellent bactericidal efficacy of stable heterojunction nanocomposites composed of single-walled carbon nanotubes (SWCNTs) and copper(II) oxide (CuO) synthesized via facile recrystallization and calcination. The bactericidal effectiveness of the fabricated nanocomposites was examined using the standard broth-dilution method and the growth-inhibition-zone analysis method, in which bacteria cultured in an incubator in tryptic soy broth medium were subjected to the prepared samples. The bactericidal activity of all of the as-synthesized samples is evident in both methods, displaying a substantial decrease in bacterial colonies and resulting in clear inhibition zones, respectively. Among the CuO-SWCNT nanocomposites, the sample subjected to calcination at 500°C for 5h was found to exhibit the best performance against Staphylococcus aureus and Escherichia coli, forming inhibition zones 182% and 162% larger than those formed by pure CuO, respectively.

Evaluation of phenotypic tests for detection of carbapenemases: New modifications with new interpretation

IntroductionThe emergence and spread of carbapenemase-producing Enterobacterales (CPE) is a worldwide public health threat. Rapid and accurate detection of CPE is essential to prevent their dissemination within health care settings. The aim of this study was to evaluate the performance of CIM, mCIM and mCIM with ammonium bicarbonate (mCIM-A) methods by using different interpretation criteria for detection of carbapenemases. MethodsOne hundred and fifty-three Klebsiella pneumoniae isolates previously characterized by molecular tests, including 133 carbapenemase producers and 20 non-carbapenemase producers, were collected in this study. CIM and mCIM tests were performed as described previously. mCIM-A by adding 50 mM ammonium bicarbonate to the bacterial suspension prepared in tryptic soy broth. The inhibition zone diameter of around meropenem disc was measured and interpreted as positive according to i) Pierce and colleagues (<19 mm), ii) EUCAST meropenem susceptibility breakpoint (<22). ResultsCIM, although seems to be good for carbapenemases other than OXA-48-like and NDM, is not satisfactory (42.3% and 83.4%, respectively) for those enzymes with any of the interpretation criteria. OXA-48-like and NDM were detected with a better performance (88.7% and 92.8, respectively) with mCIM when results were interpreted according to <22 mm zone diameter for OXA-48-like and NDM. The best results were obtained with mCIM-A using <22 mm criteria without any difference in the results of other enzymes and negative strains.Conclusions: mCIM-A method interpreted with <22 mm meropenem zone diameter seems to be preferable compared to CIM and mCIM. mCIM-A is simple and useful tool for identification of CPEs in clinical microbiology laboratories.

Susceptibility of Mature Staphylococcus Biofilms to Chinese Herbal Decoction Sanhuang Jiedu: An In Vitro Study.

Background External socking and washing with the Chinese herbal Sanhuang Jiedu decoction (SHJD) can effectively control local limb infections with bone and implant exposure. However, the antibiofilm activities of this decoction in vitro have not yet been investigated. Therefore, the aim of this study was to examine the effects and characteristics of SHJD on the mature biofilms of multidrug-resistant staphylococci on a titanium surface. Methods Biofilm-forming methicillin-resistant Staphylococcus epidermidis ATCC 35984 and S. aureus ATCC 43330, and non-biofilm-forming S. epidermidis ATCC 12228 were selected as the experimental strains. The mature biofilms were prepared on titanium surfaces. The five experimental groups were based on dilution concentrations (DC) of SHJD: the control group (biofilm incubated with 0.85% NaCl solution), the SHJD (DC:1/8) group (initial SHJD solution was diluted 1/8), the SHJD (DC:1/4) group, the SHJD (DC:1/2) group, and the SHJD (DC:1/1) group (initial SHJD solution). The effects of SHJD on the mature biofilms were observed with the bacterial spread plate method, crystal violet (CV) staining, scanning electron microscopy, and confocal laser scanning microscopy. Results After culture in tryptic soy broth for 72 h, ATCC 43300 and ATCC 35984 produced mature biofilms and ATCC 12228 did not. The optical density value of ATCC 12228 was 0.11 ± 0.02, significantly lower than that of ATCC 35984 (0.42 ± 0.05) or ATCC 43300 (0.41 ± 0.03) (P < 0.05). The mature biofilms of ATCC 43300 and ATCC 35984 clearly disintegrated when incubated for 12–24 h with SHJD (DC:1/1) or SHJD (DC:1/2), showing only scattered bacterial adhesion. In the SHJD (DC:1/4) group, although many residual bacterial colonies still clustered together, presenting a biofilm structure, it was very looser than that in the SHJD (DC:1/8) group in which the biofilm was similar to that in the control group. For ATCC 12228, only colony adhesion was observed, and the number of colonies decreased as the concentration of SHJD or the culture period increased. The quantitative results for the bacterial spread plate and CV staining showed significant differences between the SHJD groups (P < 0.05). Conclusion SHJD has antibiofilm activity against multidrug-resistant Staphylococcus strains. It weakens or disrupts already-formed mature biofilms on titanium surfaces in a concentration- and incubation time-dependent manner.

Two Type VI Secretion Systems of Enterobacter cloacae Are Required for Bacterial Competition, Cell Adherence, and Intestinal Colonization.

Enterobacter cloacae has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several E. cloacae strains revealed the presence of genes that code for expression of at least one type VI secretion system (T6SS). Here, we report that E. cloacae strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes clpV1 and hcp1 significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of E. cloacae cultures and a functional T6SS-1 was required for the secretion of this protein. A clpV2 mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent. In vivo data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in clpV1 and clpV2 genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of E. cloacae with specialized functions in the interaction with other bacteria and with host cells.

Effect of hydrogen peroxide on the dehydrogenase and quinone-reductase activity of irradiated Lactobacillus plantarum cells

The resistance of lactobacilli to oxidative stress is of great importance for their applicability as probiotics. This study aimed to evaluate the response of Lactobacillus plantarum strain ATCC® 14917™, grown in either de Man, Rogosa, and Sharpe agar (MRS medium) or tryptic soy broth (TSB medium), to 1–2 mM H2O2 after the exposure to different doses of ionising radiation. Two bacterial extracellular enzyme groups, dehydrogenases (DHAs) and quinone reductases (QRs), served as the criteria of viability and antioxidant activity, respectively. The irradiated L. plantarum culture grown in TSB showed increased QR activity at irradiation doses of 2–50 Gy, with the maximum activity at 10 Gy, compared to non-irradiated cells. The QR activity of the MRS-grown culture was increased significantly (p < 0.05) in the presence of H2O2 for the cells subjected to irradiation, compared to the control. The cultivation of L. plantarum in TSB resulted in considerable morphological changes in the 24 h culture. Our results indicate that the cultivation of L. plantarum in TSB activated the antioxidative potential of these bacteria. The most important aspect of this phenomenon is the extracellular enzyme activity that may directly influence the host organism by providing antioxidant activity.

Visualization and characterization of Enterococcus faecalis biofilm structure in bovine dentin using 2D and 3D microscopic techniques.

Bacterial biofilms are related to various dental and periodontal infectious diseases, and the characterization of this biological structure with micro-computed tomography (micro-CT) may offer valuable information for clinical and research applications. In this study, we aimed to develop a model to visualize three-dimensionally the biofilm structure on dentin using micro-CT. Dentin blocks were prepared and incubated in tryptic soy broth with Enterococcus faecalis (ATCC 29212). The control group did not receive any staining procedure, while groups 1 and 2 were stained with 100% and 50% barium sulfate, respectively. Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) were used to detect biofilm formation, barium sulfate penetration, and microbial cell density in the biofilm. Micro-computed tomography (micro-CT) (SkyScan 1172, Bruker Co., Belgium) was used to visualize biofilm formation on the dentin blocks. Biofilm thicknesses were measured from 10 different locations on the specimen surfaces, using CTAn v.1.14.4 software. Obtained data were statistically analyzed using Kruskal-Wallis and Dunn's tests. TEM photomicrographs showed that barium sulfate could penetrate the biofilm structure. CLSM analysis showed that viable and total cell densities were similar between the control and barium sulfate-treated groups (P > 0.05), indicating barium sulfate had no significant influence on cell density. In barium sulfate-treated blocks, biofilm could be discriminated from the dentin, and its thickness could be measured with micro-CT. This study showed that bacterial biofilm on dentin could be characterized by micro-CT after barium sulfate staining without causing any significant side effect on viable and total cell densities.

Antimicrobial activity as a potential factor influencing the predominance of Bacillus subtilis within the constitutive microflora of a whey reverse osmosis membrane biofilm

Current cleaning and sanitation protocols may not be adequately effective in cleaning separation membranes and can result in the formation of resilient multispecies biofilms. The matured biofilms may result in a bacterial predominance with resilient strains on membranes with a prolonged use. In our previous study, we isolated organisms such as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens from an 18-mo-old reverse osmosis membrane. The competitive exclusion studies revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigated the antimicrobial activity of the B. subtilis isolate as a potential cause of its predominance. The culture isolate was propagated in tryptic soy broth at 37°C, and microfiltered to prepare cell-free extracts (CFE) at 8-, 10-, 12-, 14-, 16-, and 18-h intervals. The CFE were freeze-dried and suspended in minimum quantities of HPLC-grade water to prepare concentrated solutions. The antimicrobial activities of CFE were tested using the agar-well assay against the biofilm constitutive microflora. The experiments were conducted in triplicates and means were compared for significant differences using a general linear mixed model procedure. The results indicated the highest antimicrobial activity of 12-h CFE of B. subtilis against other constitutive microflora such as Exiguobacterium sp., E. auranticum, and A. radioresistens, with average inhibition zone sizes of 16.5 ± 0.00, 16.25 ± 0.66, and 20.6 ± 0.00 mm, respectively. Upon treatment with proteinase K, the CFE completely lost its antimicrobial activity, establishing it to be a proteinaceous compound. The AA profiling revealed the total crude protein in CFE to be 51% (wt/wt), with its major constituent as glutamic acid (11.30% wt/wt). The freeze-dried CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min) and over a pH range of 3.0 to 6.3. The study helped us understand the role of the antimicrobial compound produced by B. subtilis as a potential cause of its predominance within the biofilm constitutive microflora.

Paraburkholderia solitsugae sp. nov. and Paraburkholderia elongata sp. nov., phenolic acid-degrading bacteria isolated from forest soil and emended description of Paraburkholderia madseniana.

Two bacterial strains, 1NT and 5NT, were isolated from hemlock forest soil using a soluble organic matter enrichment. Cells of 1NT (0.65×1.85 µm) and 5NT (0.6×1.85 µm) are Gram-stain-negative, aerobic, motile, non-sporulating and exist as single rods, diplobacilli or in chains of varying length. During growth in dilute media (≤0.1× tryptic soy broth; TSB), cells are primarily motile with flagella. At higher concentrations (≥0.3× TSB), cells of both strains increasingly form non-motile chains, and cells of 5NT elongate (0.57×~7 µm) and form especially long filaments. Optimum growth of 1NT and 5NT occurred at 25-30 °C, pH 6.5-7.0 and <0.5% salinity. Results of comparative chemotaxonomic, genomic and phylogenetic analyses revealed that 1NT and 5NT were distinct from one another and their closest related type strains: Paraburkholderia madseniana RP11T, Paraburkholderia aspalathi LMG 27731T and Paraburkholderia caffeinilytica CF1T. The genomes of 1NT and 5NT had an average nucleotide identity (91.6 and 91.3%) and in silico DNA-DNA hybridization values (45.8%±2.6 and 45.5%±2.5) and differed in functional gene content from their closest related type strains. The composition of fatty acids and patterns of substrate use, including the catabolism of phenolic acids, also differentiated strains 1NT and 5NT from each other and their closest relatives. The only ubiquinone present in strains 1NT and 5NT was Q-8. The major cellular fatty acids were C16 : 0, 3OH-C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c and summed features 2 (3OH-C14 : 0 / C16 : 1 iso I), 3 (C16 : 1 ω6c/ω7c) and 8 (C18 : 1 ω7c/ω6c). A third bacterium, strain RL16-012-BIC-B, was isolated from soil associated with shallow roots and was determined to be a strain of P. madseniana (ANI, 98.8%; 16S rRNA gene similarity, 100%). Characterizations of strain RL16-012-BIC-B (DSM 110723=LMG 31706) led to proposed emendments to the species description of P. madseniana. Our polyphasic approach demonstrated that strains 1NT and 5NT represent novel species from the genus Paraburkholderia for which the names Paraburkholderia solitsugae sp. nov. (type strain 1NT=DSM 110721T=LMG 31704T) and Paraburkholderia elongata sp. nov. (type strain 5NT=DSM 110722T=LMG 31705T) are proposed.

Characterization of a Lytic Bacteriophage vB_EfaS_PHB08 Harboring Endolysin Lys08 Against Enterococcus faecalis Biofilms.

Enterococcus faecalis is an opportunistic pathogen that causes illnesses ranging from urinary tract infections to sepsis in humans and animals. However, the overuse of antibiotics has increased rates of drug resistance among E. faecalis isolates. Bacteriophages and their derivatives have recently been identified as good candidates for the treatment of drug-resistant bacterial infections. Here, we isolated a virulent E. faecalis phage, PHB08, using the double-layer plate method. The bioactivity of the phage was determined via one-step growth curve testing and bacterial killing assays, and whole-genome sequencing was performed using the Illumina HiSeq platform. In addition, protein expression and antibiofilm assays were performed to investigate the activity of the phage lysin. Results showed that PHB08 has a 55,244-bp linear double-stranded DNA genome encoding 91 putative coding sequences. PHB08 inhibited the growth of host strain EF3964 at 37 °C in tryptic soy broth (TSB) medium, while in vegetable models, PHB08 caused a 4.69-log decrease in viable E. faecalis cells after 24 h. Both PHB08 and its endolysin lys08 showed antibiofilm activity against E. faecalis biofilms, which was enhanced by Mn2+ ions. Thus, virulent phage PHB08 and endolysin lys08 may be good candidates for reducing and/or eradicating E. faecalis infections.

Probiotic Application of Bacteriocin-Producing S. Epidermidis in A Cellulosic Pad to Treat Some Skin Infections

The aim of this study was to evaluate the possibility of using Staphylococcus epidermidis cells as a probiotic to treat some skin infections. For this purpose, S. epidermidis Y73, which is an active bacteriocin producer and non-biofilm forming isolate, was selected among 134 skin isolates through primary and secondary screening. Tryptic soya broth was selected as the best medium to support bacteriocin production, while the optimal pH and temperature for S. epidermidis Y73 growth were 7 and 37°C, respectively, which were invested in the formula preparation. Furthermore, the possibility of using this isolate as a probiotic was investigated by preparing 4 potential cellulosic pads with 4 different formulae which were all subjected to an in vitro trial to select the one which is superior to the others in terms of supporting bacteriocin production and cells viability. The shelf life of the pad was estimated and the results showed that the cells remained vital until the 20th week. The selected pad formula was used to treat artificially induced wounds on rabbit skin. The wounds were infected with Staphylococcus aureus, Kocuria rosea and Pseudomonas. aeruginosa. The symptoms in both control and treated animals were recorded and, based on the results; the healing process with the presence of the S. epidermidis Y73 pad was significantly faster compared with that for the control. This research will serve as a base for future studies on using vital cells of S. epidermidis as probiotics and, hence, make a contribution to the current literature on using live cells to treat bacterial skin infections.

Dependence of the Staphylococcal Volatilome Composition on Microbial Nutrition.

In vitro cultivation of staphylococci is fundamental to both clinical and research microbiology, but few studies, to-date, have investigated how the differences in rich media can influence the volatilome of cultivated bacteria. The objective of this study was to determine the influence of rich media composition on the chemical characteristics of the volatilomes of Staphylococcus aureus and Staphylococcus epidermidis. S. aureus (ATCC 12600) and S. epidermidis (ATCC 12228) were cultured in triplicate in four rich complex media (brain heart infusion (BHI), lysogeny broth (LB), Mueller Hinton broth (MHB), and tryptic soy broth (TSB)), and the volatile metabolites produced by each culture were analyzed using headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography—time-of-flight mass spectrometry (HS-SPME-GC×GC-TOFMS). When comparing the chemical compositions of the staph volatilomes by the presence versus absence of volatiles produced in each medium, we observed few differences. However, when the relative abundances of volatiles were included in the analyses, we observed that culturing staph in media containing free glucose (BHI and TSB) resulted in volatilomes dominated by acids and esters (67%). The low-glucose media (LB and MHB) produced ketones in greatest relative abundances, but the volatilome compositions in these two media were highly dissimilar. We conclude that the staphylococcal volatilome is strongly influenced by the nutritional composition of the growth medium, especially the availability of free glucose, which is much more evident when the relative abundances of the volatiles are analyzed, compared to the presence versus absence.

Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro

BackgroundBiofilm is known to be tolerant towards antibiotics and difficult to eradicate. Numerous studies have reported minimum biofilm eradication concentration (MBEC) values of antibiotics for many known biofilm pathogens. However, the experimental parameters applied in these studies differ considerably, and often the rationale behind the experimental design are not well described. This makes it difficult to compare the findings. To demonstrate the importance of experimental parameters, we investigated the influence of biofilm growth age, antibiotic concentration and treatment duration, and growth media on biofilm eradication. Additionally, OSTEOmycin™, a clinically used antibiotic containing allograft bone product, was tested for antibiofilm efficacy.ResultsThe commonly used Calgary biofilm device was used to grow 24 h and 72 h biofilms of Staphylococcus aureus and Pseudomonas aeruginosa, which were treated with time-dependent vancomycin (up to 3000 mg L− 1) and concentration-dependent tobramycin (up to 80 mg L− 1), respectively. Two common bacteriological growth media, tryptic soy broth (TSB) and cation-adjusted Mueller Hinton broth (CaMHB), were tested. We found for both species that biofilms were more difficult to kill in TSB than in CaMHB. Furthermore, young biofilms (24 h) were easier to eradicate than old biofilms (72 h). In agreement with vancomycin being time-dependent, extension of the vancomycin exposure increased killing of S. aureus biofilms. Tobramycin treatment of 24 h P. aeruginosa biofilms was found concentration-dependent and time-independent, however, increasing killing was indicated for 72 h P. aeruginosa biofilms. Treatment with tobramycin containing OSTEOmycin T™ removed 72 h and 168 h P. aeruginosa biofilms after 1 day treatment, while few 72 h S. aureus biofilms survived after 2 days treatment with vancomycin containing OSTEOmycin V™.ConclusionsThis study demonstrated biofilm removal efficacy was influenced by media, biofilm age and antibiotic concentration and treatment duration. It is therefore necessary to taking these parameters into consideration when designing experiments. The results of OSTEOmycin™ products indicated that simple in vitro biofilm test could be used for initial screening of antibiofilm products. For clinical application, a more clinically relevant biofilm model for the specific biofilm infection in question should be developed to guide the amount of antibiotics used for local antibiofilm treatment.