LB Medium Research Articles

Arthrobacter vasquezii sp. nov., isolated from a soil sample from Union Glacier, Antarctica.

A Gram-stain-positive, catalase-positive, non-motile bacteria, with a rod-coccus cycle (designated as EH-1B-1T) was isolated from a soil sample from Union Glacier in Ellsworth Mountains, Antarctica. Strain EH-1B-1T had an optimal growth temperature of 28 °C and grew at pH 7-10. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, C16 : 0 and anteiso-C17 : 0. The G+C content based on the whole genome sequence was 63.1 mol%. Strain EH-1B-1T was most closely related to members of the genus Arthrobacter, namely Arthrobacter subterraneus and Arthrobacter tumbae. The strain grew on tryptic soy agar, Reasoner's 2A agar, lysogeny broth agar and nutrient agar. The average nucleotide identity and digital DNA-DNA hybridization values between strain EH-1B-1T and its closest reference type strains ranged from 78 to 88 % and from 20.9 to 36.3 %, respectively. Based on phenotypic, chemotypic and genotypic evidence, it is proposed that strain EH-1B-1T represents a novel species of Arthrobacter, for which the name Arthrobacter vasquezii sp. nov. is proposed, with strain EH-1B-1T (RGM 3386T=LMG 32961T) as the type strain.

Roles of crrAB two-component regulatory system in Klebsiella pneumoniae: growth yield, survival in initial colistin treatment stage, and virulence

Alternation of the colistin resistance-regulating two-component regulatory system (crrAB) is a colistin-resistance mechanism in Klebsiella pneumoniae (K. pneumoniae), but its role in bacteria is not fully understood. Twelve colistin-susceptible K. pneumoniae clinical isolates were included in this study: six crrAB-positive and six crrAB-negative. We deleted the crrAB genes from two crrAB-positive isolates and complemented them. We measured the growth yields by determining growth curves in lysogeny broth and minimal media with or without Fe2+. In vitro selection rates for colistin resistance were determined by exposure to colistin, and survival rates against high concentrations of colistin (20 mg/L) at the early stage of growth (20 min) were investigated. Virulence was determined using a serum bactericidal assay and Galleria mellonella larval infection. The presence of crrAB was not associated with colistin resistance and did not increase the in vitro selection rate of colistin resistance after exposure. The growth yield of crrAB-positive isolates was higher in lysogeny broth media and increased when Fe2+ was added to minimal media. The crrAB-positive isolates showed higher survival rates in the early stages of exposure to high colistin concentrations. Decreased serum resistance was identified in the crrAB-deleted mutants. More G. mellonella larvae survived when infected by crrAB-deleted mutants, and higher survival rates of bacteria were identified within the larvae infected with wild-type than crrAB-deletant isolates. Through rapid response to external signals, crrAB would provide advantages for K. pneumoniae survival by increasing the final growth yield and initial survival against colistin treatment. This may partly contribute to the bacterial virulence.

Trichuris muris egg-hatching assay for anthelminthic drug discovery and characterization

Trichuriasis is a neglected tropical disease widely distributed among tropical and sub-tropical areas and associated with poverty and lack of access to safe drinking water, sanitation and hygiene. Existing drugs have limited efficacy and face a constant risk of developing resistance, necessitating the search for alternative treatments. However, drug discovery efforts are sparse and little research has been performed on anthelminthic effects on embryonated eggs, the infectious life stage of Trichuris spp.We examined bacterial species dependent egg hatching of the murine model parasite Trichuris muris and identified Escherichia coli, Pseudomonas aeruginosa and Enterobacter hormaechei effective as hatching inducers, resulting in hatching yields of 50–70%. Streptococcus salivarius, reported to be associated with reduced drug efficacy of ivermectin-albendazole coadministration in Trichuris trichiura infected patients, did not promote egg hatching in vitro. We optimized hatching conditions using E. coli grown in luria broth or brain-heart infusion media to reach consistently high hatching yields to provide a sensitive, robust and simple egg-hatching assay. Oxantel pamoate demonstrated the strongest potency in preventing hatching, with an EC50 value of 2–4 μM after 24 h, while pyrantel pamoate, levamisole and tribendimidine exhibited only moderate to weak inhibitory effects. Conversely, all tested benzimidazoles and macrolide anthelminthics as well as emodepside failed to prevent hatching (EC50 > 100 μM).Our study demonstrates that egg-hatching assays complement larval and adult stage drug sensitivity assays, to expand knowledge about effects of current anthelminthics on Trichuris spp. Further, the developed T. muris egg-hatching assay provides a simple and cheap screening tool that could potentially lead to the discovery of novel anthelminthic compounds.

Biological control of Magnaporthe oryzae using natively isolated Bacillus subtilis G5 from Oryza officinalis roots.

Rice blast, caused by Magnaporthe oryzae, is a major threat to global rice production causing significant crop losses and impacting grain quality. The annual loss of rice production due to this disease ranges from 10% to 30%. The use of biologically controlled strains, instead of chemical pesticides, to control plant diseases has become a research hotspot. In this study, an antagonistic endophytic bacterial strain was isolated from the roots of Oryza officinalis using the traditional isolation and culture methods. A phylogenetic tree based on 16S RNA and whole-genome sequencing identified isolate G5 as a strain of Bacillus subtilis. This isolate displayed strong antagonistic effects against different physiological strains of M. oryzae. After co-culture in LB medium for 7 days, the inhibition rates of the mycelial growth of four strains of M. oryzae, ZB15, WH97, Guy11, and T-39800E were 98.07 ± 0.0034%, 98.59 ± 0.0051%, 99.16 ± 0.0012%, and 98.69 ± 0.0065%, respectively. Isolate G5 significantly inhibited the formation of conidia of M. oryzae, with an inhibition rate of 97% at an OD600 of 2. Isolate G5 was able to provide 66.81% protection against rice blast under potted conditions. Whole-genome sequencing revealed that the genome size of isolate G5 was 4,065,878 bp, including 4,182 coding genes. Using the anti-SMASH software, 14 secondary metabolite synthesis gene clusters were predicted to encode antifungal substances, such as fengycin, surfactin, and bacilysin. The G5 isolate also contained genes related to plant growth promotion. These findings provide a theoretical basis for expounding the biocontrol mechanisms of this strain and suggest further development of biogenic agents that could effectively inhibit rice blast pathogen growth and reduce crop damage, while being environmentally friendly, conducive to ecological development, and a sustainable alternative to chemical pesticides. This study also enriches the relevant research on endophytes of wild rice, which proves that wild rice is a valuable microbial resource bank.

Autoinduction expression of Bst DNA polymerase using lac operon-controlled expression systems in Escherichia coli

Autoinduction simplified the steps for protein production by omitting the need to monitor the correct time for addition of inducer. This method is preferable for industrially production of protein. By using lac operon-controlled expression system, it is possible to produce the protein without the addition of IPTG as inducer. In a conventional way, IPTG was added at the exponential growth phase. By using autoinduction, IPTG could be replaced by lactose in the lac operon-controlled expression systems. Lactose was already supplemented in the medium and it was more cost efficient than IPTG. Therefore, this study aims to employ autoinduction method for expression of our protein target as it is a more convenient way and it is suitable for industrial application. In this study, DNA polymerase from Geobacillus stearothermophilus (Bst DNA polymerase), an enzyme which generally used in loop-mediated isothermal amplification (LAMP) assay, was expressed using lac operon-controlled expression systems in Escherichia coli. Bst DNA polymerase was successfully expressed using autoinduction expression method in LB medium containing 0.2% Lactose.

IDENTIFIKASI BAKTERI Escherichia Coli PADA SAUS MAKANAN ATAU JAJANAN CILOK DI SEKOLAH DASAR (SD) KECAMATAN INDRALAYA SELATAN KABUPATEN OGAN ILIR

Food poisoning often occurs in elementary school children due to the lack of personal and environmental hygiene. According to children tend to choose unhealthy foods because regardless of the physical/chemical properties of food, taste, brand, availability and environment. The environment such as personal hygiene from food vendors is one of the factors that influence food conditions. This study aims to determine whether there is Escherichia coli bacteria on cilok sauce in Junior High Schoolab So1th Indralaya District. The results of the study by identifying Escherichia coli bacteria in cilok sauce, namely the isolation test of bacteria on LB media, obtained 8 out of 10 samples contaminated with bacteria. The EMB test results obtained 3 out of 8 samples that were positive for Escherichia coli. For bacterial tests with biochemical tests, SIM, MR, citrate, 3 positive samples of Escherichia coli bacteria were obtained where there was a color change. Citrate changes from green to blue, SIM is indicated by the formation of a pink indole ring and the MR test changes the solution to a pink color.

Cell Viability Studies on Bacillus sp. under Different Storage Conditions for Usage in Improving Concrete Compressive Strength

Bacterial concrete is a possible approach toward sustainability in concrete construction through crack-healing. Including a bacterial culture as an admixture in concrete can enhance the service life of a structure through the self-healing of cracks. Incorporating bacterial cells as an admixture in concrete is a major challenge as bacteria are living organisms with a limited shelf-life. It is essential to evaluate the shelf-life of bacterial cultures to encourage the inclusion of bacteria in concrete applications. Hence, the main focus of this study was to record the cell viability of these microorganisms before addition to cementitious systems. In the first stage, three different bacterial cultures of Bacillus subtilis, Bacillus cereus, and Bacillus licheniformis were stored in Luria Bertani broth under two different conditions of room temperature and refrigeration. These stored bacterial solutions were checked for viability based on cell count after 1 day, 3 days, 7 days, 15 days, and 20 days of storage. In the second stage, the fresh bacterial cultures and the 15-day stock were added to prepare bacterial concrete and cement paste samples to assess their compressive strengths and microstructural changes, respectively. It was observed that the cell viability in terms of cell count of the selected bacterial strains attained up to 15 days when stored at room temperature. It was also observed that the compressive strength of the bacterial concrete prepared with stored bacterial cultures increased by 6% and 11% at 7 and 28 days compared with the control Portland cement concrete mix, respectively. However, the compressive strength decreased by 6% to 12% compared with the bacterial concrete prepared with fresh cultures at the same ages. Additionally, the compressive strength results were validated using microstructural analyses.

Influence of Central Metabolism Disruption on Escherichia coli Biofilm Formation.

Biofilms are widely recognized as a prominent mode of microbial growth and strategy of antimicrobial tolerance in many environments. Characteristics that are often overlooked in biofilm investigations include the examination of metabolic pathways as the assumption might be that interference with central pathways such as glycolysis would only reduce growth and thus not be meaningful. Using the Keio collection of Escherichia coli mutants, we investigated the influence of biofilm formation and planktonic growth in full strength and diluted Luria-Bertani (LB) broth using strains with a disruption of glycolysis (Δpgi), the Entner-Doudoroff pathway (Δedd), or the pentose phosphate pathway (Δgnd). Unexpectedly, in contrast to the E. coli Keio parent strain (BW25113), planktonic growth was enhanced in full strength and diluted LB broth in the metabolic mutants. Using a microtiter biofilm assay, the E. coli parent strain showed the highest crystal violet staining. However, when analyzed by culture assays, there was an increase in biofilm populations in the mutants in comparison to the parent strain. Fluorescence microscopy showed differences in colonization patterns in the strains. Given the availability of mutant collections in many model organisms, similar metabolic studies are warranted for biofilms, given their importance in nature.

Biofilm formation on endovascular aneurysm repair (EVAR) grafts—a proof of concept in vitro model

ObjectivesAn endovascular aneurysm repair (EVAR) graft is a catheter-implanted vascular prosthesis and is the preferred treatment for patients with aortic aneurysm. If an EVAR graft becomes the focus of infection, the treatment possibilities are limited because it is technically difficult to remove the graft to obtain source control. This study examines whether Pseudomonas aeruginosa and Staphylococcus aureus form biofilm on EVAR prostheses. MethodsEVAR graft sections were exposed to bacteria at 102 or 108 colony forming units (CFU)/mL in lysogeny broth and Krebs-Ringer at 37°C, bacterial biofilm formation was evaluated by scanning electron microscopy and counting CFU on the graft sections after antibiotic exposure at × 10 minimal inhibitory concentration. Bacteria were tested for tolerance to benzylpenicillin, tobramycin, and ciprofloxacin. ResultsBacterial exposure for 15 minutes established biofilms on all prosthesis fragments (6/6 replicates). After 4 hours, bacteria were firmly attached to the EVAR prostheses and resisted washing. After 18–24 hours, the median CFU/g of EVAR graft reached 5.2 × 108 (1.15 × 108–1.1 × 109) for S. aureus and 9.1 × 107 (3.5 × 107–6.25 × 108) for P. aeruginosa. Scanning electron microscopy showed bacterial attachment to the graft pieces. There was a time-dependent development of tolerance with approximately 20 (tobramycin), 560 (benzylpenicillin), and 600 (ciprofloxacin) times more S. aureus surviving antibiotic exposure in 24- compared with 0-hour-old biofilm. Five (tobramycin) and 170 times (ciprofloxacin) more P. aeruginosa survived antibiotic exposure in 24- compared with 0-hour-old biofilms. DiscussionOur results show that bacteria can rapidly adhere to and subsequently form antibiotic-tolerant biofilms on EVAR graft material in concentrations equivalent to levels seen in transient bacteraemia in vivo. Potentially, the system can be used for identifying optimal treatment combinations for infected EVAR prosthesis.

Escherichia coli O157:H7 had a high degree of acid resistance in the presence of osmolytes (glycerol, glycine or fructose) by altering its lipid membrane composition

This study aims to investigate the resistance of E. coli O157:H7 to acetic acid (AA) or malic acid (MA) by adding osmolytes, such as glycerol, glycine, glucose, and fructose, in Luria-Bertani broth without NaCl (LBW/S) or phosphate buffer (PB) stored at 25 °C. In LBW/S, a significantly (p < 0.05) higher D-value of E. coli O157:H7 was observed when treated with AA and 20% glycine (D-value: 1.18–3.44) or 40% glucose (D-value: 1.05–2.52) compared to that of AA alone (D-value: 0.40–0.47). In contrast, the addition of osmolytes (i.e. 3-40% glucose, 3–40% fructose or 20% glycine) to LBW/S acidified by MA significantly decreased D-values of E. coli O157:H7, which was enumerated by using a selective medium. Furthermore, when E. coli O157:H7 was incubated in LBW/S containing AA and osmolytes at 25 °C for 3 d, this bacterium had an increased proportion of C16:0 and C17:0 cyclo (cyclopropane acid) compared to its AA-treated counterparts. Along with the altered shift in membrane phospholipids, the addition of osmolytes into a laboratory medium in the presence of nutritive substrates may increase the resistance of E. coli O157:H7 to AA.

Enhanced antibacterial activity of dimethyl gallium quinolinolates toward drug-resistant Klebsiella pneumoniae in low iron environments

A series of dimethylgallium quinolinolate [GaMe2L] (L = 5-chloroquinolinolate, 5, 7-dichloroquinolinolate, 5, 7-dibromoquinolinolate or 5, 7-doiodoquinolinolate) complexes, shown previously to be active toward the Leishmania parasite, have been studied for their antibacterial activity toward a reference and drug resistant strain of Klebsiella pneumoniae (KP). The assays were conducted in standard iron-rich LB media and in the iron depleted RPMI and RPMI-HS media to better understand the effect of Fe concentration on the activity of the Ga complexes. In LB broth the parent quinolinols and the gallium complexes were inactive up to the highest concentration tested, 100 μM. In the more physiologically relevant ‘iron-poor’ RPMI-HS media the quinolonols remained inactive, however, the gallium complexes showed exceptional activity in the range 48–195 nM. Only in RPMI without any added HS did both the quinolinols and the gallium complexes show good activity. The significant differences in activity across the various media types suggest that the unnaturally high iron content of conventional LB media may provide false negative results for potentially potent Ga therapeutics. A protein binding assay on the organometallic gallium complexes showed a much slower uptake of Ga by Fe-binding proteins than is typically observed for gallium salts. This indicates that their greater lipophilicity and greater hydrolytic stability could account for their increased biological activity in RPMI-HS media.

Impact of Phylogenetically Diverse Bacterial Endophytes of Bergenia pacumbis on Bergenin Production in the Plant Cell Suspension Cultures.

Plant in vitro cultures are potential sources for secondary metabolites. However, low productivity is often a major drawback for industrial application. Elicitation is an important strategy to improve product formation in vitro. In this context, endophytes are of special interest as biotic elicitors due to their possible interaction with the metabolism of the host plant. A total of 128 bacterial endophytes were isolated from the medicinal plant Bergenia pacumbis and taxonomically classified using 16S rRNA gene sequencing. Five strains belonging to different genera were grown in lysogeny broth and tryptic soy broth medium and cells as well as spent media were used as elicitors in cell suspension cultures of B. pacumbis. Production of the main bioactive compound bergenin was enhanced 3-fold (964 µg/g) after treatment with cells of Moraxella sp. or spent tryptic soy broth medium of Micrococcus sp. These results indicate that elicitation of plant cell suspension cultures with endophytic bacteria is a promising strategy for enhancing the production of desired plant metabolites.

First report of Arthrobacter woluwensis causing brown blotch disease on white button mushroom (Agaricus bisporus).

Agaricus bisporus is one of the most commonly grown edible fungi in the world. In December 2021, brown blotch disease (2% incidence) was observed on the cap of A. bisporus, growing in a mushroom cultivation base in Guangxi, China. Initially, brown blotches (1-1.3 cm) appeared on the cap of A. bisporus, which expanded gradually as the cap grew. After two days, the infection penetrated inner tissues of fruiting bodies, and blotches were dark brown. For the isolation of causative agent(s), internal tissue samples of the infected stipes (5×5×5 mm) were sterilized in 75% ethanol for 30 s, rinsed three times with sterile deionized water (SDW), then, mashed in the sterile 2 ml Eppendorf tubes, 1000 µl SDW was added and the suspension was diluted into seven concentrations (10-1~10-7). Each suspension (120 µl) was spread on Luria Bertani (LB) medium and incubated for 24 hours at 28 °C. Morphological examination of the isolates was referred to Liu et al (2022). The dominant single colonies were whitish-grayish, smooth, convex. The cells were Gram-positive, non-flagellated, nonmotile, no pods or endospores formed, and no fluorescent pigments production on King's B medium (Solarbio). Amplified 16S rRNA (1351 bp; OP740790) of five colonies using universal primers 27f/1492r (Liu et al., 2022), exhibited 99.26% identity with Arthrobacter (Ar.) woluwensis. The partial sequences of the ATP synthase subunit beta gene (atpD) (677 bp; OQ262957), RNA polymerase subunit beta gene (rpoB) (848 bp; OQ262958), preprotein translocase subunit SecY gene (secY) (859 bp; OQ262959) and elongation factor Tu gene (tuf) (831 bp; OQ262960) genes of colonies were amplified using the method of Liu et al (2018), also exhibited more than 99% similarities to Ar. woluwensis. The biochemical tests for isolates (n=3) were performed via bacterial micro-biochemical reaction tubes (Hangzhou Microbial Reagent Co., LTD), and the results showed the same biochemical characteristics as Ar. woluwensis (Positive for esculin hydrolysis, urea, gelatinase, catalase, sorbitol, gluconate, salicin and arginine. Negative for citrate, nitrate reduction and rhamnose) (Funke et al., 1996). The isolates were identified as Ar. woluwensis based on morphological characteristics, biochemical tests and phylogenetic analysis. Pathogenicity tests were performed with bacterial suspensions (1 × 109 CFU/ml) after growing for 36 h in LB Broth at 28 °C, 160 rpm. 30 µl bacterial suspension was added to the cap and tissue of young A. bisporus. SDW was added as a negative control. All treatments were incubated at 20 °C and 80-85% humidity. The experiment was repeated three times with five caps and five tissues of young A. bisporus each time. Brown blotches were observed on all the parts of the inoculated caps and tissues after 24 h of inoculation. At 48 h, the inoculated caps turned dark brown while the infected tissues changed from brown to black and expanded to the entire tissue block giving a severely rotten appearance and foul odor. This disease symptoms were similar to those observed in the original samples. There were no lesions in the control group. After the pathogenicity test, the pathogen was re-isolated from the infected caps and tissues based on morphological characteristics, 16S rRNA sequences, and biochemical results, fulfilling Koch's postulates. Arthrobacter spp. are very widely distributed in the environment (Kim et al., 2008). To date, two studies have confirmed Arthrobacter spp. as a pathogen of edible fungi (Bessette, 1984; Wang et al., 2019). However, this is the first report of Ar. woluwensis causing brown blotch disease on A. bisporus. Our finding could contribute to developing phytosanitary and control treatments for this disease.

Lysogeny Broth (LB, Lennox Formulation; 1-L)

Lysogeny Broth (LB, Lennox Formulation; 1-L)

Production of Astaxanthin Using CBFD1/HFBD1 from Adonis aestivalis and the Isopentenol Utilization Pathway in Escherichia coli.

Astaxanthin is a powerful antioxidant and is used extensively as an animal feed additive and nutraceutical product. Here, we report the use of the β-carotene hydroxylase (CBFD1) and the β-carotene ketolase (HBFD1) from Adonis aestivalis, a flowering plant, to produce astaxanthin in E. coli equipped with the P. agglomerans β-carotene pathway and an over-expressed 4-methylerythritol-phosphate (MEP) pathway or the isopentenol utilization pathway (IUP). Introduction of the over-expressed MEP pathway and the IUP resulted in a 3.2-fold higher carotenoid content in LB media at 36 h post-induction compared to the strain containing only the endogenous MEP. However, in M9 minimal media, the IUP pathway dramatically outperformed the over-expressed MEP pathway with an 11-fold increase in total carotenoids produced. The final construct split the large operon into two smaller operons, both with a T7 promoter. This resulted in slightly lower productivity (70.0 ± 8.1 µg/g·h vs. 53.5 ± 3.8 µg/g·h) compared to the original constructs but resulted in the highest proportion of astaxanthin in the extracted carotenoids (73.5 ± 0.2%).

Screening for extended-spectrum beta-lactamaseEscherichia coli in recreational beach waters in Singapore.

Aims: As part of Singapore's One Health antimicrobial resistance (AMR) management, this work was designedto understand the AMR burden in recreational beach waters using extended-spectrum beta-lactamase Escherichia coli (ESBL-EC) as an indicator. Materials & methods: A total of 90 water samples were collected from six different recreational beaches over three different time periods. Only 28/90 (31.3%) water samples yielded E. coli colonies ranging from 1 to 80colony-forming units/100ml. Results & conclusion: Screening of all colonies using CHROMID® ESBL agar and Luria-Bertani broth supplemented with ceftriaxone showed that none was ESBL-EC. Further monitoring is required to understand the prevalence of ESBL-EC spatiotemporally, contributing to the national AMR surveillance program and providing timely risk assessment for exposure to ESBL-EC.

Pseudomonas aeruginosa SWUC02 Cell-Free Culture as a Potential Antimicrobial Agent Against Household Antibiotics-Resistant Staphylococcus aureus

Contamination of household environments with pathogenic bacteria poses a significant risk of foodborne illnesses. This study aimed to investigate the effectiveness of cell-free culture obtained from Pseudomonas aeruginosa SWUC02 (CF-SWUC02) against Staphylococcus aureus, a common pathogen associated with food poisoning outbreaks. The antagonistic activity of P. aeruginosa SWUC02 and CF-SWUC02 against various pathogenic bacteria, particularly S. aureus, was assessed. The active antimicrobial compounds produced by P. aeruginosa SWUC02 demonstrated resistance to protease enzymes and high temperatures. Optimal culture conditions for inhibiting S. aureus were determined as LB media supplemented with 0.01% CuCl2, inoculated with 1x105 CFU.mL-1 of P. aeruginosa SWUC02, and incubated at 32°C with agitation (100 rpm) for 12 days. Eighty-six S. aureus isolates were collected from household environments and tested for antibiotic resistance, with 55 isolates exhibiting resistance to penicillin, and 17 isolates were identified as methicillin-resistant S. aureus (MRSA). CF-SWUC02 demonstrated inhibitory effects against all S. aureus isolates, including MRSA. In conclusion, CF-SWUC02 displayed antagonistic activity against drug resistant S. aureus, suggesting its potential as a valuable resource for combating these pathogens. Furthermore, the presence of heat and protease stable antimicrobial compounds in CF-SWUC02 highlights the need for further investigation to explore their potential applications in the field of antimicrobial research.

Unraveling the potential and constraints associated with corn steep liquor as a nutrient source for industrial fermentations.

Costly complex media components such as yeast extract and peptone are still widely used in industrial bioprocesses, despite their ill-defined composition. Side stream products such as corn steep liquor (CSL) present a compelling economical alternative that contains valuable nutrients required for microbial growth, that is, nitrogen and amino acids, but also vitamins, trace elements, and other minerals. However, as a side stream product, CSL may be subject to batch-to-batch variations and compositional heterogeneity. In this study, the Respiration Activity MOnitoring System designed for shake flasks (RAMOS) and 96-well microtiter plates (μTOM) were applied to investigate the potential and constraints of CSL utilization for two model microorganisms: E. coli and B. subtilis. Considering the dry substance content of complex nutrients involved, CSL-based media are more efficient in biomass production than the common lysogeny broth (LB) medium, containing 5 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl. At a glucose to CSL (glucose/CSL, g/g) ratio of 1/1 (g/g) and 2/1 (g/g), a secondary substrate limitation occurred in E. coli and B. subtilis cultivations, respectively. The study sheds light on differences in the metabolic activity of the two applied model organisms between varying CSL batches, which relate to CSL origin and production process, as well as the effect of targeted nutrient supplementation. Through a targeted nutrient supplementation, the most limiting component of the CSL-glucose medium used for these applied model microorganisms was identified to be ammonium nitrogen. This study proves the suitability of CSL as an alternative nutrient source for E. coli and B. subtilis. The RAMOS and μTOM technique detected differences between CSL batches, allowing easy and early identification of varying batches. A consistent performance of the CSL batches in E. coli and B. subtilis cultivations was demonstrated.

The Metabolomics Changes in Luria-Bertani Broth Medium under Different Sterilization Methods and Their Effects on Bacillus Growth.

Luria-Bertani broth (LB) culture medium is a commonly used bacterial culture medium in the laboratory. The nutrient composition, concentration, and culture conditions of LB medium can influence the growth of microbial strains. The purpose of this article is to demonstrate the impact of LB liquid culture medium on microbial growth under different sterilization conditions. In this study, LB medium with four different treatments was used, as follows: A, LB medium without treatments; B, LB medium with filtration; C, LB medium with autoclaving; and D, LB medium with autoclaving and cultured for 12 h. Subsequently, the protein levels and antioxidant capacity of the medium with different treatments were measured, and the effects of the different LB medium treatments on the growth of microorganisms and metabolites were determined via 16s rRNA gene sequencing and metabolomics analysis, respectively. Firmicutes and Lactobacillus were the dominant microorganisms, which were enriched in fermentation and chemoheterotrophy. The protein levels and antioxidant capacity of the LB medium with different treatments were different, and with the increasing concentration of medium, the protein levels were gradually increased, while the antioxidant capacity was decreased firstly and then increased. The growth trend of Bacillus subtilis, Bacillus paralicheniformis, Micrococcus luteus, and Alternaria alternata in the medium with different treatments was similar. Additionally, 220 and 114 differential metabolites were found between B and C medium, and between C and D medium, which were significantly enriched in the "Hedgehog signaling pathway", "biosynthesis of plant secondary metabolites", "ABC transporters", "arginine and proline metabolism", and "linoleic acid metabolism". LB medium may be a good energy source for Lactobacillus growth with unsterilized medium, and LB medium filtered with a 0.22 μm filter membrane may be used for bacterial culture better than culture medium after high-pressure sterilization. LB medium still has the ability for antioxidation and to keep bacteria growth whether or not autoclaved, indicating that there are some substances that can resist a high temperature and pressure and still maintain their functions.

Expression of Cholera Toxin (CT) and the Toxin Co-Regulated Pilus (TCP) by Variants of ToxT in Vibrio cholerae Strains.

The expression of the two major virulence genes of Vibrio cholerae-tcpA (the major subunit of the toxin co-regulated pilus) and ctxAB (cholera toxin)-is regulated by the ToxR regulon, which is triggered by environmental stimuli during infection within the human small intestine. Special culture methods are required to induce the expression of virulence genes in V. cholerae in the laboratory setting. In the present study, induction of the expression of virulence genes by two point mutations (65th and 139th amino acids) in toxT, which is produced by the ToxR regulon and activates the transcription of the virulence genes in V. cholerae, under laboratory culture conditions has been investigated. Each of the four toxT alleles assessed displayed different transcriptional activator functions in a given V. cholerae strain. Although the ToxR regulon has been known to not be expressed by El Tor biotype V. cholerae strains cultured under standard laboratory conditions, the variant toxT alleles that we assessed in this study enabled the expression virulence genes in El Tor biotype strains grown under simple culture conditions comprising shake culture in LB medium, suggesting that the regulation of virulence gene expression may be regulated more complexly than previously thought and may involve additional factors beyond the production of ToxT by the ToxR regulon.