Isolation of Shewanella putrefaciens GRD 03 from Fish and Explication of Biofilm Adherence Potency on Different Substrates

Foodborne pathogens are the main threat and cause of food poisoning. The majority of food infections have been related to the biofilm formation of foodborne pathogens in the food industry. Shewanella putrefaciens (KX355803, GRD 03), a Gram-negative pathogen isolated from mackerel fish, was identified and recognized as a food spoilage bacterium and a strong biofilm producer. The adhesion or attachment ability of Shewanella putrefaciens was determined on steel, plastic, glass, PVC and wood. NB (Nutrient broth), LB (Luria-Bertani broth), TSB (Tryptic soy broth) and BHI (Brain heart infusion broth) were enriched with glucose and shows optimum for bacterial adhesion. In the microtiter plate method (MTP), the strong attachment was observed at 48 and 72 hours of incubation and significant differences were obtained at p < 0.05. As the incubation period increases, the OD value (Optical density) of samples also increase. Biofilm formation is the major cause cross-contamination, and shows resistance to certain disinfectants, which leads to environmental stress tolerance. This study suggested with optimum biofilm production of isolate from fish by using glucose enriched media on different substrates, also comparing different growth media provide a detailed idea about biofilm-forming ability at different incubation time intervals.

Antimicrobial Activity and Action Mechanism of Thymoquinone against Bacillus cereus and Its Spores

In this study, thymoquinone (TQ), a natural active substance, was investigated for its antibacterial activity against Bacillus cereus, and its inhibitory effect on B. cereus in reconstituted infant formula (RIF) was evaluated. In addition, the inhibitory effect of TQ on B. cereus spore germination was explored. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of TQ against eight B. cereus strains ranged from 4.0 to 8.0 μg/mL, whereas B. cereus treated with TQ displayed a longer lag phase than the untreated control. TQ exerted a good bactericidal effect on B. cereus in Luria–Bertani broth. In addition, TQ obviously reduced the intracellular ATP concentration of B. cereus, which caused depolarization of the cell membrane, increased the intracellular reactive oxygen species level, impaired the cell morphology, and destroyed proteins or inhibited proteins synthesis. This provides a mechanism for its bacteriostatic effect. TQ also inactivated B. cereus growth in RIF. Moreover, reverse transcription–quantitative polymerase chain reaction illustrated that TQ downregulated the transcription of genes related to hemolysin, non-hemolytic enterotoxin, enterotoxin, and cytotoxin K. Meanwhile, TQ displayed the ability to inhibit the germination of B. cereus spores. These findings indicate that TQ, as an effective natural antimicrobial preservative, has potential applications in controlling food contamination and foodborne diseases caused by B. cereus.

A highly efficient auxin-producing bacterial strain and its effect on plant growth

Background Various bacteria promote plant root growth in the rhizosphere, as a measure of securing and enlarging their ecological niche. These interactions are mediated by plant growth regulators (PGRs) such as auxin, and indole-3-acetic acid (IAA) is one of the physiologically active auxin. In this study, we isolated an unusual bacterial strain from food process waste with high efficiency and demonstrated its effects on plant rooting and early-stage growth. Results The efficiency of this bacterial strain in producing IAA was 16.6 mg/L/h in Luria-Bertani broth containing 0.05% l-tryptophan (Trp) at room temperature (24 ± 2 °C). Its IAA production was highly dependent on the presence of precursor, Trp. This bacterium was identified as Ignatzschineria sp. by 16S rDNA sequencing. Its bacterial culture supernatant (BCS) enhanced plant root initiation, root growth, and plant growth in the early stages. The root mass formed BCS-treated in apple mint cuttings was twofold of that formed in the control. The root number and length were 46% and 18% higher, respectively, in BCS-treated chrysanthemum cuttings than in the control. Conclusions These results show that the BCS of Ignatzschineria sp. CG20001 isolate obtained in this study can be used for agricultural applications. In addition, the novelty of this strain makes it a valuable genetic resource for biotechnological applications.

Pembentukan Biofilm Pseudomonas aeruginosa pada Beberapa Media Cair

Pseudomonas aeruginosa merupakan bakteri Gram negatif berbentuk batang bersifat patogen oportunistik yang menjadi penyebab utama infeksi nosokomial dan mampu membentuk biofilm pada media pertumbuhan, biofilm sering mengakibatkan pengobatan penyakit infeksi menjadi lebih sulit. Media pertumbuhan bakteri ada beberapa jenis, komposisi dan merek. Tujuan penelitian ini adalah untuk mengetahui kemampuan P. aeruginosa dalam membentuk biofilm pada beberapa media biakan cair. P. aeruginosa diisolasi dari sampel klinis dari rumah sakit, media cair yang digunakan adalah nutrien broth, laktosa broth, brain-heart infusion (BHI), luria bertani broth, dan tripticase soy broth. Uji pembentukan biofilm menggunakan metode microtiter plate culture technique, kemampuan pembetukan biofilm diukur berdasarakan optical density dengan menggunakan microtiter plate reader pada panjang gelombang 570nm, dengan pewarnaan crystal violet 0,1%, setelah inkubasi 24 jam pada suhu 37oC, dengan replikat 8 kali. Hasil penelitian menunjukkan bahwa P. aeruginosa memiliki kemampuan membentuk biofilm pada nutrient broth 0,926±0,081, laktosa broth 0,521±0,041, BHI 1,283±0,031, luria bertani 1,301±0,043, dan media trypticase soy broth 1,563±0,032. Pembentukan biofilm tertinggi pada trypticase soy broth, dan terendah pada laktosa broth, sedangkan pada media BHI dan luria bertani kemampuan pembentukan biofilm yang setara. Kesimpulan penelitian ini adalah P. aeruginosa memiliki kemampuan yang berbeda dalam membentuk biofilm ketika ditumbuhkan pada media cair yang berbeda.Kata kunci : Biofilm, Pseudomonas aeruginosa, media cair

Effect of cultivation media and temperature on metabolite profiles of three nematicidal Bacillus species

Summary Globally, root-knot nematode (RKN) infestations cause great financial losses. Although agrochemicals are used to manage these pests, there is increased interest in using biocontrol agents based on natural antagonistic microorganisms, such as Bacillus. These nematicidal bacteria demonstrate antagonism towards RKN through different modes of action, including specialised metabolite production. The aim of this study was to compare metabolite profiles of nematicidal Bacillus species and assess the influence of cultivation conditions on these profiles. Two hyphenated metabolomics platforms, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), were employed to profile and compare metabolite features produced during the cultivation of three nematicidal Bacillus species (Bacillus firmus, B. cereus and B. soli) in complex Luria-Bertani broth (LB) and a simpler minimal broth (MB), at three different temperatures (25, 30 and 37°C). Cultivation in complex LB as opposed to simpler MB resulted in the production of more statistically significant metabolite features. Selected temperatures in this study did not have a significant influence on metabolite profiles. Moreover, media-specific influences outweighed temperature-specific influences on metabolite profiles. Results from this study are a valuable first step in establishing suitable cultivation conditions for the production of Bacillus metabolites of interest.

Comparable Expressions of Bacillus species on Different Growth Media

An investigation was carried out using Pikovskaya Broth (PKB), Luria Bertani Broth (LBB), and Peptone Water (PW) to analyse growth expressions of constructed Bacillus subtilis sub sp and compared to a commercial Bacillus subtilis RIK 1285. The aim was to determine the effect of carbon, nitrogen and other elements at different variations on the metabolic activities under different conditions. The results obtained showed growth density of 4.1 g/ml at 70oC and 3.1 g/ml at pH 6.0; 3.3 g/ml at 70oC and 2.8 g/ml at pH 6.0; 3.8 g/ml at 60oC and 2.6 g/ml at pH 7.0 from PKB, LBB and PW respectively. The growth density of the commercial strain recorded 3.8 g/ml at 50oC and 2.8 g/ml at pH 7.0; 3.1 g/ml at 50oC and 2.3 g/ml; 3.0 at 50oC and 2.3 at pH respectively. The investigation showed importance and relevance of gene metabolic upgrade on the utilization of multiple nutrients present from one media to another. Keywords: media formulation, microbial reaction, growth promoters, growth density

A Rapid Method for Performing a Multivariate Optimization of Phage Production Using the RCCD Approach

Bacteriophages can be used in various applications, from the classical approach as substitutes for antibiotics (phage therapy) to new biotechnological uses, i.e., as a protein delivery vehicle, a diagnostic tool for specific strains of bacteria (phage typing), or environmental bioremediation. The demand for bacteriophage production increases daily, and studies that improve these production processes are necessary. This study evaluated the production of a T4-like bacteriophage vB_EcoM-UFV09 (an E. coli-infecting phage with high potential for reducing environmental biofilms) in seven types of culture media (Luria–Bertani broth and the M9 minimal medium with six different carbon sources) employing four cultivation variables (temperature, incubation time, agitation, and multiplicity of infection). For this purpose, the rotatable central composite design (RCCD) methodology was used, combining and comparing all parameters to determine the ideal conditions for starting to scale up the production process. We used the RCCD to set up the experimental design by combining the cultivation parameters in a specific and systematic way. Despite the high number of conditions evaluated, the results showed that when specific conditions were utilized, viral production was effective even when using a minimal medium, such as M9/glucose, which is less expensive and can significantly reduce costs during large-scale phage production.

Evolutionary dynamics of asexual hypermutators adapting to a novel environment

How microbes adapt to a novel environment is a central question in evolutionary biology. While adaptive evolution must be fueled by beneficial mutations, whether higher mutation rates facilitate the rate of adaptive evolution remains unclear. To address this question, we cultured Escherichia coli hypermutating populations, in which a defective methyl-directed mismatch repair pathway causes a 140-fold increase in single-nucleotide mutation rates. In parallel with wild-type E. coli, populations were cultured in tubes containing Luria-Bertani broth, a complex medium known to promote the evolution of subpopulation structure. After 900 days of evolution, in three transfer schemes with different population-size bottlenecks, hypermutators always exhibited similar levels of improved fitness as controls. Fluctuation tests revealed that the mutation rates of hypermutator lines converged evolutionarily on those of wild-type populations, which may have contributed to the absence of fitness differences. Further genome-sequence analysis revealed that, although hypermutator populations have higher rates of genomic evolution, this largely reflects the effects of genetic draft under strong linkage. Despite these linkage effects, the evolved populations exhibit parallelism in fixed mutations, including those potentially related to biofilm formation, transcription regulation, and mutation-rate evolution. Together, these results generally negate the presumed relationship between high mutation rates and high adaptive speed of evolution, providing insight into how clonal adaptation occurs in novel environments.

Insight into the Metabolic Profiles of Pb(II) Removing Microorganisms

The objective of the study was to gather insight into the metabolism of lead-removing microorganisms, coupled with Pb(II) removal, biomass viability and nitrate concentrations for Pb(II) bioremoval using an industrially obtained microbial consortium. The consortium used for study has proven to be highly effective at removing aqueous Pb(II) from solution. Anaerobic batch experiments were conducted with Luria-Bertani broth as rich growth medium over a period of 33 h, comparing a lower concentration of Pb(II) with a higher concentration at two different nutrient concentrations. Metabolite profiling and quantification were conducted with the aid of both liquid chromatography coupled with tandem mass spectroscopy (UPLC-HDMS) in a “non-targeted” fashion and high-performance liquid chromatography (HPLC) in a “targeted” fashion. Four main compounds were identified, and a metabolic study was conducted on each to establish their possible significance for Pb(II) bioremoval. The study investigates the first metabolic profile to date for Pb(II) bioremoval, which in turn can result in a clarified understanding for development on an industrial and microbial level.

Protease Production from Bacillus sp. Isolated from Gastrointestinal Tract of Catfish (Clarias sp.) with Different Medium

The need for protease in the industrial field has been increasing. Candidates for producing these enzymes can be isolated from the digestive tract of catfish (Clarias sp.). The purpose of this study was to obtain bacterial isolates that produce proteolytic from the gastrointestinal tract of catfish and determine the effect of different production media on the activity of proteolytic. The first step of this study was isolation, screening, and identification of bacteria. The second step was to test the effect of the media Luria Bertani, trypticase soy broth, and skim milk broth on proteolytic activity. Nineteen isolates were obtained from the screening process of proteolytic bacteria. Isolate no 1, was known as the best isolate in producing enzymes and was known as Bacillus sp. Tests with different growth media gave results that semi-quantitative, nutrient growth media produced the highest activity with a proteolytic index value of 2.09 ± 0.41. In addition, based on quantitative tests, the media Luria Bertani Broth produced the highest specific activity with a value of 36.479 U/mg. The conclusion of this study, Bacillus sp. from the gastrointestinal tract of catfish that cultured on the Luria Bertani Broth medium produced the best activity.