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.

Resistance-Nodulation-Cell Division (RND) Transporter AcrD Confers Resistance to Egg White in Salmonella enterica Serovar Enteritidis

The excellent survival ability of Salmonella enterica serovar Enteritidis (S. Enteritidis) in egg white leads to outbreaks of salmonellosis frequently associated with eggs and egg products. Our previous proteomic study showed that the expression of multidrug efflux RND transporter AcrD in S. Enteritidis was significantly up-regulated (4.06-fold) in response to an egg white environment. In this study, the potential role of AcrD in the resistance of S. Enteritidis to egg white was explored by gene deletion, survival ability test, morphological observation, Caco-2 cell adhesion and invasion. It was found that deletion of acrD had no apparent effect on the growth of S. Enteritidis in Luria-Bertani (LB) broth but resulted in a significant (p < 0.05) decrease in resistance of S. Enteritidis to egg white and a small number of cell lysis. Compared to the wild type, a 2-log population reduction was noticed in the ΔacrD mutant with different initial concentrations after incubation with egg white for 3 days. Furthermore, no significant difference (p > 0.05) in the adhesion and invasion was found between the wild type and ΔacrD mutant in LB broth and egg white, but the invasion ability of the ΔacrD mutant in egg white was significantly (p < 0.05) lower than that in LB broth. This indicates that acrD is involved in virulence in Salmonella. Taken together, these results reveal the importance of AcrD on the resistance of S. Enteritidis to egg white.

Jeotgalibacillus Auranticolor sp. nov., Isolated from Freshwater Collected from Baiyangdian Lake Lake, China

A novel Gram-positive, strictly aerobic, rod-shaped, orange-pigmented bacterial strain, designated R-1-5s-1T, was isolated from Baiyangdian Lake, China. Strain R-1-5s-1T grew at 15-37℃ (optimum 37℃) and pH 7-11 (optimum pH 8) in Luria-Bertani medium. Based on 16S rRNA gene sequence analysis, strain R-1-5s-1T was assigned to the genus Jeotgalibacillus and showed the closest relationships with Jeotgalibacillus salarius ASL-1T (97.69%), Jeotgalibacillus alkaliphilus JC303T (97.29%), Jeotgalibacillus marinus DSM 1297T (97.15%), Jeotgalibacillus campisalis SF-57T (97.01%), and Jeotgalibacillus spp. (≤ 97%). The predominant polar lipids were phosphatidylglycerol and diphosphatidylglycerol; the major cellular fatty acids were iso-C15:0, anteiso-C15:0, iso-C17:0, and anteiso-C17:0; and the major respiratory quinones were MK-7 and MK-8. The peptidoglycan type of the cell wall was A1a linked via L-lysine as the diamino acid. The G+C content was 43.6%, and the draft genome size of strain R-1-5s-1T was 3.4 Mbp. Between strain R-1-5s-1T and the related strain J. salarius ASL-1T, the ANI and dDDH relatedness values were 78.9% and 20.8%, respectively. Phylogenetic, chemotaxonomic, and genotypic analyses revealed that strain R-1-5s-1T is a novel species in the genus Jeotgalibacillus, for which the name Jeotgalibacillus auranticolor sp. nov. is proposed. The type strain is R-1-5s-1T (=CGMCC 1.13567T=KCTC 43038T).

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.

PRODUÇÃO DE PRODIGIOSINA POR SERRATIA MARCESCENS UCP 1549 UTILIZANDO FARELO DE MILHO COMO FONTE SUSTENTÁVEL

Os corantes microbianos têm sido bastante utilizados nas últimas décadas, devido a elevada biodiversidade microbiana existente. Serratia marcescens é uma bactéria que tem despertado interesse comercial e industrial devido ao elevado potencial de produção de um pigmento vermelho, conhecido como prodigiosina. Este pigmento é um metabolito secundário caracterizado como um alcalóide tripirrol que possui ações antimicrobiana e anticancerígena. O objetivo deste trabalho foi produzir prodigiosina por S. marcescens UCP 1549 a partir da metabolização de farelo de milho como substrato sustentável. Para isso, S. marcescens foi crescida em meio Luria Bertani e posteriormente, células jovens foram transferidas para o meio de produção constituído por farelo de milho (1%) e base de sais. A influência do volume do meio de produção no rendimento de biomassa e prodigiosina foi investigada através do escalonamento da fermentação em frascos de 500, 1000 e 1500 mL. A biomassa produzida foi quantificada através de gravimetria, enquanto que a extração do pigmento presente na biomassa foi realizado utilizando um sistema de solventes clorofórmio: metanol, sendo o rendimento do pigmento expresso em mg.g de biomassa. A identificação do pigmento vermelho produzido foi realizada através de espectrofotometria e cromatografia em camada delgada. O pigmento extraído foi aplicado na coloração de sabonetes. De acordo com os resultados obtidos, a máxima produção de biomassa (9,52 g/L) ocorreu no meio de produção contendo 1500 mL. Por outro lado, o máximo rendimento do pigmento vermelho bruto (30,97 mg.g de biomassa) ocorreu no meio de produção com volume de 500 mL. O valor do fator de retenção (Rf) do pigmento vermelho foi de0.9 com um pico máximo de absorbância foi detectado no comprimento de onda de 535 nm, confirmando assim a produção de prodigiosina por S. marcescens. A prodigiosina foi utilizada eficientemente como corante de sabonete em barra, o que sugere o seu potencial de uso na indústria de cosméticos.

Carotenoid Cocktail Produced by An Antarctic Soil Flavobacterium with Biotechnological Potential

Carotenoids are highly important in pigmentation, and its content in farmed crustaceans and fish correlates to their market value. These pigments also have a nutritional role in aquaculture where they are routinely added as a marine animal food supplement to ensure fish development and health. However, there is little information about carotenoids obtained from Antarctic bacteria and its use for pigmentation improvement and flesh quality in aquaculture. This study identified carotenoids produced by Antarctic soil bacteria. The pigmented strain (CN7) was isolated on modified Luria–Bertani (LB) media and incubated at 4 °C. This Gram-negative bacillus was identified by 16S rRNA analysis as Flavobacterium segetis. Pigment extract characterization was performed through high-performance liquid chromatography (HPLC) and identification with liquid chromatography–mass spectrometry (LC–MS). HPLC analyses revealed that this bacterium produces several pigments in the carotenoid absorption range (six peaks). LC–MS confirms the presence of one main peak corresponding to lutein or zeaxanthin (an isomer of lutein) and several other carotenoid pigments and intermediaries in a lower quantity. Therefore, we propose CN7 strain as an alternative model to produce beneficial carotenoid pigments with potential nutritional applications in aquaculture.

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.

Heat shock transformation and expression of the plasmid containing cytolethal distending toxin of Campylobacter fetus subsp venerealis in Escherichia coli

The cytolethal distending toxins (cdt) is a multi-subunit toxin consisted of three subunit encoded cdtA, cdtB and cdtC. The cdt played an important role as a virulence factor of Campylobacter infection, including C. fetus subsp venerealis. The cdtA which responsible for binding the cdt to cell membrane, was cloned in plasmid expression and inserted into bacterial cells of Escherichia coli BL21(DE3). The research was conducted to evaluate the transformation using the heat shock method of a plasmid containing cdtA3 gene and the protein expression induced by various concentration of IPTG. Transformation was done using the heat shock method at 42oC for 90 second. Evaluation of the transformation was observed on the presence of E. coli BL21(DE3) colonies on Luria Bertani agar containing Ampicillin antibiotic with 100 µg/mL dosage. The recombinant protein was expressed using IPTG-induction with various concentration (0.1mM, 0.25mM, 0.5mM, 0.75mM and 1 mM). The result showed that the transformation and IPTG-induction 0.1 mM produced higher concentration of protein than other concentration applied. The protein characterization was observed with SDS PAGE and cdtA3 protein was detected on 23,4 kDa.