Luria-Bertani Research Articles

A Study of Naturally-occurring L-glutamic Acid Producing Bacteria From Tropical Soil and Aquatic Environments.

Microbial means of L-glutamic acid production haveadvantage of directly yielding optical and biological active form of the aminoacid. The production of L-glutamic acid was investigated using bacterialisolates obtained from tropical soil and water samples obtained from Lagos,Nigeria. L–glutamic acid producing bacterial strains were isolated andidentified using the Analytical Profile Index (API) Test 50CHB/20E kit. TheL-Glutamic acid production was tested on Luria Bertani Broth and determined byqualitative and quantitative approaches. Six (6) isolates were L-Glutamateproducers of which the best producing isolates were Paenibacillus alvei and Corynebacteriumglucuronolyticum – seminale giving a yield higher than 0.7 g/L. The best L-glutamic acid producing isolateswere further tested to determine the effects of a variety of carbon andnitrogen sources as well as varying concentration of biotin on the yield of theamino acid produced. Glucose gave yields of 1.19 g/L and 1.05 g/L of L-glutamicacid for Paenibacillus alvei and Corynebacterium glucuronolyticum–seminale respectively,while ammonium chloride and optimum biotin concentration at 2µg gave yields of1.15 g/L and 2.09 g/L as well as 1.59 g/L and 1.75 g/L respectively. Thesebacterial strains therefore offer tremendous promise as candidates for furthermanipulation for enhanced production of L-glutamic acid using indigenousbacteria from the tropical environment

Persistence of Salmonella Typhimurium LT2 in Soil Enhanced after Growth in Lettuce Medium.

The persistence of Salmonella in the environment is influenced by a multitude of biotic and abiotic factors. In addition, its persistence can be influenced by preadaptation before the introduction into the environment. In order to study how preadaptation changes the survival of Salmonella in soil and therefore its potential to colonize the phytosphere, we developed a new medium based on lettuce material [lettuce medium (LM)]. Salmonella enterica serovar Typhimurium strain LT2 was used as a model for Salmonella in this study. LT2 was inoculated into soil microcosms after pregrowth in Luria Bertani (LB) broth or in LM. Survival of LT2 in soil was monitored over 56 days by plate counts and quantification of the Typhimurium-specific gene STM4497 using qPCR in total community DNA for which primers and TaqMan probe were designed in this study. Significantly enhanced persistence was observed for LT2 pregrown in LM compared to LT2 pregrown in LB, indicating a preadaptation effect. Surprisingly, no improved survival could be observed for S. Typhimurium strain 14028s and S. enterica serovar Senftenberg after pregrowth on LM. This indicates a high strain specificity of preadaptation. Results from previous studies suggested that biofilm formation could enhance the survival of human pathogens in various environments and might contribute to enhanced survival on plants. In vitro biofilm assays with several Salmonella strains revealed a strain-specific effect of LM on the biofilm formation. While LM significantly improved the biofilm formation of S. Senftenberg, the biofilm formation of LT2 was better in LB. This indicates that the better survival of LM-pregrown LT2 in soil was not linked to an improved ability to form biofilms but was likely due to other factors. Most importantly, this study showed that the medium used to pregrow Salmonella can influence its survival in soil and its biofilm formation which might influence the fate of Salmonella in soil.

MOLECULAR EXPRESSION OF WINGLESS-TYPE MMTV INTEGRATION SITE FAMILY MEMBER 4 GENE USING Escherichia coli BL21

This research was conducted to find out the Wnt4 recombinant proteins which expressed by Escherichia coli (E. coli) BL21 carrying the recombinant DNA wnt4 (E. coli transformation). Research materials were E. coli BL21 transformation and E. coli BL21 non-transformation (negative control). The expression of recombinant protein was conducted by culturing E. coli for 24 hours in Luria-Bertani (LB) media with isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Recombinant protein was isolated by sonication of pellet bacteria. Protein analysis performed by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that recombinant protein with a molecular weight of 33 kDa has been expressed by E. coli BL21 transformation successfully.

Heterologous expression and biochemical characterization of assimilatory nitrate and nitrite reductase reveals adaption and potential of Bacillus megaterium NCT-2 in secondary salinization soil

Large accumulation of nitrate in soil has resulted in “salt stress” and soil secondary salinization. Bacillus megaterium NCT-2 which was isolated from secondary salinization soil showed high capability of nitrate reduction. The genes encoding assimilatory nitrate and nitrite reductase from NCT-2 were cloned and over-expressed in Escherichia coli. The optimum co-expression condition was obtained with E. coli BL21 (DE3) and 0.1mM IPTG for 10h when expression was carried out at 20°C and 120rpm in Luria-Bertani (LB) medium. The molecular mass of nitrate reductase was 87.3kDa and 80.5kDa for electron transfer and catalytic subunit, respectively. The large and small subunit of nitrite reductase was 88kDa and 11.7kDa, respectively. The purified recombinant enzymes showed broad activity range of temperature and pH. The maximum activities were obtained at 35°C and 30°C, pH 6.2 and 6.5, which was similar to the condition of greenhouse soils. Maximum stimulation of the enzymes occurred with addition of Fe3+, while Cu2+ caused the maximum inhibition. The optimum electron donor was MV+Na2S2O4+EDTA and MV+Na2S2O4, respectively. Kinetic parameters of Km and Vmax were determined to be 670μM and 58U/mg for nitrate reductase, and 3100μM and 5.2U/mg for nitrite reductase. Results of quantitative real-time PCR showed that the maximum expression levels of nitrate and nitrite reductase were obtained at 50mM nitrate for 8h and 12h, respectively. These results provided information on novel assimilatory nitrate and nitrite reductase and their properties presumably revealed adaption of B. megaterium NCT-2 to secondary salinization condition. This study also shed light on the role played by the nitrate assimilatory pathway in B. megaterium NCT-2.

In vitro transfer of methicillin resistance determinants mecA from methicillin resistant Staphylococcus aureus (MRSA) to methicillin susceptible Staphylococcus aureus (MSSA)

BackgroundStaphylococcus aureus more than any other human pathogen is a better model for the study of the adaptive evolution of bacterial resistance to antibiotics, as it has demonstrated a remarkable ability in its response to new antibiotics. This study was designed to investigate the in vitro transfer of mecA gene from methicillin resistant S. aureus to methicillin susceptible S. aureus.ResultThe recipient transconjugants were resistant to erythromycin, cefpodoxime and were mecA positive. PCR amplification of mecA after mix culture plating on Luria Bertani agar containing 100 μg/mL showed that 75% of the donor and 58.3% of the recipient transconjugants were mecA positive. Additionally, 61.5% of both the donor cells and recipient transconjugants were mecA positive, while 46.2% and 41.75% of both donor and recipient transconjugants were mecA positive on LB agar containing 50 μg/mL and 30 μg/mL respectively.ConclusionIn this study, the direction of transfer of phenotypic resistance as well as mecA was observed to have occurred from the donor to the recipient strains. This study affirmed the importance of horizontal transfer events in the dissemination of antibiotics resistance among different strains of MRSA.

PRODUÇÃO DE BIOSSURFACTANTES POR CEPAS BACTERIANAS DE ORIGEM MARINHA UTILIZANDO QUEROSENE COMO FONTE DE CARBONO

Este trabalho teve como objetivo avaliar a capacidade de bactérias marinhas, isoladas de região portuária de Fortaleza, Ceará, em produzir compostos biossurfactantes utilizando querosene comercial como fonte de carbono. Foram utilizadas 22 cepas bacterianas, sendo 14 provenientes de amostras de água e 8 de sedimento. Os microrganismos foram crescidos por um período de 72 horas em meio de cultura Luria Bertani (LB) tendo como única fonte de carbono o querosene na concentração de 2%. Com o sobrenandante (líquido metabólico - LM) das culturas bacterianas foram realizados os seguintes testes de avaliação da produção de biossurfactantes: colapso da gota, atividade de emulsificação, capacidade de formação e estabilidade de emulsão. Foi avaliada a capacidade dos isolados em produzir hemólise em meio Ágar Sangue, indicativo da produção de biossurfacatntes. Do total de isolados, 36% obtiveram forte atividade no teste do colapso da gota, 50% produziram boas emulsões (acima de 40%), apresentando boa estabilidade até 168 horas de observação e 63% dos isolados produziram halos de hemólise. As cepas obtiveram bons resultados nos testes realizados, indicando um potencial em degradar querosene comercial, utilizando-o como fonte de carbono e produzindo compostos biossurfactantes/bioemulsificantes.

First report of Erwinia psidii associated with papaya dieback disease in Malaysia

Aims: The outbreak of papaya dieback disease in Malaysia has been reported since 2003. Several reports previously confirmed Erwinia papayae and E. mallotivora to be the causal pathogen of the disease. The present study aimed to identify the causal pathogen of papaya dieback disease in Sabah. Methodology and results: Infected tissues of papaya dieback disease were collected from Kota Belud, Sabah and the bacterium responsible for the infection was isolated on Luria Bertani (LB) agar and nutrient agar (NA). Seven isolates with similar characteristics to Erwinia were isolated, subjected to the Koch’s Postulates test and then identified using 16S rRNA sequencing technique. The bacterium was identified to be E. psidii, a common pathogen to guava but not to papaya. Conclusion, Significance and Impact of study: This report serves as the first confirmation of the E. psidii in causing papaya dieback disease, suggesting the possibility of this bacterium undergoing host shifting to papaya plants and the possibility of becoming another major threat to the papaya industry in the future.

Antimicrobial activity of Micrococcus luteus Cartenoid pigment

Cartenoids are group of pigments ,with enormous types different structurally and functionally, have colors range from red to yellow found in a wide variety of plants, fungi, algae and bacteria. The animals took from food because they cannot made it, on contrary, the plants and microbes produce them due to subjection to environment. The aim of the study is to isolate and characterize the cartenoid pigment from Micrococcus luteus. The pigment extraction was done by acetone, then was characterized with Ultra Violet –Visible spectroscopy (UV–Vis) and Fourier Transform Infrared (FTIR) spectroscopy. Then, it was tested for antibacterial activity against five different bacterial isolates and antifungal activity tests against six different fungal isolates by well diffusion method. The results found that, the extracted pigment having antibacterial activity and antifungal activity and having the ability to absorb UVA rays within the range of 300-500 nm. There were no significant difference in antimicrobial effect of pigment , even when the extraction and isolation were done by two culture medium (Nutrient Broth and Luria Bertani Broth). There were considerable inhibition percentages of adhesion after subjection to Cartenoid pigment ranged between (5.71, 23.84) % for Klebsiella spp. and Pseudomonas aeruginosa respectively and all the 11 isolate changed from Biofilm producer to non-producer. The isolated compound can be used against different bacterial and fungal infections. So they had a great future in medicine, cosmetics and as a sun protecting agent.

Effect of the Medium Composition on the Zn2+ Lixiviation and the Antifouling Properties of a Glass with a High ZnO Content.

The dissolution of an antimicrobial ZnO-glass in the form of powder and in the form of sintered pellets were studied in water, artificial seawater, biological complex media such as common bacterial/yeast growth media (Luria Bertani (LB), yeast extract, tryptone), and human serum. It has been established that the media containing amino acids and proteins produce a high lixiviation of Zn2+ from the glass due to the ability of zinc and zinc oxide to react with amino acids and proteins to form complex organic compounds. The process of Zn2+ lixiviation from the glass network has been studied by X-ray photoelectron spectroscopy (XPS). From these results we can state that the process of lixiviation of Zn2+ from the glass network is similar to the one observed in sodalime glasses, where Na+ is lixiviated to the media first and the fraction of Zn that acts as modifiers (~2/3) is lixiviated in second place. After the subsequent collapse of the outer surface glass layer (about 200–300 nm thick layer) the dissolution process starts again. Antifouling properties against different bacteria (S. epidermidis, S. aureus, P. aeruginosa, E. coli, and M. lutea) have also been established for the glass pellets.

Isolasi dan Karakterisasi Fitase pada Mikrobia yang Terdapat pada Pupuk Kompos, Rumen Sapi, Ragi dan Tanah Sawah

<p>Penelitian ini bertujuan untuk mengisolasi dan mengkarakterisasi fitase dari berbagai sumber yang diasumsikan kaya akan senyawa fosfat komplek, seperti: pupuk kompos, isi rumen sapi dan ragi. Isolasi mikrobia dari pupuk kompos, isi rumen sapi dan ragi. Isolasi bakteri penghasil fitase dilakukan dengan menggunakan media LB (<em>Luria Bertani</em>) dengan inkubasi pada suhu 37<sup>o</sup>C selama 16 jam, sedangkan isolasi mikrobia dari ragi menggunakan media PDA (<em>Pottato Dextrose Agar</em>). Ekstrak enzim kasar pada supernatan diperoleh dengan cara sentrifugasi pada kecepatan 5.000 rpm selama 5 menit. Ekstrak enzim kasar dikarakterisasi secara fisik meliputi pH dan temperatur optimum dan pengaruh efektor logam terhadap aktivitas enzim relatif. Hasil penelitian menunjukkan bahwa isolat B1 dari kompos P88 mepunyai aktivitas relatif optimum pada pH 5, suhu 50<sup>o</sup>C dan dengan aktifator Zn<sup>2+</sup> pada konsentrasi 10<sup>-3 </sup>M dan 10<sup>-4</sup>, Isolat B2 dari rumen sapi mepunyai aktivitas relatif optimum pada pH 5, suhu 50<sup>o</sup>C dan dengan aktifator Zn<sup>2+ </sup>pada konsentrasi 10<sup>-3</sup> M dan Mg<sup>2+</sup> pada konsentrasi 10<sup>-4</sup> M. Isolat B3 dari ragi kecap mepunyai aktivitas relatif optimum pada pH 5, suhu 60<sup>o</sup>C dan dengan aktifator Mg<sup>2+</sup> pada konsentrasi 10<sup>-3 </sup>M dan Fe<sup>2+</sup> pada 10<sup>-4</sup> M dan B4 dari ragi tempe mepunyai aktivitas relatif optimum pada pH 5, suhu 50<sup>o</sup>C dan dengan aktifator Mg<sup>2+</sup> pada konsentrasi 10<sup>-3 </sup>M dan Ca<sup>2+</sup> pada konsentrasi 10<sup>-4</sup> M.</p><p> </p><p>Kata kunci : fitase, pH optimum, temperatur optimum, efektor logam, isolat</p>

Synthetic cystic fibrosis sputum medium diminishes Burkholderia cenocepacia antifungal activity against Aspergillus fumigatus independently of phenylacetic acid production.

Phenylacetic acid (PAA), an intermediate of phenylalanine degradation, is emerging as a signal molecule in microbial interactions with the host. In this work, we explore the presence of phenylalanine and PAA catabolism in 3 microbial pathogens of the cystic fibrosis (CF) lung microbiome: Pseudomonas aeruginosa, Burkholderia cenocepacia, and Aspergillus fumigatus. While in silico analysis of B. cenocepacia J2315 and A. fumigatus Af293 genome sequences showed complete pathways from phenylalanine to PAA, the P. aeruginosa PAO1 genome lacked several coding genes for phenylalanine and PAA catabolic enzymes. High-performance liquid chromatography analysis of supernatants from B. cenocepacia K56-2 detected PAA when grown in Luria-Bertani medium but not in synthetic cystic fibrosis sputum medium (SCFM). However, we were unable to identify PAA production by A. fumigatus or P. aeruginosa in any of the conditions tested. The inhibitory effect of B. cenocepacia on A. fumigatus growth was evaluated using agar plate interaction assays. Inhibition of fungal growth by B. cenocepacia was lessened in SCFM but this effect was not dependent on bacterial production of PAA. In summary, while we demonstrated PAA production by B. cenocepacia, we were not able to link this metabolite with the B. cenocepacia - A. fumigatus microbial interaction in CF nutritional conditions.

Dagaa (Rastrinoebola argentea) protein hydrolysate as a nitrogen source in microbial culture media

Dagaa is a small pelagic fish found in Lake Victoria. It is used as a nutrient source in animal feeds and human consumption. Utilization of fish protein hydrolysate as a nutrient in culture media has been explored in several fish species and dagaa protein hydrolysate (DPH) can also be a candidate due to its rich protein and lipid content. Potential of DPH as a nitrogenous source in microbial growth media was assayed using the following bacteria; Escherichia coli, Pseudomonas aeroginosa, Bacillus subtilis and Rhodobacter capsulatus and two fungi, Metarhizium anisopliae and Beauveria bassiana. Cell growth was evaluated using turbidity and biomass in liquid cultures. DPH formulated media produced similar cell growth when compared to positive controls made up of Luria Bertani (LB) for bacteria and Sabouraud’s dextrose broth (SDB) for fungi. This indicates that formulated DPH media can be considered as an alternative media for microbial culture.

Methods of Isolating Actinomycetes from the Soils of Menengai Crater in Kenya.

This study was carried out to investigate the best method isolating actinomycetes from the soils of Menengai crater. Based on land terrain and differences in vegetation, the study area was divided into regions A, B, C and D. Soil samples were collected from 8 sampling points from each region. The samples were separately mixed to form composite samples. To isolate actinomycetes, three isolation media such as starch casein (SC), Luria Bertani (M1) and starch nitrate (SN) agar were used. Prior to isolation, the soil samples were heat for 1h at 121°C. In serial dilution method, serial dilution upto 10-6 was carried out. On the other hand, in membrane filtration the pre-treated soil samples were sprinkled on the membrane filters previously placed on the isolation media and the membranes were removed after 4 d of incubation. Direct inoculation involved application of 2-3 mg of soil on the culture media. Characterization of the isolates was carried out by cultural, morphological and biochemical means. There was a significant difference in the number of actinomycetes isolated using the three media (F=7.252999 P=0.000897). The number of actinomycetes isolated using serial dilution, membrane filtration and direct inoculation were also significantly different (F=8.683374 P=0.000237). Likewise, the number of actinomycetes isolated from region A, B C, and D varied significantly (F= 27.50 P=0.000).

Antimicrobial Properties of Actinomycetes Isolated from Menengai Crater in Kenya

A study was carried out to isolate and screen actinomycetes for antimicrobials from Menengai Crater in Kenya. The actinomycetes were isolated using starch casein agar, Luria Bertani agar and starch nitrate agar. Primary screening for antagonism was carried out using perpendicular method while secondary screening was done using agar disk technique. Extraction of the antimicrobials was carried out using ethyl acetate. Sensitivity testing of the crude extracts against Staphylococcus aureus, Bacillus subtilis, Escherichia faecalis, Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Xanthomonas campestris, Erwinia carotovora, Candida albicans, Alternaria alternate and Fusarium oxysporum was carried out using agar well technique. Biochemical tests and carbon source requirements were used in characterization of the selected antimicrobial producers. M1 was the best agar medium for isolation of actinomycetes. The number of actinomycetes from regions A, B, C, and D in the crater varied significantly (F = 27.50 P = 0.000). Out of the 156 actinomycetes isolates, 20 isolates were positive for both primary and secondary screening for antimicrobials. There was no significant difference in the zones of inhibition in primary screening of the actinomycetes for antagonistic properties against the test pathogens (F = 1.6957 P = 0.0838). The zones of inhibition after secondary screening varied significantly (F = 2.4473 P = 0.0089). Likewise, there was a significant difference (F = 6.6046 P = 0.001338) in the zones of inhibition after exposing the pathogens to ethyl extracts of the selected antagonistic actinomycetes. There is need to purify and characterize the antimicrobials obtained from the present study.

Overproduction of Pyocyanin in Pseudomonas aeruginosa by Supplementation of Pathway Precursor Shikimic acid and Evaluation of its Activity

Pyocyanin is a fluorescent phenazine compound with enormous biotechnological applications. In this study, the production of pyocyanin by P. aeruginosa PAO1 was investigated under different growth conditions. Optimization of growth media was carried out using nutrient broth, tryptic soy broth, Pseudomonas broth and Luria bertani broth. The effect of incubation time, and temperature on production of pyocyanin was investigated. Subsequently, shikimic acid pathway mediated pyocyanin overproduction was targeted using Illicium verum hot water extract as a source of shikimic acid for phenazine biosynthesis. Pyocyanin was purified and tested for its activity against glioma cells. Optimum yield of pyocyanin was obtained in nutrient broth at 37 oC and pH 7.2. We also found that supplementation of nutrient media with Illicium verum extract increased the pyocyanin production. Pyocyanin exhibited cytotoxicity of 66.34% at 200µg/ml concentration against glioma cells.

Bacillus velezensis YP2의 겨자채 흰가루병의 생물적 방제

Bacillus velezensis YP2 균주는 쌈채소에서 분리한 Cercospora sp. 1~3, Septoria sp., Colletotrichum sp., Sclerotinia sclerotiorum, Phoma sp., Botrytis cinerea에 대하여 균사생육을 억제하였다. B. velezensis YP2 균주의 LB배양액을 10배 희석액은 적겨자와 청겨자에 발생하는 Erysiphe cruciferarm에 의한 흰가루병을 91.8%와 80.9% 방제효과을 보였다. B. velezensis YP2 균주의 배양액을 10배 희석하여 5일 간격 4회 처리구가 적겨자 흰가루병을 70.6%, 7일 간격 3회 처리구는 65.0%, 10일 간격 2회 처리구는 49.5% 방제하였다. 또한 B. velezensis YP2 균주는 적겨자 종자 발아를 증가시켜고 생육을 촉진하였다. 이상의 결과에서 YP2 균주는 겨자채 흰가루병 방제에 매우 효과적이었다. Bacillus velezensis YP2 inhibited the mycelial growth of several plant pathogens including Cercespora spp., Septoria sp., Phoma sp., Botrytis cinerea and Sclerotinia scleotiorum occurring in leafy vegetables. Control efficacy for powdery mildew caused by Erysiphe cruciferarm on red leaf mustard and cheong mustard by treatment of spraying with 10-fold diluted Luria-Bertani (LB) broth of B. velezensis YP2 was 91.8% and 80.9%, respectively. When B. velezensis YP2 was treated four times with five-day interval, three times at seven-day interval and two times at ten day interval in the greenhouse test, the control effect of red leaf mustard powdery mildew was 70.6%, 65.0% and 40.9%, respectively. Also B. velezensis YP2 could promote the seed germination and plant growth of led leaf mustard. The results showed that the culture broth of B. velezensis YP2 was very effective to control the powdery mildew of leaf mustard.

Toxicity and transformation of graphene oxide and reduced graphene oxide in bacteria biofilm.

Impact of graphene based material (GNMs) on bacteria biofilm has not been well understood yet. In this study, we compared the impact of graphene oxide (GO) and reduced graphene oxide (rGO) on biofilm formation and development in Luria-Bertani (LB) medium using Escherichia coli and Staphylococcus aureus as models. GO significantly enhanced the cell growth, biofilm formation, and biofilm development even up to a concentration of 500mg/L. In contrast, rGO (≥50mg/L) strongly inhibited cell growth and biofilm formation. However, the inhibitory effects of rGO (50mg/L and 100mg/L) were attenuated in the mature phase (>24h) and eliminated at 48h. GO at 250mg/L decreased the reactive oxygen species (ROS) levels in biofilm and extracellular region at mature phase. ROS levels were significantly increased by rGO at early phase, while they returned to the same levels as control at mature phase. These results suggest that oxidative stress contributed to the inhibitory effect of rGO on bacterial biofilm. We further found that supplement of extracellular polymeric substances (EPS) in the growth medium attenuated the inhibitory effect of rGO on the growth of developed biofilm. XPS results showed that rGO were oxidized to GO which can enhance the bacterial growth. We deduced that the elimination of the toxicity of rGO at mature phase was contributed by EPS protection and the oxidation of rGO. This study provides new insights into the interaction of GNMs with bacteria biofilm.

Effect of culture media on chemical stability and antibacterial activity of allicin

Minimum Inhibitory Concentration (MIC) of allicin were determined for 11 bacterial strains using 6 culture media including Todd Hewitt Broth (THB), Brain Heart Infusion (BHI) Broth, Tryptic Soy Broth (TSB), Mueller Hinton Broth (MHB), Luria Bertani (LB) Broth, and Brucella Broth (BB) at 37°C. The chemical stability of allicin as a function of media was measured by HPLC. The MICs for most individual isolates grown at the same temperature varied with the media used. Higher MICs were obtained in BB, THB, BHI, TSB/MHB and LB with geometric mean values of 272.7, 145.2, 90.5, 77.3/72.6 and 46.7μgml−1 respectively. In media with higher MICs a big percent of allicin disappeared within the first 4h of incubation and apparently allicin had shorter half-life compared to those with lower MIC values. The results showed that temperature directly influences the stability of allicin and shorter half-life was found at 37°C.We conclude that the type of culture media, markedly influences the chemical stability of allicin and accordingly its MIC in a given bacterial species.

EVALUATION OF DIFFERENT CULTURE MEDIA FOR ENHANCED PRODUCTION OF PSEUDOMONAS AERUGINOSA (MTCC NO 2453) BIOMASS AND ITS PROTEINS

Objective: Microorganisms, especially bacteria and its proteins have proven to be potential anti-cancer agents as they selectively attack the tumor cells or tumor micro-environments. The extract of Pseudomonas aeruginosa found to contain proteins that have shown promising anticancer activity. In this work, it was attempted to increase the biomass and trigger the total protein fraction of Pseudomonas aeruginosa (MTCC 2453).Methods: The organism was cultivated in three different such as Luria-Bertani (LB) broth, minimal medium9 (M9), super broth medium (SB) and asparagine-proline (AP) broth. Asparagine proline broth was selected as it has shown high cell growth rate. The media was further optimized by the addition of NaHCO3 and copper sulphate to trigger the protein production. Optimized Aspergine proline broth has achieved highest cell biomass. After the shake flask culture, the overnight grown culture in optimized AP medium was further grown in a 5 L bioreactor by fed-batch cultivation to achieve higher cell densities.Results: The highest protein production was achieved at 40 ° C. Highest biomass and protein content was observed at pH 8 while lowest biomass was produced at pH 2. A gradual increase in biomass content observed from 12 h towards to 48 h.Conclusion: High biomass and proteins content and of Pseudomonas aeruginosa (MTCC 2453) can be produced in optimized asparagine-proline broth. Further the extract is purified to produce novel anti-cancer proteins.

Repurposing the anticancer drug mitomycin C for the treatment of persistent Acinetobacter baumannii infections

Acinetobacter baumannii is an emergent opportunistic bacterial pathogen responsible for recalcitrant infections owing to its high intrinsic tolerance to most antibiotics; therefore, suitable strategies to treat these infections are needed. One plausible approach is the repurposing of drugs that are already in use. Among them, anticancer drugs may be especially useful due their cytotoxic activities and ample similarities between bacterial infections and growing tumours. In this work, the effectiveness of four anticancer drugs on the growth of A. baumannii ATTC BAA-747 was evaluated, including the antimetabolite 5-fluorouracil and three DNA crosslinkers, namely cisplatin, mitomycin C (MMC) and merphalan. MMC was the most effective drug, having a minimum inhibitory concentration for 50% of growth in Luria–Bertani medium at ca. 7 µg/mL and completely inhibiting growth at 25 µg/mL. Hence, MMC was tested against a panel of 21 clinical isolates, including 18 multidrug-resistant (MDR) isolates, 3 of which were sensitive only to colistin. The minimum inhibitory concentrations and minimum bactericidal concentrations of MMC in all tested strains were found to be similar to those of A. baumannii ATCC BAA-747, and MMC also effectively killed stationary-phase, persister and biofilm cells. Moreover, MMC was able to increase survival of the insect larvae Galleria mellonella against an otherwise lethal A. baumannii infection from 0% to ≥53% for the antibiotic-sensitive A. baumannii ATCC BAA-747 strain and the MDR strains A560 and A578. Therefore, MMC is highly effective at killing the emergent opportunistic pathogen A. baumannii.