Application of Plackett–Burman experimental design and Box and Wilson design to improve broad-spectrum antimicrobial compound

386-394The present study is aimed at optimization of process parameters for production of antimicrobial compound from a potent antibiotic producing Streptomyces isolate. Among a total of 130 actinomycetes, isolate 2A showed antimicrobial activity against all the test organisms including drug resistant bacteria. 16S rRNA gene sequence analysis showed that strain 2A belongs to the genus Streptomyces and exhibited highest 16S rRNA gene sequence similarity (99.8%) with Streptomyces flavotricini NBRC12823T (AB184173<span style="font-family:Arial;mso-bidi-font-family:Mangal;mso-bidi-font-weight: bold" lang="EN-GB">). Comparison of physiological characteristics and DNA-DNA hybridization (manifested 36.7% similarity) with S. flavotricini suggests that 2A is a novel strain (GenBank acc. no. GQ906975). The traditional one-variable-at-a-time optimization strategy and statistically based experimental designs, Plackett-Burman design (PBD) and Box and Wilson design, were applied to provide an efficient approach for optimization. Statistical analysis using PBD demonstrated that KNO3, K2HPO4 and NaCl had significant positive influences on the production. In optimized medium, antimicrobial compound production was increased by 1.5-fold as compared to the basal production medium. In addition, the optimization enhanced the antimicrobial activity spectrum. The crude extract showed ability<span style="font-family:TimesNewRoman;mso-fareast-font-family:Calibri; mso-bidi-font-family:TimesNewRoman" lang="EN-GB"> to inhibit all tested bacteria with MIC ranging from 15.6-125 μg mL‐1<span style="font-family:TimesNewRoman;mso-fareast-font-family:Calibri; mso-bidi-font-family:TimesNewRoman" lang="EN-GB"> and MBC from 31.2-500 μg mL‐1. </span

Biocontrol Potential of Streptomyces hydrogenans strain DH16 Towards Alternaria brassicicola to Control Damping Off and Black Leaf Spot of Raphanus sativus

Biocontrol agents and their bioactive metabolites provide one of the best alternatives to decrease the use of chemical pesticides. In light of this, the present investigation reports the biocontrol potential of Streptomyces hydrogenans DH16 and its metabolites towards Alternaria brassicicola, causal agent of black leaf spot and damping off of seedlings of crucifers. In vitro antibiosis of strain against pathogen revealed complete suppression of mycelial growth of pathogen, grown in potato dextrose broth supplemented with culture supernatant (20% v/v) of Streptomyces hydrogenans DH16. Microscopic examination of the fungal growth showed severe morphological abnormalities in the mycelium caused by antifungal metabolites. In vivo studies showed the efficacy of streptomycete cells and culture supernatant as seed dressings to control damping off of Raphanus sativus seedlings. Treatment of pathogen infested seeds with culture supernatant (10%) and streptomycete cells significantly improved seed germination (75-80%) and vigour index (1167-1538). Furthermore, potential of cells and culture supernatant as foliar treatment to control black leaf spot was also evaluated. Clearly visible symptoms of disease were observed in the control plants with 66.81% disease incidence and retarded growth of root system. However, disease incidence reduced to 6.78 and 1.47% in plants treated with antagonist and its metabolites, respectively. Additionally, treatment of seeds and plants with streptomycete stimulated various growth traits of plants over uninoculated control plants in the absence of pathogen challenge. These results indicate that S. hydrogenans and its culture metabolites can be developed as biofungicides as seed dressings to control seed borne pathogens, and as sprays to control black leaf spot of crucifers

Purification and characterization of actinomycins from Streptomyces strain M7 active against methicillin resistant Staphylococcus aureus and vancomycin resistant Enterococcus

Abstract Background The increased rate of resistance among two highly concerned pathogens i.e. methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) necessitates the discovery of novel anti-MRSA and anti-VRE compounds. In microbial drug discovery, Streptomyces are well known source of two-thirds of natural antibiotics used clinically. Hence, screening of new strains of streptomycetes is the key step to get novel bioactive compounds with antimicrobial activity against drug resistant bacteria. Results In the present study, Streptomyces antibioticus strain M7, possessing potent antibacterial activity against different pathogenic bacteria, was isolated from rhizospheric soil of Stevia rebudiana. 16S rRNA sequence of M7 (1418 bp) showed 96.47–100% similarity with different Streptomyces spp. and the maximum similarity (100%) was observed with Streptomyces antibioticus NBRC 12838T (AB184184). Phylogenetic analysis using neighbor joining method further validated its similarity with Streptomyces antibioticus NBRC 12838 T (AB184184) as it formed clade with the latter and showed high boot strap value (99%). Antibacterial metabolites isolated from the fermentation broth were characterized using NMR, FT-IR and LC-MS as actinomycins V, X2 and D. The purified actinomycins exhibited potent antibacterial activities against test bacteria viz. B. subtilis, K. pneumoniae sub sp. pneumoniae, S. aureus, S. epidermidis, S. typhi, E. coli, MRSA and VRE. Among these actinomycins, actinomycin X2 was more effective as compared to actinomycins D and V. The minimum inhibitory concentration values of purified compounds against a set of test bacterial organisms viz. VRE, MRSA, E. coli (S1-LF), K. pneumoniae sub sp. pneumoniae and B. subtilis ranged between 1.95 and 31.25 μg/ml. Conclusions This study demonstrates that actinomycins V, X2 and D produced by S. antibioticus strain M7 hold the potential to be used against multidrug resistant bacteria, particularly VRE and MRSA

Investigating the plant growth promoting and biocontrol potentiality of endophytic Streptomyces SP. SP5 against early blight in Solanum lycopersicum seedlings

Abstract Background Early blight (EB), caused by Alternaria solani, is one of the alarming diseases that restrict tomato production globally. Existing cultural practices and fungicide applications are not enough to control early blight diseases. Therefore, the study aimed to isolate, identify, and characterize an endophytic Streptomyces exhibiting the potential to control early blight in tomato and also promote plant growth. Results From a Citrus jambhiri leaf, an endophytic Streptomyces sp. with antagonistic activity against Alternaria solani, Colletotrichum acutatum, Cladosporium herbarum, Alternaria brassicicola, Alternaria sp., Fusarium oxysporum and Fusarium sp. was isolated. It was identified as a Streptomyces sp. through 16S ribosomal DNA sequence analysis and designated as SP5. It also produced indole acetic acid which was confirmed by Salkowski reagent assay, TLC and HPLC analysis. Treatment of pathogen infected plants with Streptomyces sp. SP5 antagonists (culture cells/culture supernatant/solvent extract/ acetone precipitates) decreased the early blight disease incidence and significantly increased the various agronomic traits. Conclusion The present study concluded that Streptomyces sp. SP5 possessed antifungal activity against different fungal phytopathogens and had significant potential to control early blight disease and promote plant growth

Purification and biological analysis of antimicrobial compound produced by an endophytic Streptomyces sp.

Abstract Fungal phytopathogens and drug-resistant bacteria are two significant challenges in agriculture and public health, respectively. As a result, new sources of antimicrobial compounds are urgently needed. Taking into consideration these aspects, the present study was carried out to explore the antimicrobial activity of Streptomyces sp. SP5 against drug-resistant bacteria, especially methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus  and fungal phytopathogens. MRSA and VRE are both types of antibiotic-resistant bacteria that pose significant challenges to public health. In vitro analysis of the metabolites of Streptomyces sp. SP5 exhibited broad-spectrum antimicrobial activity against drug-resistant bacteria and phytopathogenic fungi. Further chemical investigation of the diethyl ether extract led to the isolation and purification of an antimicrobial compound. The structure of the purified compound was elucidated by performing detailed spectroscopic analysis including MS, IR, and NMR. The compound was identified as plicacetin. Plicacetin is a nucleoside antibiotic that has been reported for antibacterial activity against Gram-positive bacterium Mycobacterium tuberculosis. According to our knowledge, the present study is the first to demonstrate the antimicrobial properties of plicacetin against Fusarium oxysporum, Alternaria brassicicola, Fusarium solani, VRE and Bacillus subtilis. The outcome of the current study endorses that compound produced by Streptomyces sp. SP5 can be used as an antimicrobial agent against fungal phytopathogens and drug-resistant bacteria

Purification and characterization of a novel lipopeptide from Streptomyces amritsarensis sp. nov. active against methicillin-resistant Staphylococcus aureus

Nowadays antimicrobial lipopeptides are being widely exploited for developing potential therapeutic agents for treating bacterial infections. In the present study, we have purified and characterized an antimicrobial lipopeptide produced by Streptomyces amritsarensis sp. nov. (= MTCC 11845(T) = JCM 19660(T)). The lipopeptide was purified using silica gel chromatography, size exclusion chromatography and reverse phase- HPLC. The MS/MS analysis of the lipopeptide revealed that it has amino acid sequence as Ala-Thr-Gly-Ser-His-Gln and a long chain fatty acid tail with six times repeated the molecular mass of 161 Da which is corresponding to -C(12)H(19). Based on the molecular mass (878.5 Da) and amino acid composition, the lipopeptide was identified as a novel lipopeptide. The MIC values of purified lipopeptide against Bacillus subtilis (MTCC 619), Staphylococcus epidermidis (MTCC 435), Mycobacterium smegmatis (MTCC 6) and clinical strain, Methicillin Resistant Staphylococcus aureus (MRSA) were found to be 10, 15, 25 and 45 μg/ml, respectively. It was completely stable at 70°C for 1 h and retained 81.8% activity after autoclaving (121°C for 15 min). It did not show any change in its activity profile between pH 5.0 - 9.0 and is stable to trypsin, proteinase K and lipase enzymes. It was found to be non-mutagenic against Salmonella typhimurium (TA98) and did not show cytotoxicity when checked against Chinese hamster ovary (CHO) cell line. In addition to antibacterial activity it also exhibits biosurfactant activity

Antimicrobial Activity of Actinomycetes Against Multidrug Resistant Staphylococcus aureus , Escherichia coli and Various Other Pathogens

Purpose: The rapid emergence of drug resistance among pathogenic bacteria, especially multidrugresistant bacteria, underlines the need to look for new antibiotics. Methods: In the present study, 134 different actinomycetes, isolated from the soil samples collected from different localities of Punjab and Himachal Pradesh, were screened for antimicrobial activity against various test organisms including multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli in order to identify potential antibiotic producers. Results: Among these isolates, 51 (38 %) showed antimicrobial activity against one or more test organisms and six exhibited promising broad-spectrum activity against all the tested organisms. The observed cultural, morphological, physiological and biochemical characteristics confirmed that these isolates are species of the genus, Streptomyces. Conclusion: Further studies on the bioactive metabolites from these cultures will be useful for discovering novel compounds of clinical and agricultural use

Additional file 1 of Investigating the plant growth promoting and biocontrol potentiality of endophytic Streptomyces SP. SP5 against early blight in Solanum lycopersicum seedlings

Additional file 1