443 research outputs found

    Mechanisms of resistance to β-lactam antibiotics in Streptomycetes

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    The most successful antimicrobial agents in clinical use are of microbial origin and of these the greatest variety has been found in the genus Streptomyces. However, the resistance of the pathogenic microbes to the commonly used antibiotics is increasing as a result of the wide-spread and long-term use of these antibiotics. Therefore, understanding the strategies that bacteria use to become resistant is of crucial need. Streptomycetes are Gram positive bacteria, commonly found in soil and are known antibiotic-producers. The focus of this thesis was to underpin the mechanism of resistance to penicillin G in isolated strains of streptomycetes that exhibit elevated resistance to penicillin G and to characterise these organisms. Moreover, to investigate the interaction between penicillin G and PBPs in Streptomyces strains and investigate the relationship between growth rate and penicillin G resistance in Streptomyces in vitro. Ninety six Streptomycetes were isolated and characterized. Morphological examination and the16s rRNA sequences of these strains indicated that strains belong to the species Streptomyces. The MICs and MBCs for penicillin G for the isolated Streptomyces strains were measured by plate culture. Some strains showed growth up to 400 μg/ml with penicillin G, which indicate that the strains were highly resistant against penicillin G. Some strains were unable to grow at penicillin concentrations above 200μg/ml. Also, The MICs of penicillin G for isolated Streptomyces strains were measured using a novel OxoPlates® system in 96-well culture format employing Mueller-Hinton broth culture. The MICs of all strains ranged from 1-100 μg /ml. Results indicate that the sensitivity of Streptomyces strains of penicillin G is not directly related to β-lactamase production in the panel of isolates examined. There was no correlation between the MICs of penicillin G and the growth rate in these isolates. Likewise, there was no association between the position of beta-lactamase producing and non-beta-lactamase producing strains on the phylogenetic tree and their beta-lactamase xii activity. Beta-lactamase producing and non-producing strains refers to the same ancestral origin clade. Additionally, the comparative analysis of 16S rRNA gene sequence and phylogenetic relationship of strain (W43) revealed that the isolate clustered with (W76) Streptomyces lividans strain YLA0. Bocillin (a penicillin binding protein stain) staining in β-lactamase producing strains showed staining throughout the mycelia whereas in non β-lactamase producing strains staining only occurred in certain parts of the mycelia. Bocillin also revealed that in spores PBPs were located on both poles of the spores. Streptomyces coelicolor has the ability to grow at high concentrations of penicillin G up to 640 μg/ml in continuous culture. It also has the capacity to grow at very low amounts of dissolved oxygen in continuous culture. Significantly, there was a correlation between the growth rate of S. coelicolor and the resistant to penicillin G. S. Coelicolor was more sensitive to penicillin G at a high dilution rate. Furthermore, our strategy of using the Bug-Lab for monitoring the progress of S. Coelicolor 1147 in continuous culture, even at low concentrations of cells in real time was successful

    Antibiotic resistance in the environment, with particular reference to MRSA

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    The introduction of β-lactam antibiotics (penicillins and cephalosporins) in the 1940s and 1950s probably represents the most dramatic event in the battle against infection in human medicine. Even before widespread global use of penicillin, resistance was already recorded. E. coli producing a penicillinase was reported in Nature in 1940 (Abraham, 1940) and soon after a similar penicillinase was discovered in Staphylococcus aureus (Kirby, 1944). The appearance of these genes, so quickly after the discovery and before the widespread introduction of penicillin, clearly shows that the resistance genes pre-dated clinical use of the antibiotic itself

    Dissolution of the Disparate:Co-ordinate Regulation in Antibiotic Biosynthesis

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    Discovering new antibiotics is vital to combat the growing threat of antimicrobial resistance. Most currently used antibiotics originate from the natural products of actinomycete bacteria, particularly Streptomyces species, that were discovered over 60 years ago. However, genome sequencing has revealed that most antibiotic-producing microorganisms encode many more natural products than previously thought. Biosynthesis of these natural products is tightly regulated by global and cluster situated regulators (CSRs), most of which respond to unknown environmental stimuli, and this likely explains why many biosynthetic gene clusters (BGCs) are not expressed under laboratory conditions. One approach towards novel natural product discovery is to awaken these cryptic BGCs by re-wiring the regulatory control mechanism(s). Most CSRs bind intergenic regions of DNA in their own BGC to control compound biosynthesis, but some CSRs can control the biosynthesis of multiple natural products by binding to several different BGCs. These cross-cluster regulators present an opportunity for natural product discovery, as the expression of multiple BGCs can be affected through the manipulation of a single regulator. This review describes examples of these different mechanisms, including specific examples of cross-cluster regulation, and assesses the impact that this knowledge may have on the discovery of novel natural products

    The war against bacteria, from the past to present and beyond

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    Introduction: : The human defense against microorganisms dates back to the ancient civilizations, with attempts to use substances from vegetal, animal or inorganic origin to fight infections. Today, the emerging threat of multidrug-resistant bacteria highlights the consequences of antibiotics inappropriate use, and the urgent need of novel effective molecules. Methods and materials: We extensively researched on more recent data within the databases PubMed, Medline, Web of Science, Elsevier's EMBASE, Cochrane Review for the modern pharmacology in a period ranging from 1987 until 2021. Furthermore, the historical evolution was defined through a detailed analysis of past studies on the significance of medical applications in the ancient therapeutic field. Areas covered: To examine history of antibiotics development and discovery, the most relevant biochemical aspects of their mode of action, and the biomolecular mechanisms conferring bacterial resistance to antibiotics. Expert opinion: : The list of pathogens showing low sensitivity or full resistance to most currently available antibiotics is growing worldwide. Long after the "golden age" of antibiotic discovery, the most novel molecules should be carefully reserved to treat serious bacterial infections of susceptible bacteria. A correct diagnostic and therapeutic procedure can slow down the spreading of nosocomial and community infections sustained by multidrug-resistant bacterial strains

    An Introduction to Actinobacteria

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    Actinobacteria, which share the characteristics of both bacteria and fungi, are widely distributed in both terrestrial and aquatic ecosystems, mainly in soil, where they play an essential role in recycling refractory biomaterials by decomposing complex mixtures of polymers in dead plants and animals and fungal materials. They are considered as the biotechnologically valuable bacteria that are exploited for its secondary metabolite production. Approximately, 10,000 bioactive metabolites are produced by Actinobacteria, which is 45% of all bioactive microbial metabolites discovered. Especially Streptomyces species produce industrially important microorganisms as they are a rich source of several useful bioactive natural products with potential applications. Though it has various applications, some Actinobacteria have its own negative effect against plants, animals, and humans. On this context, this chapter summarizes the general characteristics of Actinobacteria, its habitat, systematic classification, various biotechnological applications, and negative impact on plants and animals

    Assessment of antibiotic production by some marine Streptomyces isolated from the Nahoon Beach

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    Rapidly emerging strains of bacteria resistant to most advanced antibiotics have become issues of very important public health concern. Research currently directed towards marine actinomycetes presents a vast potential for new compounds that could be able to safely and effectively target resistant species. In this regard, ten putative Streptomyces strains isolated from the Nahoon beach were selected and assessed for antibiotic production and activity against a wide range of bacteria including reference strains, environmental strain and clinical isolates. The ethyl acetate extracts of the putative Streptomyces isolates showed activities against at least 6 and up to 26 of the 32 test bacteria. Inhibition zones were found to range between 9-32 mm diameters at a concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) of the crude extracts ranged from 0.039 - 10 mg/ml and the least minimum bactericidal concentration (MBC) demonstrated was 0.625 mg/ml against a reference strain Staphylococcus aureus ATCC 6538. Time kill kinetics of all extracts revealed bacteristatic and bactericidal activities. Average Log reductions in viable cell counts for all the extracts ranged from 0.86 Log10 and 3.99 Log10 cfu/ml after 3 h interaction and 0.01 Log10 and 4.86 Log10 after 6 h interaction at MIC, 2 × MIC, 3 × MIC and 4 × MIC concentrations. Most of the extracts were speedily bactericidal at 3 × MIC and 4 × MIC resulting in over 50 % elimination of most of the test bacteria within 3 h and 6 h interaction. The partial characterization of the crude extracts by IR spectral analysis revealed possibility of terpenoid, long chain fatty acids and secondary amine derivatives compounds in the extracts. It is therefore recommended that further investigation should address the relationship between the structure of the active component of the extracts and the broad spectrum activity, as well as a rapid method for large scale production and purification and whether this group of antibiotics has any application in managing human infectious disease

    Analysis of blm: a gene of unclear function involved in the biosynthesis of clavulanic acid in Streptomyces clavuligerus

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    Streptomyces clavuligerus produces the medicinally important specialized metabolite clavulanic acid (CA), but detailed understanding of the roles played by many genes involved in CA biosynthesis is still lacking. These include the overlapping blm (orf12), orf13 and orf14 genes, disruptions of which cause defective CA production in S. clavuligerus. I investigated the potential polarity associated with disrupting blm in S. clavuligerus, along with the cellular localization of Blm. This was accomplished using complementation of prepared blm deletion mutants and western analysis of strains expressing epitope (FLAG) tagged copies of the protein. blm-like genes from other microorganisms containing sequenced CA gene clusters were examined for their ability to complement a S. clavuligerus blm in-frame mutant. Results demonstrate that the blm mutation is not associated with polar effects and that Blm is a cytoplasmic protein, suggesting that it could either play a regulatory or enzymatic role during CA production in S. clavuligerus

    Characterization of Antibacterial Compounds from Marine Sponge-associated Streptomyces spp. against Some Pathogenic Bacteria

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    The increasing trend of antibiotic resistance among pathogenic bacteria is a worldwide problem. Streptomyces produce a number of bioactive compounds such as antibacterial. This study aimed to investigate the effect of different media and incubation time in increasing the antibacterial activity of marine sponge-associated Streptomyces spp. and characterize antibacterial compounds of marine sponge-associated Streptomyces spp. against pathogenic bacteria. Among the three tested media and some days of incubation times, Streptomyces spp. produce more antibacterial activity when grown using modified molasses medium at 15 days incubation. The ethyl acetate extracts of Dbi28t exhibited a significant inhibitory zone against Extended Spectrum β-Lactamase (ESBL)-producing Escherichia coli, Providencia rettgeri then followed by Proteus mirabilis and Pseudomonas putida and the results were higher than some commercial antibiotics. This study has identified nine antibacterial compounds in Dbi28t using Liquid Chromatography-tandem Mass Spectrometric (LC-MS/MS) analysis, with the most abundance belonging to pumilacidin A, then followed by pumilacidin B, surfactin B, surfactin A, phenazostatin B, chalcomycin B, neopyrrolomycin C, saquayamycin A and saphenamycin. This work provides the first report from a Streptomyces sp. Dbi28t produced pumilacidin, surfactin and other bioactive compounds with the modified molasses medium for optimization of characterization of its antibacterial compounds

    Enzymatic resistance to the lipopeptide surfactin as identified through imaging mass spectrometry of bacterial competition

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    Many species of bacteria secrete natural products that inhibit the growth or development of competing species. In turn, competitors may develop or acquire resistance to antagonistic molecules. Few studies have investigated the interplay of these countervailing forces in direct competition between two species. We have used an imaging mass spectrometry (IMS) approach to track metabolites exchanged between Bacillus subtilis and Streptomyces sp. Mg1 cultured together. Surfactin is a cyclic lipopeptide produced by B. subtilis that inhibits the formation of aerial hyphae by streptomycetes. IMS analysis exposed an addition of 18 mass units to surfactin in the agar proximal to Streptomyces sp. Mg1 but not other streptomycetes tested. The spatially resolved change in the mass of surfactin indicated hydrolysis of the molecule. We observed that the aerial growth of Streptomyces sp. Mg1 was resistant to inhibition by surfactin, which suggests that hydrolysis was a mechanism of resistance. To identify possible enzymes from Streptomyces sp. Mg1 with surfactin hydrolase activity, we isolated secreted proteins and identified candidates by mass spectrometry. We purified one candidate enzyme that hydrolyzed surfactin in vitro. We tested the role of this enzyme in surfactin resistance by deleting the corresponding gene from the S. Mg1 genome. We observed that aerial growth by the ΔsfhA mutant strain was now sensitive to surfactin. Our results identify an enzyme that hydrolyzes surfactin and confers resistance to aerial growth inhibition, which demonstrates the effective use of an IMS approach to track natural product modifications during interspecies competition

    Another Brick in the Wall: the role of the actinobacterial cell wall in antibiotic resistance, phylogeny and development

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    Streptomyces are multicellular, Gram-positive bacteria in the phylum of actinobacteria which produce a high amount of bioactive natural products of which the expression is tightly coordinated with the life cycle. This thesis shows the identification of S. roseifaciens, a novel species with an uncommon, verticillate spore morphology and a unique household of SsgA-like proteins. Analyses of the peptidoglycan composition show that S. coelicolor show a pattern of 3-3 cross-linking befitting a tip-growing organism and change in composition between vegetative mycelium and spores. Kitasatosporae carry meso-DAP in the peptidoglycan of vegetative mycelium and LL-DAP in the peptidoglycan of spores. In line with this difference, the peptidoglycan architecture of these two growth stages undergoes such radical changes that they would seem to be from different species. S. coelicolor is naturally vancomycin resistant, but the addition of D-alanine and disruption in a single gene increases vancomycin sensitivity by a thousandfold. A knockout mutant of the alanine racemase, alr, requires exogenous addition of D-alanine. The Alr crystal structure of S. coelicolor and the D-cycloserine producer S. lavendulae were compared as to look for possible mechanisms for D-cycloserine resistance. Microbial Biotechnolog
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