Adaptive laboratory evolution triggers pathogen-dependent broad-spectrum antimicrobial potency in Streptomyces

Background In the present study, adaptive laboratory evolution was used to stimulate antibiotic production in a Streptomyces strain JB140 (wild-type) exhibiting very little antimicrobial activity against bacterial pathogens. The seven different competition experiments utilized three serial passages (3 cycles of adaptation-selection of 15 days each) in which Streptomyces strain (wild-type) was challenged repeatedly to one (bi-culture) or two (tri-culture) or three (quadri-culture) target pathogens. The study demonstrates a simple laboratory model to study the adaptive potential of evolved phenotypes and genotypes in Streptomyces to induce antibiotic production. Results Competition experiments resulted in the evolution of the wild-type Streptomyces strain JB140 into the seven unique mutant phenotypes that acquired the ability to constitutively exhibit increased antimicrobial activity against three bacterial pathogens Salmonella Typhi (NCIM 2051), Staphylococcus aureus (NCIM 2079), and Proteus vulgaris (NCIM 2027). The mutant phenotypes not only effectively inhibited the growth of the tested pathogens but were also observed to exhibit improved antimicrobial responses against one clinical multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC 1021) isolate. In contrast to the adaptively evolved mutants, only a weak antimicrobial activity was detected in the wild-type parental strain. To get molecular evidence of evolution, RAPD profiles of the wild-type Streptomyces and its evolved mutants were compared which revealed significant polymorphism among them. Conclusion The competition-based adaptive laboratory evolution method can constitute a platform for evolutionary engineering to select improved phenotypes (mutants) with increased antibacterial profiles against targeted pathogens.

Investigation of the microbial community structure and diversity in the environment surrounding a veterinary antibiotic production factory

The wastewater discharged from the veterinary antibiotic production factory, due to its antibiotic composition, partly affects the microbial community and distribution in the surrounding environment.

Behaviors of Homologous Antibiotic Resistance Genes in a Cephalosporin WWTP, Subsequent WWTP and the Receiving River

High concentrations of antibiotics in antibiotic production wastewater can cause the widespread transmission of antibiotic resistance genes (ARGs). Here, we collected a set of time series samples from a cephalosporin production wastewater treatment plant (X-WWTP), the subsequent municipal WWTP (Y-WWTP) and the receiving stream. Using a functional gene microarray, GeoChip 5.0, which contains multiple homologous probes for 18 ARG and 13 antibiotic metabolism gene (AMG) families, we found that more than 50% of homologous probes for 20 gene families showed a relative abundance higher in X-WWTP, while only 10–20% showed lower relative abundance. The different response patterns of homologous ARG (hARGs) within the same ARG family imply environmental selection pressures are only responsible for the ARG enrichment and spread of some specific instead of all ARG-containing microorganisms, which contradicted the traditionally held belief that environmental selection pressures, especially antibiotic concentration, select for all ARG-containing microorganisms thereby selecting different hARGs in the same ARG family in an undifferentiated way. Network results imply that hARGs from three β_lactamase families enriched under the selection pressure of high cephalosporin antibiotic concentrations in X-WWTP formed positively correlated homologous ARG clusters (pohARGCs). The pohARGCs were also enhanced in the sediment of the receiving stream. The enrichment of hARGs from three β_lactamase families was likely through microorganisms belonging to the Betaproteobacteria genus.

Antibiotic Activity of Actinomycetes Isolated from Young Tectona Grandis (L.) Wood and Pith

Actinomycetes are a source for novel bioactive compounds and justify obtaining new species from various sources. Hardwoods such as Tectona grandis (L.) have not been studied for actinomycete isolation. We aim to isolate endophytic actinomycetes from young Tectona grandis wood and pith and screen for antibiotic activity. Five young wood were cut and surface sterilized using ethanol and hypochlorous acid. The wood and pith of each sample are placed in eight plates of Humic acid-vitamin B (HV), Tap water Yeast extract (TWYE), and Yeast Extract Casein Digest (YECD) medium and incubated at 27°C for four weeks. Actinomycetes were isolated from such medium, observed every week, and transferred to an International Streptomyces Project-2 (ISP-2) medium for identification and antibiotic production tested against Staphylococcus aureus, Helicobacter pylori, and Escherichia coli using the Kirby-Bauer method. Seven actinomycetes were isolated from the wood, primarily from YECD and TWYE media, with varying morphological characteristics. One isolate having maroon-colored aerial and vegetative mycelium with grey spores showed moderate antibacterial activity against S. aureus and H. pylori (13.49±1.03 mm and 14.9±0.7 mm, respectively), while two other actinomycetes showed weak activity against these bacteria. However, none of the actinomycetes show any activity against E. coli. Tectona grandis (L.) is a potential source for novel actinomycetes with an antibiotic activity which warrants further exploration

Reconstruction of a global gene regulatory network for Streptomyces coelicolor: Curation, inference, and assessment

Streptomyces coelicolor A3(2) is a model microorganism for the study of Streptomycetes, antibiotic production, and secondary metabolism in general. However, little effort to globally study its transcription has been made even though S. coelicolor has an outstanding variety of regulators among bacteria. We manually curated 29 years of literature and databases to assemble a meta-curated experimentally-validated gene regulatory network (GRN) with 5386 genes and 9707 regulatory interactions (~41% of the total expected interactions). This provides the most extensive and up-to-date reconstruction available for the regulatory circuitry of this organism. We found a low level of direct experimental validation for the regulatory interactions reported in the literature and curated in this work. Only ~6% (533/9687) are supported by experiments confirming the binding of the transcription factor to the upstream region of the target gene, the so-called "strong" evidence. To tackle network incompleteness, we performed network inference using several methods (including two proposed here) for motif detection in DNA sequences and GRN inference from transcriptomics. Further, we contrasted the structural properties and functional architecture of the networks to assess the predictions' reliability, finding the inference from DNA sequence data to be the most trustworthy. Finally, we show two possible applications of the inferred and the curated network. The inferred one allowed us to identify putative novel transcription factors for the key Streptomyces antibiotic regulatory proteins (SARPs). The curated one allows us to study the conservation of the system-level components between S. coelicolor and Corynebacterium glutamicum. There we identified the basal machinery as the common signature between the two organisms. The curated networks were deposited in Abasy Atlas (https://abasy.ccg.unam.mx/) while the inferences are available as Supplementary Material.

Diguanylate cyclase and phosphodiesterase interact to maintain the specificity of c-di-GMP signaling in the regulation of antibiotic synthesis in Lysobacter enzymogenes

Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. The large number of c-di-GMP-related diguanylate cyclases (DGCs), phosphodiesterases (PDEs) and effectors are responsible for the complexity and dynamics of c-di-GMP signaling. Some of these components deploy various methods to avoid undesired crosstalk to maintain signaling specificity. Synthesis of the antibiotic HSAF ( H eat S table A ntifungal F actor) in Lysobacter enzymogenes is regulated by a specific c-di-GMP signaling pathway that includes a PDE LchP and a c-di-GMP effector Clp (also a transcriptional regulator). In the present study, from among 19 DGCs, we identified a diguanylate cyclase, LchD, which participates in this pathway. Subsequent investigation indicates that LchD and LchP physically interact and that the catalytic center of LchD is required for both the formation of the LchD-LchP complex and HSAF production. All the detected phenotypes support that LchD and LchP dispaly local c-di-GMP signaling to regulate HSAF biosynthesis. Although direct evidence is lacking, our investigation, which shows that the interaction between a DGC and a PDE maintains the specificity of c-di-GMP signaling, suggests the possibility of the existence of local c-di-GMP pools in bacteria. Importance Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. Signaling of c-di-GMP is complex and dynamic, and it is mediated by a large number of components, including c-di-GMP synthases (diguanylate cyclases. DGCs), c-di-GMP degrading enzymes (phosphodiesterases, PDEs), and c-di-GMP effectors. These components deploy various methods to avoid undesired crosstalk to maintain signaling specificity. In the present study, we identified a DGC that interacted with a PDE to specifically regulate antibiotic biosynthesis in L. enzymogenes . We provide direct evidence to show that the DGC and PDE form a complex, and also indirect evidence to argue that they may balance a local c-di-GMP pool to control the antibiotic production. The results represent an important finding regarding the mechanism of a pair of DGC and PDE to control the expression of specific c-di-GMP signaling pathways.

Metaproteomics reveals insights into microbial structure, interactions, and dynamic regulation in defined communities as they respond to environmental disturbance

Background Microbe-microbe interactions between members of the plant rhizosphere are important but remain poorly understood. A more comprehensive understanding of the molecular mechanisms used by microbes to cooperate, compete, and persist has been challenging because of the complexity of natural ecosystems and the limited control over environmental factors. One strategy to address this challenge relies on studying complexity in a progressive manner, by first building a detailed understanding of relatively simple subsets of the community and then achieving high predictive power through combining different building blocks (e.g., hosts, community members) for different environments. Herein, we coupled this reductionist approach with high-resolution mass spectrometry-based metaproteomics to study molecular mechanisms driving community assembly, adaptation, and functionality for a defined community of ten taxonomically diverse bacterial members of Populus deltoides rhizosphere co-cultured either in a complex or defined medium. Results Metaproteomics showed this defined community assembled into distinct microbiomes based on growth media that eventually exhibit composition and functional stability over time. The community grown in two different media showed variation in composition, yet both were dominated by only a few microbial strains. Proteome-wide interrogation provided detailed insights into the functional behavior of each dominant member as they adjust to changing community compositions and environments. The emergence and persistence of select microbes in these communities were driven by specialization in strategies including motility, antibiotic production, altered metabolism, and dormancy. Protein-level interrogation identified post-translational modifications that provided additional insights into regulatory mechanisms influencing microbial adaptation in the changing environments. Conclusions This study provides high-resolution proteome-level insights into our understanding of microbe-microbe interactions and highlights specialized biological processes carried out by specific members of assembled microbiomes to compete and persist in changing environmental conditions. Emergent properties observed in these lower complexity communities can then be re-evaluated as more complex systems are studied and, when a particular property becomes less relevant, higher-order interactions can be identified.

Comparative Bacterial Metagenomics of Cnidoscolus aconitifolius (Mill.) I. M. Johnston and Other Leafy Vegetables

Aims: Vegetables provide a favourable habitat for diverse populations of microorganisms. Some vegetables, especially the ones used in salads are ready-to-eat food products and some phyllosphere bacteria might contribute to the prolonged presence of human food-borne pathogens in these vegetables. Methodology: Phyllosphere bacteria associated with Cnidoscolus aconitifolius were evaluated using a culture-independent approach, Illumina MiSeq platform of 16S rRNA gene sequencing and then compared with publicly available data obtained from Spinacia oleracea (spinach) and Lactuca sativa (lettuce) on GenBank. Results: The results from this study showed that the three vegetables harbor diverse bacterial organisms. Eighty-three (83) Operational Taxonomic Units (OTUs) assigned to five phyla were obtained from C. aconitifolius phyllosphere. The most predominant phyla across studied vegetables were: Proteobacteria (74.79%), Actinobacteria (8.69%) and Firmicutes (7.37%). Potential human pathogenic species such as Bacillus spp., Enterococcus spp., Staphylococcus spp., Klebsiella spp., and Pseudomonas spp. were also present in lettuce and spinach. Bacteria with potential for antibiotic production, anti-microbial and antibiotic resistant genes belong to the families Bacillaceae, Streptomycetaceae, Pseudomonaceae, Enterobacteriaceae, Staphylococcaceae, Enterococcaceae and Streptococcaceae. The most abundant taxa obtained from this study were Pseudomonas, Erwinia, Brachybacterium, Megasphaera, Janthinobacterium, Sphingomonas and Lactobacillus. Conclusion: Our result successfully determined the relative abundance of potential human and plant pathogens in the leafy vegetables and also showed the bacterial community structure in the studied vegetables.

Spike Formation Is a Turning Point Determining Wheat Root Microbiome Abundance, Structures and Functions

Root selection of their associated microbiome composition and activities is determined by the plant’s developmental stage and distance from the root. Total gene abundance, structure and functions of root-associated and rhizospheric microbiomes were studied throughout wheat growth season under field conditions. On the root surface, abundance of the well-known wheat colonizers Proteobacteria and Actinobacteria decreased and increased, respectively, during spike formation, whereas abundance of Bacteroidetes was independent of spike formation. Metagenomic analysis combined with functional co-occurrence networks revealed a significant impact of plant developmental stage on its microbiome during the transition from vegetative growth to spike formation. For example, gene functions related to biofilm and sensorial movement, antibiotic production and resistance and carbons and amino acids and their transporters. Genes associated with these functions were also in higher abundance in root vs. the rhizosphere microbiome. We propose that abundance of transporter-encoding genes related to carbon and amino acid, may mirror the availability and utilization of root exudates. Genes related to antibiotic resistance mechanisms were abundant during vegetative growth, while after spike formation, genes related to the biosynthesis of various antibiotics were enriched. This observation suggests that during root colonization and biofilm formation, bacteria cope with competitor’s antibiotics, whereas in the mature biofilm stage, they invest in inhibiting new colonizers. Additionally, there is higher abundance of genes related to denitrification in rhizosphere compared to root-associated microbiome during wheat growth, possibly due to competition with the plant over nitrogen in the root vicinity. We demonstrated functional and phylogenetic division in wheat root zone microbiome in both time and space: pre- and post-spike formation, and root-associated vs. rhizospheric niches. These findings shed light on the dynamics of plant–microbe and microbe–microbe interactions in the developing root zone.