Antibiotic-producing Actinobacteria Research Articles

From the inside out: Were the cuticular Pseudonocardia bacteria of fungus-farming ants originally domesticated as gut symbionts?

The mutualistic interaction specificity between attine ants and antibiotic-producing Actinobacteria has been controversial because Pseudonocardia strains cannot always be isolated from worker cuticles across attine ant species, while other actinobacteria can apparently replace Pseudonocardia and also inhibit growth of Escovopsis mycopathogens. Here we report that across field samples of Panamanian species: (i) Cuticular Pseudonocardia were largely restricted to species in the crown of the attine phylogeny and their appearance likely coincided with the first attines colonizing Central/North America. (ii) The phylogenetically basal attines almost always had cuticular associations with other Actinobacteria than Pseudonocardia. (iii) The sub-cuticular glands nourishing cuticular bacteria appear to be homologous throughout the phylogeny, consistent with an ancient general attine-Actinobacteria association. (iv) The basal attine species investigated always had Pseudonocardia as gut symbionts while Pseudonocardia presence appeared mutually exclusive between cuticular and gut microbiomes. (v) Gut-associated Pseudonocardia were phylogenetically ancestral while cuticular symbionts formed a derived crown group within the Pseudonocardia phylogeny. We further show that laboratory colonies often secondarily acquire cuticular Actinobacteria that they do not associate with in the field, suggesting that many previous studies were uninformative for questions of co-adaptation in the wild. An exhaustive literature survey showed that published studies concur with our present results, provided that they analyzed field colonies and that Actinobacteria were specifically isolated from worker cuticles shortly after field collection. Our results offer several testable hypotheses for a better overall understanding of attine-Pseudonocardia interaction dynamics and putative coevolution throughout the Americas.

Exploring the Antimicrobial and Antioxidant Activities of Streptomyces sp. EIZ2 Isolated from Moroccan Agricultural Soil

Antibiotics play a crucial role in preserving and improving public health, saving millions of lives every year. However, their effectiveness is currently under threat due to the ability of bacteria to adapt and develop resistance to these treatments. Therefore, this study was carried out on two soil samples collected in two areas of Arba Aounate, Sidi Bennour province, Morocco, to identify natural antibiotic-producing Actinobacteria capable of combating multi-drug-resistant (MDR) bacteria. A primary screening revealed that of the 50 isolates, 16 exhibited antimicrobial activity against Pseudomonas aeruginosa ATCC 27,853, Staphylococcus aureus ATCC 25,923, Escherichia coli ATCC 25,922, and Candida albicans ATTC 60193. A secondary screening showed that of the 16 isolates, only EIZ1 and EIZ2 isolates displayed outstanding antimicrobial and antifungal activity against 6 MDR bacteria, including Escherichia coli 19L2418, Listeria monocytogenes, Proteus sp. 19K1313, Klebsiella pneumoniae 19K 929, Proteus vulgaris 16C1737, and Klebsiella pneumoniae 20B1572. These two isolates were also characterized culturally, morphologically, physiologically, and biochemically. Afterward, the amplification of 16S rRNA revealed that isolate EIZ2 was 99.06% strongly related to the genus Streptomyces. Furthermore, this extract exhibits strong antioxidant activity against DPPH and ABTS free radicals and elevated ferric-reducing antioxidant power. A significant (p < 0.0001) positive correlation was observed between antioxidant activities and total phenolic and flavonoid contents. A GC–MS analysis of the ethyl acetate extract revealed the presence of 10 compounds, mainly diethyl phthalate (97%) and benzeneacetic acid (94%). This research demonstrates that Streptomyces sp. strain EIZ2 represents a potential source of antimicrobial and antioxidant compounds. These compounds could offer considerable potential as therapeutic agents, paving the way for future developments in medical applications.

Antimicrobial Profile of Actinomycin D Analogs Secreted by Egyptian Desert Streptomyces sp. DH7.

Egyptian deserts are an underexplored ecological niche, especially the Sinai Peninsula. In our recent study, we explored this extreme environment and shed light on the bioactive capabilities of desert Actinobacteria isolated from Sinai. Fifty desert Actinobacteria were isolated from the Sinai desert using mineral salt media, basal media, and starch casein media. The filtrate of Streptomyces sp. DH 7 displayed a high inhibitory effect against multidrug-resistant Staphylococcus aureus (MRSA) strains. The 16S rDNA sequencing confirmed that isolate DH7 belongs to the genus Streptomyces. The NJ phylogenetic tree showed relatedness to the Streptomyces flavofuscus strain NRRL B-2594 and Streptomyces pratensis strain ch24. The minimum inhibitory concentrations against MRSA were 16 and 32 μg/μL. Chemical investigation of the ethyl acetate extract of Streptomyces sp. DH7 led to the isolation and purification of natural products 1–4. Structure elucidation of the purified compounds was performed using detailed spectroscopic analysis including 1 and 2D NMR, and ESI-MS spectrometry. To the best of our knowledge, this is the first report for the isolation of compounds 1–4 from a natural source, while synthetic analogs were previously reported in the literature. Compounds 3–4 were identified as actinomycin D analogues and this is the first report for the production of actinomycin D analogs from the Sinai desert with an inhibitory effect against MRSA. We indorse further study for this analog that can develop enhanced antimicrobial activities. We confirm that the desert ecosystems in Egypt are rich sources of antibiotic-producing Actinobacteria.

Vanishing benefits - The loss of actinobacterial symbionts at elevated temperatures.

Only a few insect species are known to engage in symbiotic associations with antibiotic-producing Actinobacteria and profit from this kind of protection against pathogens. However, it still remains elusive how widespread the symbiotic interactions with Actinobacteria in other organisms are and how these partnerships benefit the hosts in terms of the growth and survival. We characterized a drastic temperature-induced change in the occurrence of Actinobacteria in the gut of the terrestrial isopod Porcellio scaber reared under two different temperature (15 °C and 22 °C) and oxygen conditions (10% and 22% O2) using 16S rRNA gene sequencing. We show that the relative abundance of actinobacterial gut symbionts correlates with increased host growth at lower temperature. Actinobacterial symbionts were almost completely absent at 22 °C under both high and low oxygen conditions. In addition, we identified members of nearly half of the known actinobacterial families in the isopod microbiome, and most of these include members that are known to produce antibiotics. Our study suggests that hosting diverse actinobacterial symbionts may provide conditions favorable for host growth. These findings show how a temperature-driven decline in microbiome diversity may cause a loss of beneficial functions with negative effects on ectotherms.

Convergent evolution of complex structures for ant–bacterial defensive symbiosis in fungus-farming ants

Evolutionary adaptations for maintaining beneficial microbes are hallmarks of mutualistic evolution. Fungus-farming "attine" ant species have complex cuticular modifications and specialized glands that house and nourish antibiotic-producing Actinobacteria symbionts, which in turn protect their hosts' fungus gardens from pathogens. Here we reconstruct ant-Actinobacteria evolutionary history across the full range of variation within subtribe Attina by combining dated phylogenomic and ultramorphological analyses. Ancestral-state analyses indicate the ant-Actinobacteria symbiosis arose early in attine-ant evolution, a conclusion consistent with direct observations of Actinobacteria on fossil ants in Oligo-Miocene amber. qPCR indicates that the dominant ant-associated Actinobacteria belong to the genus Pseudonocardia Tracing the evolutionary trajectories of Pseudonocardia-maintaining mechanisms across attine ants reveals a continuum of adaptations. In Myrmicocrypta species, which retain many ancestral morphological and behavioral traits, Pseudonocardia occur in specific locations on the legs and antennae, unassociated with any specialized structures. In contrast, specialized cuticular structures, including crypts and tubercles, evolved at least three times in derived attine-ant lineages. Conspicuous caste differences in Pseudonocardia-maintaining structures, in which specialized structures are present in worker ants and queens but reduced or lost in males, are consistent with vertical Pseudonocardia transmission. Although the majority of attine ants are associated with Pseudonocardia, there have been multiple losses of bacterial symbionts and bacteria-maintaining structures in different lineages over evolutionary time. The early origin of ant-Pseudonocardia mutualism and the multiple evolutionary convergences on strikingly similar anatomical adaptations for maintaining bacterial symbionts indicate that Pseudonocardia have played a critical role in the evolution of ant fungiculture.

Elucidation of the genetic mechanisms contributing to moenomycin resistance in actinobacteria

Aim. Moenomycins are phosphoglycolipid antibiotics produced almost exclusively by representatives of genus Streptomyces. These antibiotics directly inhibit peptidoglycan glycosyltransferases and are extremely active against cocci. Here we studied how antibiotic-producing actinobacteria protect themselves from toxic action of moenomycins. Methods. Microbiological and molecular genetic approaches were combined to reveal intrinsic levels and distribution of moenomycin resistance across actinobacteria genera, and to pinpoint genes contributing to moenomycin resistance in model strain Streptomyces coelicolor M145. Results. Out of 51 actinobacterial species (90 % of which Streptomyces) being tested, only Streptomyces albus J1074 turned out to be highly susceptible to moenomycin A, although resistant variants can be facilely raised. Several classes of mutations increased level of susceptibility of S. coelicolor to moenomycin, although in no case the latter was equal to what we observed in J1074 strain. Conclusions. Moenomycin resistance is widespread across actinobacteria, and it most likely is caused by a combination factors, such as richly decorated cell wall and organization of divisome apparatus. It is possible that moenomycin resistance mechanisms operating in actinobacteria and pathogenic cocci are different.
 Keywords: moenomycin, antibiotic resistance, peptidoglycan.

Design, development and application of whole-cell based antibiotic-specific biosensor

Synthetic biology techniques hold great promise for optimising the production of natural products by microorganisms. However, evaluating the phenotype of a modified bacterium represents a major bottleneck to the engineering cycle – particularly for antibiotic-producing actinobacteria strains, which grow slowly and are challenging to genetically manipulate. Here, we report the generation and application of antibiotic-specific whole-cell biosensor derived from TetR transcriptional repressor for use in identifying and optimising antibiotic producers. The constructed biosensor was successfully used to improve production of polyketide antibiotic pamamycin. However, an initial biosensor based on native genetic elements had inadequate dynamic and operating ranges. To overcome these limitations, we fine-tuned biosensor performance through alterations of the promoter and operator of output module and the ligand affinity of transcription factor module, which enabled us to deduce recommendations for building and application of actinobacterial biosensors.

High-Throughput Sequencing Analysis of the Actinobacterial Spatial Diversity in Moonmilk Deposits.

Moonmilk are cave carbonate deposits that host a rich microbiome, including antibiotic-producing Actinobacteria, making these speleothems appealing for bioprospecting. Here, we investigated the taxonomic profile of the actinobacterial community of three moonmilk deposits of the cave “Grotte des Collemboles” via high-throughput sequencing of 16S rRNA amplicons. Actinobacteria was the most common phylum after Proteobacteria, ranging from 9% to 23% of the total bacterial population. Next to actinobacterial operational taxonomic units (OTUs) attributed to uncultured organisms at the genus level (~44%), we identified 47 actinobacterial genera with Rhodoccocus (4 OTUs, 17%) and Pseudonocardia (9 OTUs, ~16%) as the most abundant in terms of the absolute number of sequences. Streptomycetes presented the highest diversity (19 OTUs, 3%), with most of the OTUs unlinked to the culturable Streptomyces strains that were previously isolated from the same deposits. Furthermore, 43% of the OTUs were shared between the three studied collection points, while 34% were exclusive to one deposit, indicating that distinct speleothems host their own population, despite their nearby localization. This important spatial diversity suggests that prospecting within different moonmilk deposits should result in the isolation of unique and novel Actinobacteria. These speleothems also host a wide range of non-streptomycetes antibiotic-producing genera, and should therefore be subjected to methodologies for isolating rare Actinobacteria.

Evidence for Widespread Associations between Neotropical Hymenopteran Insects and Actinobacteria.

The evolutionary success of hymenopteran insects has been associated with complex physiological and behavioral defense mechanisms against pathogens and parasites. Among these strategies are symbiotic associations between Hymenoptera and antibiotic-producing Actinobacteria, which provide protection to insect hosts. Herein, we examine associations between culturable Actinobacteria and 29 species of tropical hymenopteran insects that span five families, including Apidae (bees), Vespidae (wasps), and Formicidae (ants). In total, 197 Actinobacteria isolates were obtained from 22 of the 29 different insect species sampled. Through 16S rRNA gene sequences of 161 isolates, we show that 91% of the symbionts correspond to members of the genus Streptomyces with less common isolates belonging to Pseudonocardia and Amycolatopsis. Electron microscopy revealed the presence of filamentous bacteria with Streptomyces morphology in brood chambers of two different species of the eusocial wasps. Four fungal strains in the family Ophiocordycipitacea (Hypocreales) known to be specialized insect parasites were also isolated. Bioassay challenges between the Actinobacteria and their possible targeted pathogenic antagonist (both obtained from the same insect at the genus or species level) provide evidence that different Actinobacteria isolates produced antifungal activity, supporting the hypothesis of a defensive association between the insects and these microbe species. Finally, phylogenetic analysis of 16S rRNA and gyrB demonstrate the presence of five Streptomyces lineages associated with a broad range of insect species. Particularly our Clade I is of much interest as it is composed of one 16S rRNA phylotype repeatedly isolated from different insect groups in our sample. This phylotype corresponds to a previously described lineage of host-associated Streptomyces. These results suggest Streptomyces Clade I is a Hymenoptera host-associated lineage spanning several new insect taxa and ranging from the American temperate to the Neotropical region. Our work thus provides important insights into the widespread distribution of Actinobacteria and hymenopteran insects associations, while also pointing at novel resources that could be targeted for the discovery of active natural products with great potential in medical and biotechnological applications.

Dissemination of antibiotic resistance genes from antibiotic producers to pathogens

It has been hypothesized that some antibiotic resistance genes (ARGs) found in pathogenic bacteria derive from antibiotic-producing actinobacteria. Here we provide bioinformatic and experimental evidence supporting this hypothesis. We identify genes in proteobacteria, including some pathogens, that appear to be closely related to actinobacterial ARGs known to confer resistance against clinically important antibiotics. Furthermore, we identify two potential examples of recent horizontal transfer of actinobacterial ARGs to proteobacterial pathogens. Based on this bioinformatic evidence, we propose and experimentally test a ‘carry-back’ mechanism for the transfer, involving conjugative transfer of a carrier sequence from proteobacteria to actinobacteria, recombination of the carrier sequence with the actinobacterial ARG, followed by natural transformation of proteobacteria with the carrier-sandwiched ARG. Our results support the existence of ancient and, possibly, recent transfers of ARGs from antibiotic-producing actinobacteria to proteobacteria, and provide evidence for a defined mechanism.

Bacterial community composition and diversity in an ancestral ant fungus symbiosis.

Fungus-farming ants (Hymenoptera: Formicidae, Attini) exhibit some of the most complex microbial symbioses because both macroscopic partners (ants and fungus) are associated with a rich community of microorganisms. The ant and fungal microbiomes are thought to serve important beneficial nutritional and defensive roles in these symbioses. While most recent research has investigated the bacterial communities in the higher attines (e.g. the leaf-cutter ant genera Atta and Acromyrmex), which are often associated with antibiotic-producing Actinobacteria, very little is known about the microbial communities in basal lineages, labeled as 'lower attines', which retain the ancestral traits of smaller and more simple societies. In this study, we used 16S amplicon pyrosequencing to characterize bacterial communities of the lower attine ant Mycocepurus smithii among seven sampling sites in central Panama. We discovered that ant and fungus garden-associated microbiota were distinct from surrounding soil, but unlike the situation in the derived fungus-gardening ants, which show distinct ant and fungal microbiomes, microbial community structure of the ants and their fungi were similar. Another surprising finding was that the abundance of actinomycete bacteria was low and instead, these symbioses were characterized by an abundance of Lactobacillus and Pantoea bacteria. Furthermore, our data indicate that Lactobacillus strains are acquired from the environment rather than acquired vertically.

Broad Escovopsis-inhibition activity of Pseudonocardia associated with Trachymyrmex ants.

Attine ants maintain an association with antibiotic-producing Actinobacteria found on their integuments. Evidence supports these bacteria as auxiliary symbionts that help ants to defend the fungus gardens against pathogens. Using Pseudonocardia strains isolated from Trachymyrmex ants, we tested whether the inhibitory capabilities of such strains are restricted to Escovopsis parasites that infect gardens of this ant genus. Twelve Pseudonocardia strains were tested in in vitro bioassays against Escovopsis strains derived from fungus gardens of Trachymyrmex (n = 1) and leaf-cutting ants (n = 3). Overall, significant differences were observed in the mycelial growth among each Escovopsis strain in the presence of Pseudonocardia. Particularly, Escovopsis from Acromyrmex and Trachymyrmex were the most inhibited strains in comparison to Escovopsis isolated from Atta. This result suggests that Pseudonocardia isolated from Trachymyrmex possibly secrete antimicrobial compounds effective against diverse Escovopsis strains. The fact that Trachymyrmex ants harbour Pseudonocardia strains with broad spectrum of activity and its defensive role on attine gardens are discussed.

Analysis of the bacterial communities associated with two ant–plant symbioses

Insect fungiculture is practiced by ants, termites, beetles, and gall midges and it has been suggested to be widespread among plant–ants. Some of the insects engaged in fungiculture, including attine ants and bark beetles, are known to use symbiotic antibiotic-producing actinobacteria to protect themselves and their fungal cultivars against infection. In this study, we analyze the bacterial communities on the cuticles of the plant–ant genera Allomerus and Tetraponera using deep sequencing of 16S rRNA. Allomerus ants cultivate fungus as a building material to strengthen traps for prey, while Tetraponera ants cultivate fungus as a food source. We report that Allomerus and Tetraponera microbiomes contain >75% Proteobacteria and remarkably the bacterial phyla that dominate their cuticular microbiomes are very similar despite their geographic separation (South America and Africa, respectively). Notably, antibiotic-producing actinomycete bacteria represent a tiny fraction of the cuticular microbiomes of both Allomerus and Tetraponera spp. and instead they are dominated by γ-proteobacteria Erwinia and Serratia spp. Both these phyla are known to contain antibiotic-producing species which might therefore play a protective role in these ant–plant systems.

Exploring the Potential for Actinobacteria as Defensive Symbionts in Fungus-Growing Termites

In fungus-growing termites, fungi of the subgenus Pseudoxylaria threaten colony health through substrate competition with the termite fungus (Termitomyces). The potential mechanisms with which termites suppress Pseudoxylaria have remained unknown. Here we explore if Actinobacteria potentially play a role as defensive symbionts against Pseudoxylaria in fungus-growing termites. We sampled for Actinobacteria from 30 fungus-growing termite colonies, spanning the three main termite genera and two geographically distant sites. Our isolations yielded 360 Actinobacteria, from which we selected subsets for morphological (288 isolates, grouped in 44 morphotypes) and for 16S rRNA (35 isolates, spanning the majority of morphotypes) characterisation. Actinobacteria were found throughout all sampled nests and colony parts and, phylogenetically, they are interspersed with Actinobacteria from origins other than fungus-growing termites, indicating lack of specificity. Antibiotic-activity screening of 288 isolates against the fungal cultivar and competitor revealed that most of the Actinobacteria-produced molecules with antifungal activity. A more detailed bioassay on 53 isolates, to test the specificity of antibiotics, showed that many Actinobacteria inhibit both Pseudoxylaria and Termitomyces, and that the cultivar fungus generally is more susceptible to inhibition than the competitor. This suggests that either defensive symbionts are not present in the system or that they, if present, represent a subset of the community isolated. If so, the antibiotics must be used in a targeted fashion, being applied to specific areas by the termites. We describe the first discovery of an assembly of antibiotic-producing Actinobacteria occurring in fungus-growing termite nests. However, due to the diversity found, and the lack of both phylogenetic and bioactivity specificity, further work is necessary for a better understanding of the putative role of antibiotic-producing bacteria in the fungus-growing termite mutualistic system.

Fungus-growing Allomerus ants are associated with antibiotic-producing actinobacteria

Fungus-growing attine ants use natural-product antibiotics produced by mutualist actinobacteria as 'weedkillers' in their fungal gardens. Here we report for the first time that fungus-growing Allomerus ants, which lie outside the tribe Attini, are associated with antifungal-producing actinobacteria, which offer them protection against non-cultivar fungi isolated from their ant-plants.

Preliminary In Vitro Insights into the Use of Natural Fungal Pathogens of Leaf-cutting Ants as Biocontrol Agents

Leaf-cutting ants are one of the main herbivores of the Neotropics, where they represent an important agricultural pest. These ants are particularly difficult to control because of the complex network of microbial symbionts. Leaf-cutting ants have traditionally been controlled through pesticide application, but there is a need for alternative, more environmentally friendly, control methods such as biological control. Potential promising biocontrol candidates include the microfungi Escovopsis spp. (anamorphic Hypocreales), which are specialized pathogens of the fungi the ants cultivate for food. These pathogens are suppressed through ant behaviors and ant-associated antibiotic-producing Actinobacteria. In order to be an effective biocontrol agent, Escovopsis has to overcome these defenses. Here, we evaluate, using microbial in vitro assays, whether defenses in the ant-cultivated fungus strain (Leucoagaricus sp.) and Actinobacteria from the ant pest Acromyrmex lundii have the potential to limit the use of Escovopsis in biocontrol. We also explore, for the first time, possible synergistic biocontrol between Escovopsis and the entomopathogenic fungus Lecanicillium lecanii. All strains of Escovopsis proved to overgrow A.lundii cultivar in less than 7days, with the Escovopsis strain isolated from a different leaf-cutting ant species being the most efficient. Escovopsis challenged with a Streptomyces strain isolated from A. lundii did not exhibit significant growth inhibition. Both results are encouraging for the use of Escovopsis as a biocontrol agent. Although we found that L. lecanii can suppress the growth of the cultivar, it also had a negative impact on Escovopsis, making the success of simultaneous use of these two fungi for biocontrol of A. lundii questionable.

Insect Symbioses: A Case Study of Past, Present, and Future Fungus-growing Ant Research

Fungus-growing ants (Attini: Formicidae) engage in an obligate mutualism with fungi they cultivate for food. Although biologists have been fascinated with fungus-growing ants since the resurgence of natural history in the modern era, the early stages of research focused mainly on the foraging behavior of the leaf-cutters (the most derived attine lineage). Indeed, the discovery that the ants actually use leaf fragments to manure a fungus did not come until the 1800s. More recently, three additional microbial symbionts have been described, including specialized microfungal parasites of the ant's fungus garden, antibiotic-producing actinobacteria that help protect the fungus garden from the parasite, and a black yeast that parasitizes the ant-actinobacteria mutualism. The fungus-growing ant symbiosis serves as a particularly useful model system for studying insect-microbe symbioses, because, to date, it contains four well-characterized microbial symbionts, including mutualists and parasites that encompass micro-fungi, macro-fungi, yeasts, and bacteria. Here, we discuss approaches for studying insect-microbe symbioses, using the attine ant-microbial symbiosis as our framework. We draw attention to particular challenges in the field of symbiosis, including the establishment of symbiotic associations and symbiont function. Finally, we discuss future directions in insect-microbe research, with particular focus on applying recent advances in DNA sequencing technologies.