Regulation of Antibiotic Resistance Genes on Agricultural Land Is Dependent on Both Choice of Organic Amendment and Prevalence of Predatory Bacteria.

Antibiotic resistance genes (ARGs) are widespread in the environment, and soils, specifically, are hotspots for microorganisms with inherent antibiotic resistance. Manure and sludge used as fertilizers in agricultural production have been shown to contain vast amounts of ARGs, and due to continued applications, ARGs accumulate in agricultural soils. Some soils, however, harbor a resilience capacity that could depend on specific soil properties, as well as the presence of predatory bacteria that are able to hydrolyse living bacteria, including bacteria of clinical importance. The objectives of this study were to (i) investigate if the antibiotic resistance profile of the soil microbiota could be differently affected by the addition of cow manure, chicken manure, and sludge, and (ii) investigate if the amendments had an effect on the presence of predatory bacteria. The three organic amendments were mixed separately with a field soil, divided into pots, and incubated in a greenhouse for 28 days. Droplet digital PCR (ddPCR) was used to quantify three ARGs, two predatory bacteria, and total number of bacteria. In this study, we demonstrated that the choice of organic amendment significantly affected the antibiotic resistance profile of soil, and promoted the growth of predatory bacteria, while the total number of bacteria was unaffected.

Encapsulated predatory bacteria efficiently protect potato tubers from soft rot disease.

Soft rot Pectobacteriaceae (SRP) are a group of destructive Gram-negative phytopathogens that can infect a wide range of plant hosts, including potatoes. There are no effective control agents available against SRP, making their management challenging. We have developed a novel approach to protect potato tubers against SRP. It makes use of encapsulated predatory Bdellovibrio bacteriovorus bacteria that upon release from a polymeric carrier, prey upon SRP. We applied a carrageenan-trehalose-based formulation containing a B. bacteriovorus HD100 predator to prevent soft-rot disease development in potato tubers, under various conditions. The dried formulation exhibited very high stability over an eighteen-month period at room temperature (˜25ºC), in contrast to unencapsulated suspensions of the predator, in which viability decreased rapidly below detection level. The rehydrated formulation was as efficient as freshly grown unencapsulated predators, and provided high protection in potted potato tubers, displaying an average of 50% reduction in disease parameters (e.g. tissue decay and disease index) under controlled conditions at 7-days post-inoculation and planting. The protective effect provided by this formulation was maintained in longer-term trials (28 days) conducted in larger vessels within a net-house under natural climate conditions, highlighting its potential for practical application in the field.

Flagellar stator genes control a trophic shift from obligate to facultative predation and biofilm formation in a bacterial predator.

The bacterial predator Bdellovibrio bacteriovorus is considered to be obligatorily prey (host)-dependent (H-D), and thus unable to form biofilms. However, spontaneous host-independent (H-I) variants grow axenically and can form robust biofilms. A screen of 350 H-I mutants revealed that single mutations in stator genes fliL or motA were sufficient to generate flagellar motility-defective H-I strains able to adhere to surfaces but unable to develop biofilms. The variants showed large transcriptional shifts in genes related to flagella, prey-invasion, and cyclic-di-GMP (CdG), as well as large changes in CdG cellular concentration relative to the H-D parent. The introduction of the parental fliL allele resulted in a full reversion to the H-D phenotype, but we propose that specific interactions between stator proteins prevented functional complementation by fliL paralogs. In contrast, specific mutations in a pilus-associated protein (Bd0108) mutant background were necessary for biofilm formation, including secretion of extracellular DNA (eDNA), proteins, and polysaccharides matrix components. Remarkably, fliL disruption strongly reduced biofilm development. All H-I variants grew similarly without prey, showed a strain-specific reduction in predatory ability in prey suspensions, but maintained similar high efficiency in prey biofilms. Population-wide allele sequencing suggested additional routes to host independence. Thus, stator and invasion pole-dependent signaling control the H-D and the H-I biofilm-forming phenotypes, with single mutations overriding prey requirements, and enabling shifts from obligate to facultative predation, with potential consequences on community dynamics. Our findings on the facility and variety of changes leading to facultative predation also challenge the concept of Bdellovibrio and like organisms being obligate predators. IMPORTANCE: The ability of bacteria to form biofilms is a central research theme in biology, medicine, and the environment. We show that cultures of the obligate (host-dependent) "solitary" predatory bacterium Bdellovibrio bacteriovorus, which cannot replicate without prey, can use various genetic routes to spontaneously yield host-independent (H-I) variants that grow axenically (as a single species, in the absence of prey) and exhibit various surface attachment phenotypes, including biofilm formation. These routes include single mutations in flagellar stator genes that affect biofilm formation, provoke motor instability and large motility defects, and disrupt cyclic-di-GMP intracellular signaling. H-I strains also exhibit reduced predatory efficiency in suspension but high efficiency in prey biofilms. These changes override the requirements for prey, enabling a shift from obligate to facultative predation, with potential consequences on community dynamics.

Unbalanced predatory communities and a lack of microbial degraders characterize the microbiota of a highly sewage-polluted Eastern-Mediterranean stream.

Wastewater pollution of water resources takes a heavy toll on humans and on the environment. In highly polluted water bodies, self-purification is impaired, as the capacity of the riverine microbes to regenerate the ecosystem is overwhelmed. To date, information on the composition, dynamics and functions of the microbial communities in highly sewage-impacted rivers is limited, in particular in arid and semi-arid environments. In this year-long study of the highly sewage-impacted Al-Nar/Kidron stream in the Barr al-Khalil/Judean Desert east of Jerusalem, we show, using 16S and 18S rRNA gene-based community analysis and targeted qPCR, that both the bacterial and micro-eukaryotic communities, while abundant, exhibited low stability and diversity. Hydrolyzers of organics compounds, as well as nitrogen and phosphorus recyclers were lacking, pointing at reduced potential for regeneration. Furthermore, facultative bacterial predators were almost absent, and the obligate predators Bdellovibrio and like organisms were found at very low abundance. Finally, the micro-eukaryotic predatory community differed from those of other freshwater environments. The lack of essential biochemical functions may explain the stream's inability to self-purify, while the very low levels of bacterial predators and the disturbed assemblages of micro-eukaryote predators present in Al-Nar/Kidron may contribute to community instability and disfunction.

Self-demise of soft rot bacteria by activation of microbial predators by pectin-based carriers.

Soft rot pectobacteria (SRP) are phytopathogens of the genera Pectobacterium and Dickeya that cause soft rots on a wide range of crops and ornamental plants. SRP produce plant cell wall degrading enzymes (PCWDEs), including pectinases. Bdellovibrio and like organisms are bacterial predators that can prey on a variety of Gram-negative species, including SRP. In this research, a low methoxyl pectin (LMP)-based immobilization system for B. bacteriovorus is established. It takes advantage that pectin residues induce PCWDE secretion by the pathogens, bringing upon the release of the encapsulated predators. Three commercial LMPs differing in the degree of esterification (DE) and amidation (DA) were tested as potential carriers, by examining their effect on SRP growth, enzymes secretion and substrate breakdown. A clear advantage was observed for pectin 5 CS with the lowest DE and DA content. The degradation of 5 CS pectin-based carriers was further optimized by reducing cross-linker and pectin concentration, by adding gelatin and by dehydration. This resulted in SRP-induced disintegration of the carrier within 72 h. The released encapsulated predator caused a large decrease in SRP population while its own significantly increased, demonstrating the efficiency of this system in which the pathogen brings about its own demise.

Biological control of soft rot in potato by κ-carrageenan carriers encapsulated microbial predators.

The Pectobacterium and Dickeya pectinolytic bacteria are phytopathogens responsible for several macerating diseases on a wide range of crops and ornamental plants. Recently, bacterial predators belonging to the Bdellovibrio and like organisms (BALOs) were shown to efficiently prey on these rot-causing bacteria and reduce soft rot-induced potato slice maceration. In the current research, our novel approach aimed at developing and studying a κ-carrageenan-based encapsulation system for fast-release of entrapped B. bacteriovorus HD100 in high numbers to prevent bacterial soft-rot infections. κ-carrageenan-dried carriers swelled and dissolved upon immersion in water due to a loss of potassium ions which are the main cross-linking agents. Survival rates of the predators after drying were higher for immobilized bdelloplasts (e.g., predator inside the host) compared to attack phase (host-searching, AP) cells, and with the addition of the osmoprotectant trehalose to the carriers. Released encapsulated predators preyed efficiently on soft rot bacteria, with bdelloplasts performing better as compared to AP cells. However, predation dynamics were influenced by the type of added osmoprotectant. Carrageenan-trehalose carriers encapsulating predators were able to reduce soft-rot disease in situ using a potato slice assay. To our knowledge, this research is the first to explore the potential of encapsulated BALOs against phytopathogens. KEY POINTS: • Dissolution of the carriers was affected by potassium concentration in the system. • Encapsulation of bdelloplasts with trehalose best maintained the predator viability. • The encapsulated predators efficiently controlled soft rot in vitro and in situ.

Identifying protein function and functional links based on large-scale co-occurrence patterns.

OBJECTIVE: The vast majority of known proteins have not been experimentally tested even at the level of measuring their expression, and the function of many proteins remains unknown. In order to decipher protein function and examine functional associations, we developed "Cliquely", a software tool based on the exploration of co-occurrence patterns. COMPUTATIONAL MODEL: Using a set of more than 23 million proteins divided into 404,947 orthologous clusters, we explored the co-occurrence graph of 4,742 fully sequenced genomes from the three domains of life. Edge weights in this graph represent co-occurrence probabilities. We use the Bron-Kerbosch algorithm to detect maximal cliques in this graph, fully-connected subgraphs that represent meaningful biological networks from different functional categories. MAIN RESULTS: We demonstrate that Cliquely can successfully identify known networks from various pathways, including nitrogen fixation, glycolysis, methanogenesis, mevalonate and ribosome proteins. Identifying the virulence-associated type III secretion system (T3SS) network, Cliquely also added 13 previously uncharacterized novel proteins to the T3SS network, demonstrating the strength of this approach. Cliquely is freely available and open source. Users can employ the tool to explore co-occurrence networks using a protein of interest and a customizable level of stringency, either for the entire dataset or for a one of the three domains-Archaea, Bacteria, or Eukarya.

Interactions between Bdellovibrio and like organisms and bacteria in biofilms: beyond predator-prey dynamics.

Bdellovibrio and like organisms (BALOs) prey on Gram-negative bacteria in the planktonic phase as well as in biofilms, with the ability to reduce prey populations by orders of magnitude. During the last few years, evidence has mounted for a significant ecological role for BALOs, with important implications for our understanding of microbial community dynamics as well as for applications against pathogens, including drug-resistant pathogens, in medicine, agriculture and aquaculture, and in industrial settings for various uses. However, our understanding of biofilm predation by BALOs is still very fragmentary, including gaps in their effect on biofilm structure, on prey resistance, and on evolutionary outcomes of both predators and prey. Furthermore, their impact on biofilms has been shown to reach beyond predation, as they are reported to reduce biofilm structures of non-prey cells (including Gram-positive bacteria). Here, we review the available literature on BALOs in biofilms, extending known aspects to potential mechanisms employed by the predators to grow in biofilms. Within that context, we discuss the potential ecological significance and potential future utilization of the predatory and enzymatic possibilities offered by BALOs in medical, agricultural and environmental applications.

Secretion systems play a critical role in resistance to predation by Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus, a Gram-negative predatory bacterium belonging to the Bdellovibrio and like organisms (BALOs), predates on Gram-negative bacteria. BALO strains differ in prey range but so far, the genetic basis of resistance against BALO predation is hardly understood. We developed a loss-of-function approach to screen for sensitive mutants in a library of strain M6, a predation-resistant strain of the plant pathogen Acidovorax citrulli. The screen is based on tracking the growth of a B. bacteriovorus strain expressing the fluorescent reporter Tdtomato in mutant pools to reveal predation-sensitive variants. Two independent loci were identified in mutant strains exhibiting significant levels of susceptibility to the predator. Genes in the two loci were analysed using both protein sequence homology and protein structure modeling. Both were secretion-related proteins and thus associated to the bacterial cell wall. Successful complementation of gspK, a gene encoding for a minor pseudopilin protein confirmed the involvement of the type II secretion system in A. citrulli M6 resistance. This proof of concept study shows that our approach can identify key elements of the BALO-prey interaction, and it validates the hypothesis that mutational changes in a single gene can drastically impact prey resistance to BALO predation.

Community and single cell analyses reveal complex predatory interactions between bacteria in high diversity systems.

A fundamental question in community ecology is the role of predator-prey interactions in food-web stability and species coexistence. Although microbial microcosms offer powerful systems to investigate it, interrogating the environment is much more arduous. Here, we show in a 1-year survey that the obligate predators Bdellovibrio and like organisms (BALOs) can regulate prey populations, possibly in a density-dependent manner, in the naturally complex, species-rich environments of wastewater treatment plants. Abundant as well as rarer prey populations are affected, leading to an oscillating predatory landscape shifting at various temporal scales in which the total population remains stable. Shifts, along with differential prey range, explain co-existence of the numerous predators through niche partitioning. We validate these sequence-based findings using single-cell sorting combined with fluorescent hybridization and community sequencing. Our approach should be applicable for deciphering community interactions in other systems.

Sex-Dependent Effects of the Microbiome on Foraging and Locomotion in Drosophila suzukii.

There is growing evidence that symbiotic microbes can influence multiple nutrition-related behaviors of their hosts, including locomotion, feeding, and foraging. However, how the microbiome affects nutrition-related behavior is largely unknown. Here, we demonstrate clear sexual dimorphism in how the microbiome affects foraging behavior of a frugivorous fruit fly, Drosophila suzukii. Female flies deprived of their microbiome (axenic) were consistently less active in foraging on fruits than their conventional counterparts, even though they were more susceptible to starvation and starvation-induced locomotion was notably more elevated in axenic than conventional females. Such behavioral change was not observed in male flies. The lag of axenic female flies but not male flies to forage on fruits is associated with lower oviposition by axenic flies, and mirrored by reduced food seeking observed in virgin females when compared to mated, gravid females. In contrast to foraging intensity being highly dependent on the microbiome, conventional and axenic flies of both sexes showed relatively consistent and similar fruit preferences in foraging and oviposition, with raspberries being preferred among the fruits tested. Collectively, this work highlights a clear sex-specific effect of the microbiome on foraging and locomotion behaviors in flies, an important first step toward identifying specific mechanisms that may drive the modulation of insect behavior by interactions between the host, the microbiome, and food.

Hidden Resistome: Enrichment Reveals the Presence of Clinically Relevant Antibiotic Resistance Determinants in Treated Wastewater-Irrigated Soils.

Treated-wastewater (TW) irrigation transfers antibiotic-resistant bacteria (ARB) to soil, but persistence of these bacteria is generally low due to resilience of the soil microbiome. Nonetheless, wastewater-derived bacteria and associated antibiotic resistance genes (ARGs) may persist below detection levels and potentially proliferate under copiotrophic conditions. To test this hypothesis, we exposed soils from microcosm, lysimeter, and field experiments to short-term enrichment in copiotroph-stimulating media. In microcosms, enrichment stimulated growth of multidrug-resistant Escherichia coli up to 2 weeks after falling below detection limits. Lysimeter and orchard soils irrigated in-tandem with either freshwater or TW were subjected to culture-based, qPCR and shotgun metagenomic analyses prior, and subsequent, to enrichment. Although native TW- and freshwater-irrigated soil microbiomes and resistomes were similar to each other, enrichment resulted in higher abundances of cephalosporin- and carbapenem-resistant Enterobacteriaceae and in substantial differences in the composition of microbial communities and ARGs. Enrichment stimulated ARG-harboring Bacillaceae in the freshwater-irrigated soils, whereas in TWW-irrigated soils, ARG-harboring γ-proteobacterial families Enterobacteriaceae and Moraxellaceae were more profuse. We demonstrate that TW-derived ARB and associated ARGs can persist at below detection levels in irrigated soils and believe that similar short-term enrichment strategies can be applied for environmental antimicrobial risk assessment in the future.

Effects of the prey landscape on the fitness of the bacterial predators Bdellovibrio and like organisms.

Bdellovibrio and like organisms (BALOs) are obligate predatory bacteria commonly encountered in the environment. In dual predator-prey cultures, prey accessibility ensures optimal feeding and replication and rapid BALO population growth. However, the environmental prey landscape is complex, as it also incorporates non-prey cells and other particles. These may act as decoys, generating unproductive encounters which in turn may affect both predator and prey population dynamics. In this study, we hypothesized that increasing decoy:prey ratios would bring about increasing costs on the predator's reproductive fitness. We also tested the hypothesis that different BALOs and decoys would have different effects. To this end, we constructed prey landscapes including periplasmic or epibiotic predators including two types of decoy under a large range of initial decoy:prey ratio, and mixed cultures containing multiple predators and prey. We show that as decoy:prey ratios increase, the maximal predator population sizes is reduced and the time to reach it significantly increases. We found that BALOs spent less time handling non-prey (including superinfection-immune invaded prey) than prey cells, and did not differentiate between efficient and less efficient prey. This may explain why in multiple predator and prey cultures, less preferred prey appear to act as decoy.

Shifting microbiomes complement life stage transitions and diet of the bird parasite Philornis downsi from the Galapagos Islands.

Domestication disconnects an animal from its natural environment and diet, imposing changes in the attendant microbial community. We examine these changes in Philornis downsi (Muscidae), an invasive parasitic fly of land birds in the Galapagos Islands. Using a 16S rDNA profiling approach we studied the microbiome of larvae and adults of wild and laboratory-reared populations. These populations diverged in their microbiomes, significantly more so in larval than in adult flies. In field-collected second-instar larvae, Klebsiella (70.3%) was the most abundant taxon, while in the laboratory Ignatzschineria and Providencia made up 89.2% of the community. In adults, Gilliamella and Dysgonomonas were key members of the core microbiome of field-derived females and males but had no or very low representation in the laboratory. Adult flies harbour sex-specific microbial consortia in their gut, as male core microbiomes were significantly dominated by Klebsiella. Thus, P. downsi microbiomes are dynamic and shift correspondingly with life cycle and diet. Sex-specific foraging behaviour of adult flies and nest conditions, which are absent in the laboratory, may contribute to shaping distinct larval, and adult male and female microbiomes. We discuss these findings in the context of microbe-host co-evolution and the implications for control measures.

A walk on the dirt: soil microbial forensics from ecological theory to the crime lab.

Forensics aims at using physical evidence to solve investigations with science-based principles, thus operating within a theoretical framework. This however is often rather weak, the exception being DNA-based human forensics that is well anchored in theory. Soil is a most commonly encountered, easily and unknowingly transferred evidence but it is seldom employed as soil analyses require extensive expertise. In contrast, comparative analyses of soil bacterial communities using nucleic acid technologies can efficiently and precisely locate the origin of forensic soil traces. However, this application is still in its infancy, and is very rarely used. We posit that understanding the theoretical bases and limitations of their uses is essential for soil microbial forensics to be judiciously implemented. Accordingly, we review the ecological theory and experimental evidence explaining differences between soil microbial communities, i.e. the generation of beta diversity, and propose to integrate a bottom-up approach of interactions at the microscale, reflecting historical contingencies with top-down mechanisms driven by the geographic template, providing a potential explanation as to why bacterial communities map according to soil types. Finally, we delimit the use of soil microbial forensics based on the present technologies and ecological knowledge, and propose possible venues to remove existing bottlenecks.

To hunt or to rest: prey depletion induces a novel starvation survival strategy in bacterial predators.

The small size of bacterial cells necessitates rapid adaption to sudden environmental changes. In Bdellovibrio bacteriovorus, an obligate predator of bacteria common in oligotrophic environments, the non-replicative, highly motile attack phase (AP) cell must invade a prey to ensure replication. AP cells swim fast and respire at high rates, rapidly consuming their own contents. How the predator survives in the absence of prey is unknown. We show that starvation for prey significantly alters swimming patterns and causes exponential decay in prey-searching cells over hours, until population-wide swim-arrest. Swim-arrest is accompanied by changes in energy metabolism, enabling rapid swim-reactivation upon introduction of prey or nutrients, and a sweeping change in gene expression and gene regulation that largely differs from those of the paradigmatic stationary phase. Swim-arrest is costly as it imposes a fitness penalty in the form of delayed growth. We track the control of the swim arrest-reactivation process to cyclic-di-GMP (CdG) effectors, including two motility brakes. CRISPRi transcriptional inactivation, and in situ localization of the brakes to the cell pole, demonstrated their essential role for effective survival under prey-induced starvation. Thus, obligate predators evolved a unique CdG-controlled survival strategy, enabling them to sustain their uncommon lifestyle under fluctuating prey supply.

Elucidating the Diversity and Potential Function of Nonribosomal Peptide and Polyketide Biosynthetic Gene Clusters in the Root Microbiome.

Polyketides (PKs) and nonribosomal peptides (NRPs) are two microbial secondary metabolite (SM) families known for their variety of functions, including antimicrobials, siderophores, and others. Despite their involvement in bacterium-bacterium and bacterium-plant interactions, root-associated SMs are largely unexplored due to the limited cultivability of bacteria. Here, we analyzed the diversity and expression of SM-encoding biosynthetic gene clusters (BGCs) in root microbiomes by culture-independent amplicon sequencing, shotgun metagenomics, and metatranscriptomics. Roots (tomato and lettuce) harbored distinct compositions of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) relative to the adjacent bulk soil, and specific BGC markers were both enriched and highly expressed in the root microbiomes. While several of the highly abundant and expressed sequences were remotely associated with known BGCs, the low similarity to characterized genes suggests their potential novelty. Low-similarity genes were screened against a large set of soil-derived cosmid libraries, from which five whole BGCs of unknown function were retrieved. Three clusters were taxonomically affiliated with Actinobacteria, while the remaining were not associated with known bacteria. One Streptomyces-derived BGC was predicted to encode a polyene with potential antifungal activity, while the others were too novel to predict chemical structure. Screening against a suite of metagenomic data sets revealed higher abundances of retrieved clusters in roots and soil samples. In contrast, they were almost completely absent in aquatic and gut environments, supporting the notion that they might play an important role in root ecosystems. Overall, our results indicate that root microbiomes harbor a specific assemblage of undiscovered SMs.IMPORTANCE We identified distinct secondary-metabolite-encoding genes that are enriched (relative to adjacent bulk soil) and expressed in root ecosystems yet almost completely absent in human gut and aquatic environments. Several of the genes were distantly related to genes encoding antimicrobials and siderophores, and their high sequence variability relative to known sequences suggests that they may encode novel metabolites and may have unique ecological functions. This study demonstrates that plant roots harbor a diverse array of unique secondary-metabolite-encoding genes that are highly enriched and expressed in the root ecosystem. The secondary metabolites encoded by these genes might assist the bacteria that produce them in colonization and persistence in the root environment. To explore this hypothesis, future investigations should assess their potential role in interbacterial and bacterium-plant interactions.