Drastic Genome Reduction in an Herbivore's Pectinolytic Symbiont.

Pectin, an integral component of the plant cell wall, is a recalcitrant substrate against enzymatic challenges by most animals. In characterizing the source of a leaf beetle's (Cassida rubiginosa) pectin-degrading phenotype, we demonstrate its dependency on an extracellular bacterium housed in specialized organs connected to the foregut. Despite possessing the smallest genome (0.27 Mb) of any organism not subsisting within a host cell, the symbiont nonetheless retained a functional pectinolytic metabolism targeting the polysaccharide's two most abundant classes: homogalacturonan and rhamnogalacturonan I. Comparative transcriptomics revealed pectinase expression to be enriched in the symbiotic organs, consistent with enzymatic buildup in these structures following immunostaining with pectinase-targeting antibodies. Symbiont elimination results in a drastically reduced host survivorship and a diminished capacity to degrade pectin. Collectively, our findings highlight symbiosis as a strategy for an herbivore to metabolize one of nature's most complex polysaccharides and a universal component of plant tissues.

De novo sequencing, diploid assembly, and annotation of the black carpenter ant, Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing.

The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America. From a single individual I used long-read nanopore sequencing to assemble a phased diploid genome of 306 Mb and 60X coverage, with quality assessed by a 97.0% BUSCO score, improving upon other ant assemblies. The mitochondrial genome reveals minor rearrangements from other ants. The reads also allowed assembly of parasitic and symbiont genomes. I include a complete Wolbachia bacterial assembly with a size of 1.2 Mb, as well as a commensal symbiont Blochmannia pennsylvanicus, at 791 kb. DNA methylation and hydroxymethylation were measured at base-pair resolution level from the same reads and confirmed extremely low levels seen in the Formicidae family. There was moderate heterozygosity, with 0.16% of bases being biallelic from the parental haplotypes. Protein prediction yielded 14 415 amino acid sequences with 95.8% BUSCO score and 86% matching to previously known proteins. All assemblies were derived from a single MinION flow cell generating 20 Gb of sequence for a cost of $1047 including consumable reagents. Adding fixed costs for equipment brings the total for an ant-sized genome to less than $5000. All analyses were performed in 1 week on a single desktop computer.

Creating reference animal genomes is typically a large, expensive process. Here I sequenced the genome of the black carpenter ant for only $1000 as a sole researcher in just one week. Along with the nuclear genome, I assembled the mitochondrial genome and two commensal bacteria species living within the ant. Nanopore technology also enabled epigenetic measurements from the same ant and replicated other studies showing very low DNA methylation. The reference genome compared favorably to other ant species in continuity and protein prediction accuracy. This method will allow other low-resource labs to create high quality genome assemblies with a low cost.

Frequency of bystander exposure to antibiotics for enteropathogenic bacteria among young children in low-resource settings.

Children in low-resource settings carry enteric pathogens asymptomatically and are frequently treated with antibiotics, resulting in opportunities for pathogens to be exposed to antibiotics when not the target of treatment (i.e., bystander exposure). We quantified the frequency of bystander antibiotic exposures for enteric pathogens and estimated associations with resistance among children in eight low-resource settings. We analyzed 15,697 antibiotic courses from 1,715 children aged 0 to 2 y from the MAL-ED birth cohort. We calculated the incidence of bystander exposures and attributed exposures to respiratory and diarrheal illnesses. We associated bystander exposure with phenotypic susceptibility of E. coli isolates in the 30 d following exposure and at the level of the study site. There were 744.1 subclinical pathogen exposures to antibiotics per 100 child-years. Enteroaggregative Escherichia coli was the most frequently exposed pathogen, with 229.6 exposures per 100 child-years. Almost all antibiotic exposures for Campylobacter (98.8%), enterotoxigenic E. coli (95.6%), and typical enteropathogenic E. coli (99.4%), and the majority for Shigella (77.6%), occurred when the pathogens were not the target of treatment. Respiratory infections accounted for half (49.9%) and diarrheal illnesses accounted for one-fourth (24.6%) of subclinical enteric bacteria exposures to antibiotics. Bystander exposure of E. coli to class-specific antibiotics was associated with the prevalence of phenotypic resistance at the community level. Antimicrobial stewardship and illness-prevention interventions among children in low-resource settings would have a large ancillary benefit of reducing bystander selection that may contribute to antimicrobial resistance.

Diverse gut pathogens exploit the host engulfment pathway via a conserved mechanism.

Macrophages clear infections by engulfing and digesting pathogens within phagolysosomes. Pathogens escape this fate by engaging in a molecular arms race; they use WxxxE motif-containing "effector" proteins to subvert the host cells they invade and seek refuge within protective vacuoles. Here, we define the host component of the molecular arms race as an evolutionarily conserved polar "hot spot" on the PH domain of ELMO1 (Engulfment and Cell Motility protein 1), which is targeted by diverse WxxxE effectors. Using homology modeling and site-directed mutagenesis, we show that a lysine triad within the "patch" directly binds all WxxxE effectors tested: SifA (Salmonella), IpgB1 and IpgB2 (Shigella), and Map (enteropathogenic Escherichia coli). Using an integrated SifA-host protein-protein interaction network, in silico network perturbation, and functional studies, we show that the major consequences of preventing SifA-ELMO1 interaction are reduced Rac1 activity and microbial invasion. That multiple effectors of diverse structure, function, and sequence bind the same hot spot on ELMO1 suggests that the WxxxE effector(s)-ELMO1 interface is a convergence point of intrusion detection and/or host vulnerability. We conclude that the interface may represent the fault line in coevolved molecular adaptations between pathogens and the host, and its disruption may serve as a therapeutic strategy.

Impact of operational conditions on drinking water biofilm dynamics and coliform invasion potential.

Biofilms within drinking water distribution systems serve as a habitat for drinking water microorganisms. However, biofilms can negatively impact drinking water quality by causing water discoloration and deterioration and can be a reservoir for unwanted microorganisms. In this study, we investigated whether indicator organisms for drinking water quality, such as coliforms, can settle in mature drinking water biofilms. Therefore, a biofilm monitor consisting of glass rings was used to grow and sample drinking water biofilms. Two mature drinking water biofilms were characterized by flow cytometry, ATP measurements, confocal laser scanning microscopy, and 16S rRNA sequencing. Biofilms developed under treated chlorinated surface water supply exhibited lower cell densities in comparison with biofilms resulting from treated groundwater. Overall, the phenotypic as well as the genotypic characteristics were significantly different between both biofilms. In addition, the response of the biofilm microbiome and possible biofilm detachment after minor water quality changes were investigated. Limited changes in pH and free chlorine addition, to simulate operational changes that are relevant for practice, were evaluated. It was shown that both biofilms remained resilient. Finally, mature biofilms were prone to invasion of the coliform, Serratia fonticola. After spiking low concentrations (i.e., ±100 cells/100 mL) of the coliform to the corresponding bulk water samples, the coliforms were able to attach and get established within the mature biofilms. These outcomes emphasize the need for continued research on biofilm detachment and its implications for water contamination in distribution networks. IMPORTANCE: The revelation that even low concentrations of coliforms can infiltrate into mature drinking water biofilms highlights a potential public health concern. Nowadays, the measurement of coliform bacteria is used as an indicator for fecal contamination and to control the effectiveness of disinfection processes and the cleanliness and integrity of distribution systems. In Flanders (Belgium), 533 out of 18,840 measurements exceeded the established norm for the coliform indicator parameter in 2021; however, the source of microbial contamination is mostly unknown. Here, we showed that mature biofilms, are susceptible to invasion of Serratia fonticola. These findings emphasize the importance of understanding and managing biofilms in drinking water distribution systems, not only for their potential to influence water quality, but also for their role in harboring and potentially disseminating pathogens. Further research into biofilm detachment, long-term responses to operational changes, and pathogen persistence within biofilms is crucial to inform strategies for safeguarding drinking water quality.

Antibiotic resistance profiles of sentinel bacteria isolated from aquaculture in Cambodia.

The misuse of antibiotics and the emergence of antimicrobial resistance (AMR) is a concern in the aquaculture industry because it contributes to global health risks and impacts the environment. This study analyzed the AMR of sentinel bacteria associated with striped catfish (Pangasisanodon hypophthalmus) and giant snakehead (Channa micropeltes), the two main fish species reared in the pond culture in Cambodia. Phenotypic and genotypic characterization of the recovered isolates from fish, water, and sediment samples revealed the presence of bacteria, such as 22 species belonging to families Aeromonadaceae, Enterobacteriaceae, and Pseudomonadaceae. Among 48 isolates, Aeromonas caviae (n = 2), Aeromonas hydrophila (n = 2), Aeromonas ichthiosmia (n = 1), Aeromonas salmonicida (n = 4) were detected. A. salmonicida and A. hydrophilla are known as fish pathogens that occur worldwide in both fresh and marine water aquaculture. Antibiotic susceptibility testing revealed antibiotic resistance patterns of 24 (50 %) isolates among 48 isolates with higher multiple antibiotic resistance index (> 0.2). All the isolates of Enterobacteriaceae were susceptible to ciprofloxacin. Ciprofloxacin is a frontline antibiotic that is not recommended to use in aquaculture. Therefore, its use has to be strictly controlled. This study expands our knowledge of the AMR status in aquaculture farms which is very limited in Cambodia.

Multiple origins of obligate nematode and insect symbionts by a clade of bacteria closely related to plant pathogens.

Obligate symbioses involving intracellular bacteria have transformed eukaryotic life, from providing aerobic respiration and photosynthesis to enabling colonization of previously inaccessible niches, such as feeding on xylem and phloem, and surviving in deep-sea hydrothermal vents. A major challenge in the study of obligate symbioses is to understand how they arise. Because the best studied obligate symbioses are ancient, it is especially challenging to identify early or intermediate stages. Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila flies. We have found that Haoronymphium and its sister species harbor a maternally inherited intracellular bacterial symbiont. We never find the symbiont in nematode-free flies, and virtually all nematodes in the field and the laboratory are infected. Treating nematodes with antibiotics causes a severe reduction in fly infection success. The association is recent, as more distantly related insect-parasitic tylenchid nematodes do not host these endosymbionts. We also report that the Howardula nematode symbiont is a member of a widespread monophyletic group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, and that is sister to Pectobacterium, a lineage of plant pathogenic bacteria. Comparative genomic analysis of this group, which we name Candidatus Symbiopectobacterium, shows signatures of genome erosion characteristic of early stages of symbiosis, with the Howardula symbiont's genome containing over a thousand predicted pseudogenes, comprising a third of its genome.

Spatial and morphological reorganization of endosymbiosis during metamorphosis accommodates adult metabolic requirements in a weevil.

Bacterial intracellular symbiosis (endosymbiosis) is widespread in nature and impacts many biological processes. In holometabolous symbiotic insects, metamorphosis entails a complete and abrupt internal reorganization that creates a constraint for endosymbiont transmission from larvae to adults. To assess how endosymbiosis copes-and potentially evolves-throughout this major host-tissue reorganization, we used the association between the cereal weevil Sitophilus oryzae and the bacterium Sodalis pierantonius as a model system. S. pierantonius are contained inside specialized host cells, the bacteriocytes, that group into an organ, the bacteriome. Cereal weevils require metabolic inputs from their endosymbiont, particularly during adult cuticle synthesis, when endosymbiont load increases dramatically. By combining dual RNA-sequencing analyses and cell imaging, we show that the larval bacteriome dissociates at the onset of metamorphosis and releases bacteriocytes that undergo endosymbiosis-dependent transcriptomic changes affecting cell motility, cell adhesion, and cytoskeleton organization. Remarkably, bacteriocytes turn into spindle cells and migrate along the midgut epithelium, thereby conveying endosymbionts to midgut sites where future mesenteric caeca will develop. Concomitantly, endosymbiont genes encoding a type III secretion system and a flagellum apparatus are transiently up-regulated while endosymbionts infect putative stem cells and enter their nuclei. Infected cells then turn into new differentiated bacteriocytes and form multiple new bacteriomes in adults. These findings show that endosymbiosis reorganization in a holometabolous insect relies on a synchronized host-symbiont molecular and cellular "choreography" and illustrates an adaptive feature that promotes bacteriome multiplication to match increased metabolic requirements in emerging adults.

Predicting microbial growth dynamics in response to nutrient availability.

Developing mathematical models to accurately predict microbial growth dynamics remains a key challenge in ecology, evolution, biotechnology, and public health. To reproduce and grow, microbes need to take up essential nutrients from the environment, and mathematical models classically assume that the nutrient uptake rate is a saturating function of the nutrient concentration. In nature, microbes experience different levels of nutrient availability at all environmental scales, yet parameters shaping the nutrient uptake function are commonly estimated for a single initial nutrient concentration. This hampers the models from accurately capturing microbial dynamics when the environmental conditions change. To address this problem, we conduct growth experiments for a range of micro-organisms, including human fungal pathogens, baker's yeast, and common coliform bacteria, and uncover the following patterns. We observed that the maximal nutrient uptake rate and biomass yield were both decreasing functions of initial nutrient concentration. While a functional form for the relationship between biomass yield and initial nutrient concentration has been previously derived from first metabolic principles, here we also derive the form of the relationship between maximal nutrient uptake rate and initial nutrient concentration. Incorporating these two functions into a model of microbial growth allows for variable growth parameters and enables us to substantially improve predictions for microbial dynamics in a range of initial nutrient concentrations, compared to keeping growth parameters fixed.

Plant defenses interact with insect enteric bacteria by initiating a leaky gut syndrome.

Plants produce suites of defenses that can collectively deter and reduce herbivory. Many defenses target the insect digestive system, with some altering the protective peritrophic matrix (PM) and causing increased permeability. The PM is responsible for multiple digestive functions, including reducing infections from potential pathogenic microbes. In our study, we developed axenic and gnotobiotic methods for fall armyworm (Spodoptera frugiperda) and tested how particular members present in the gut community influence interactions with plant defenses that can alter PM permeability. We observed interactions between gut bacteria with plant resistance. Axenic insects grew more but displayed lower immune-based responses compared with those possessing Enterococcus, Klebsiella, and Enterobacter isolates from field-collected larvae. While gut bacteria reduced performance of larvae fed on plants, none of the isolates produced mortality when injected directly into the hemocoel. Our results strongly suggest that plant physical and chemical defenses not only act directly upon the insect, but also have some interplay with the herbivore's microbiome. Combined direct and indirect, microbe-mediated assaults by maize defenses on the fall armyworm on the insect digestive and immune system reduced growth and elevated mortality in these insects. These results imply that plant-insect interactions should be considered in the context of potential mediation by the insect gut microbiome.

Monte Carlo simulation evaluation of tigecycline dosing for bacteria with raised minimum inhibitory concentrations in non-critically ill adults.

PURPOSE: Tigecycline is one of few antibiotics active against multidrug-resistant bacteria; however, the assessment of dosing strategies to optimize its activity is needed. The purpose was to use Monte Carlo Simulation (MCS) to determine if safe tigecycline dosing options attaining breakpoints for pharmacokinetic/pharmacodynamic (PK-PD) targets in non-critically ill adults could be identified. METHODS: Publications that evaluated tigecycline dosing regimens and provided mean PK variables of interest (minimum 2 of: elimination rate constant or half-life and volume of distribution or clearance), with SDs, were included. Weighted mean (±SDs) for each PK parameter were determined. Food and Drug Administration minimum inhibitory concentration (MIC) tigecycline breakpoints for susceptible (MIC ≤ 2 µg/mL), intermediate (MIC 4 µg/mL), and resistant (MIC ≥ 8 µg/mL) Enterobacteriaceae were used. MCS probability distributions for PK-PD target attainment of AUC for total tigecycline plasma concentration from 0 to 24 h following an intravenous dose (AUCtotal, 0-24h) to MIC ratios of ≥ 18, 7, and 4.5 were generated, with success defined as ≥ 80% probability of target attainment at a given MIC. RESULTS: Ten studies (n = 442) were eligible. Tigecycline 150 mg IV q12h for ward patients with resistant bacteria up to a MIC of 0.48, 1, and 2 µg/mL for an AUCtotal, 0-24h/MIC target attainment of 18, 7, and 4.5, respectively, may be appropriate. CONCLUSION: Bacterial infections with tigecycline MICs ≥ 0.48-2 µg/mL, depending on AUCtotal, 0-24h/MIC target, may require treatment with alternate antibiotics due to target attainment failure.

Kosakonia radicincitans and Cryptococcus laurentii controlled Penicillium expansum rot and decreased patulin production at 4 and 25 °C.

In the present work, we evaluated the effects of a mixture of biocontrol agents against two toxigenic strains of Penicillium expansum isolated in Argentine Patagonia from pome fruits. The two strains, INTA-5 and INTA-10, were previusly selected among ten strains coming from the Alto Valle (Rio Negro-Argentina) for their high production of patulin. For the biocontrol, Kosakonia radicincitans, Cryptococcus laurentii, and Rhodosporidium fluviale were tested in vitro experiments on Potato Dextrose Agar (PDA) dishes against the INTA-5 and INTA-10 strains. The bacterium K. radicincitans and the yeast C. laurentii were selected to be used in a mixture due to their capacity to control the fungus and reduce the mycotoxin severely. In vitro assays with the mixture showed a high antagonism against P. expansum INTA-5 and INTA-10, at 21 d of incubation at 25 °C and a patulin reduction of 98%. The mixture of microorganisms was also effective in apples stored at 25 °C for 10 d and 4 °C for 30 d. At cold storage, the mixture controlled moderately the development of rot and decreased patulin concentration. At 25 °C, the pathogen's optimal growth temperature, the mixture of Biological Control Agent (BCAs) assured both the control of rot and decrease of patulin concentration. The combination of two microorganisms, with different requirements and abilities, resulted in a mix with a strong antagonism against P. expansum with the capability to decrease the patulin concentration. Treatment with the selected mixture could be a good option for controlling strains with different behaviours and in different environmental conditions.

In Planta Colonization and Role of T6SS in Two Rice Kosakonia Endophytes.

Endophytes live inside plants and are often beneficial. Kosakonia is a novel bacterial genus that includes many diazotrophic plant-associated isolates. Plant-bacteria studies on two rice endophytic Kosakonia beneficial strains were performed, including comparative genomics, secretome profiling, in planta tests, and a field release trial. The strains are efficient rhizoplane and root endosphere colonizers and localized in the root cortex. Secretomics revealed 144 putative secreted proteins, including type VI secretory system (T6SS) proteins. A Kosakonia T6SS genomic knock-out mutant showed a significant decrease in rhizoplane and endosphere colonization ability. A field trial using rice seed inoculated with Kosakonia spp. showed no effect on plant growth promotion upon nitrogen stress and microbiome studies revealed that Kosakonia spp. were significantly more present in the inoculated rice. Comparative genomics indicated that several protein domains were enriched in plant-associated Kosakonia spp. This study highlights that Kosakonia is an important, recently classified genus involved in plant-bacteria interaction.

Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis tolB Mutant.

Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of tolB, which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming Buttiauxella agrestis type strain JCM 1090. The ΔtolB mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the ΔtolB mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of ΔtolB mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the B. agrestis ΔtolB mutant to form unique vesicles.IMPORTANCE Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, Buttiauxella agrestis JCM 1090T ΔtolB mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications.

Potential Control of Potato Soft Rot Disease by the Obligate Predators Bdellovibrio and Like Organisms.

Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. affect a wide range of crops, including potatoes, a major food crop. As of today, farmers mostly rely on sanitary practices, water management, and plant nutrition for control. We tested the bacterial predators Bdellovibrio and like organisms (BALOs) to control potato soft rot. BALOs are small, motile predatory bacteria found in terrestrial and aquatic environments. They prey on a wide range of Gram-negative bacteria, including animal and plant pathogens. To this end, BALO strains HD100, 109J, and a ΔmerRNA derivative of HD100 were shown to efficiently prey on various rot-causing strains of Pectobacterium and Dickeya solani BALO control of maceration caused by a highly virulent strain of Pectobacterium carotovorum subsp. brasilense was then tested in situ using a potato slice assay. All BALO strains were highly effective at reducing disease, up to complete prevention. Effectivity was concentration dependent, and BALOs applied before P. carotovorum subsp. brasilense inoculation performed significantly better than those applied after the disease-causing agent, maybe due to in situ consumption of glucose by the prey, as glucose metabolism by live prey bacteria was shown to prevent predation. Dead predators and the supernatant of BALO cultures did not significantly prevent maceration, indicating that predation was the major mechanism for the prevention of the disease. Finally, plastic resistance to predation was affected by prey and predator population parameters, suggesting that population dynamics affect prey response to predation.IMPORTANCE Bacterial soft rot diseases caused by Pectobacterium spp. and Dickeya spp. are among the most important plant diseases caused by bacteria. Among other crops, they inflict large-scale damage to potatoes. As of today, farmers have few options to control them. The bacteria Bdellovibrio and like organisms (BALOs) are obligate predators of bacteria. We tested their potential to prey on Pectobacterium spp. and Dickeya spp. and to protect potato. We show that different BALOs can prey on soft rot-causing bacteria and prevent their growth in situ, precluding tissue maceration. Dead predators and the supernatant of BALO cultures did not significantly prevent maceration, showing that the effect is due to predation. Soft rot control by the predators was concentration dependent and was higher when the predator was inoculated ahead of the prey. As residual prey remained, we investigated what determines their level and found that initial prey and predator population parameters affect prey response to predation.

More Is Not Always Better: Coinfections with Defensive Symbionts Generate Highly Variable Outcomes.

Animal-associated microbes are highly variable, contributing to a diverse set of symbiont-mediated phenotypes. Given that host and symbiont genotypes, and their interactions, can impact symbiont-based phenotypes across environments, there is potential for extensive variation in fitness outcomes. Pea aphids, Acyrthosiphon pisum, host a diverse assemblage of heritable facultative symbionts (HFS) with characterized roles in host defense. Protective phenotypes have been largely studied as single infections, but pea aphids often carry multiple HFS species, and particular combinations may be enriched or depleted compared to expectations based on chance. Here, we examined the consequences of single infection versus coinfection with two common HFS exhibiting variable enrichment, the antiparasitoid Hamiltonella defensa and the antipathogen Regiella insecticola, across three host genotypes and environments. As expected, single infections with either H. defensa or R. insecticola raised defenses against their respective targets. Single infections with protective H. defensa lowered aphid fitness in the absence of enemy challenge, while R. insecticola was comparatively benign. However, as a coinfection, R. insecticola ameliorated H. defensa infection costs. Coinfected aphids continued to receive antiparasitoid protection from H. defensa, but protection was weakened by R. insecticola in two clones. Notably, H. defensa eliminated survival benefits conferred after pathogen exposure by coinfecting R. insecticola Since pathogen sporulation was suppressed by R. insecticola in coinfected aphids, the poor performance likely stemmed from H. defensa-imposed costs rather than weakened defenses. Our results reveal a complex set of coinfection outcomes which may partially explain natural infection patterns and suggest that symbiont-based phenotypes may not be easily predicted based solely on infection status.IMPORTANCE The hyperdiverse arthropods often harbor maternally transmitted bacteria that protect against natural enemies. In many species, low-diversity communities of heritable symbionts are common, providing opportunities for cooperation and conflict among symbionts, which can impact the defensive services rendered. Using the pea aphid, a model for defensive symbiosis, we show that coinfections with two common defensive symbionts, the antipathogen Regiella and the antiparasite Hamiltonella, produce outcomes that are highly variable compared to single infections, which consistently protect against designated enemies. Compared to single infections, coinfections often reduced defensive services during enemy challenge yet improved aphid fitness in the absence of enemies. Thus, infection with multiple symbionts does not necessarily create generalist aphids with "Swiss army knife" defenses against numerous enemies. Instead, particular combinations of symbionts may be favored for a variety of reasons, including their abilities to lessen the costs of other defensive symbionts when enemies are not present.

Honokiol Restores Polymyxin Susceptibility to MCR-1-Positive Pathogens both In Vitro and In Vivo.

The emergence of the plasmid-mediated colistin resistance gene mcr-1 has led to serious multidrug-resistant (MDR) Enterobacteriaceae infections, which are a great threat to the clinic. This study aims to find an inhibitor of MCR-1 to reestablish the use of polymyxins against MDR Enterobacteriaceae infections. Here, we determined that the natural compound honokiol could enhance the efficacy of polymyxins against MDR Enterobacteriaceae infections by a checkerboard MIC assay, a time-kill assay, a combined disk test, Western blotting, molecular simulation dynamics, and mouse infection models. The MIC results indicated that honokiol can recover the sensitivity of polymyxins against MCR-1-positive Klebsiella pneumoniae and Escherichia coli (with a fractional inhibitory concentration index ranging from 0.09 ± 0.00 to 0.27 ± 0.06). Based on time-kill curve analysis, all of the tested bacteria were killed within 1 h following the combined therapy with honokiol and polymyxins. Molecular simulation dynamics results suggested that honokiol directly binds to the MCR-1 active region, reducing the biological activity of MCR-1. The combination of honokiol and polymyxins could increase the 40% protection rate and reduce the bacterial load on the thigh muscles of mice. Our study indicates that honokiol is a predominant natural compound whose combination therapy with polymyxins is very promising in future treatment options for MCR-1-positive Enterobacteriaceae infections.IMPORTANCE In the present study, honokiol could effectively inhibit the activity of MCR-1 and showed almost no cytotoxicity to MH-S cells. According to our results, the combination of honokiol and polymyxin had a clear synergistic effect against MCR-1-positive Enterobacteriaceae in vitro Combination therapy also showed a powerful therapeutic effect in vivo, which can significantly improve mouse livability, reduced the load of bacteria, and reduced pathological change. This combined therapy of small molecule compounds and antibiotics may not continue to induce new bacterial resistance, due to the fact that MCR-1 targeted by honokiol is not indispensable for the bacterial viability; on the other hand, it can reduce the dosage of combined antibiotics, and it is also a promising alternative therapy for the treatment of drug-resistant infections in the future.

A fitness cost resulting from Hamiltonella defensa infection is associated with altered probing and feeding behaviour in Rhopalosiphum padi.

Many herbivorous arthropods, including aphids, frequently associate with facultative endosymbiotic bacteria, which influence arthropod physiology and fitness. In aphids, endosymbionts can increase resistance against natural enemies, enhance aphid virulence and alter aphid fitness. Here, we used the electrical penetration graph technique to uncover physiological processes at the insect-plant interface affected by endosymbiont infection. We monitored the feeding and probing behaviour of four independent clonal lines of the cereal-feeding aphid Rhopalosiphum padi derived from the same multilocus genotype containing differential infection (+/-) with a common facultative endosymbiont, Hamiltonella defensa Aphid feeding was examined on a partially resistant wild relative of barley known to impair aphid fitness and a susceptible commercial barley cultivar. Compared with uninfected aphids, endosymbiont-infected aphids on both plant species exhibited a twofold increase in the number of plant cell punctures, a 50% reduction in the duration of each cellular puncture and a twofold higher probability of achieving sustained phloem ingestion. Feeding behaviour was also altered by host plant identity: endosymbiont-infected aphids spent less time probing plant tissue, required twice as many probes to reach the phloem and showed a 44% reduction in phloem ingestion when feeding on the wild barley relative compared with the susceptible commercial cultivar. Reduced feeding success could explain the 22% reduction in growth of H. defensa-infected aphids measured on the wild barley relative. This study provides the first demonstration of mechanisms at the aphid-plant interface contributing to physiological effects of endosymbiont infection on aphid fitness, through altered feeding processes on different quality host plants.

Rapid biodegradation and biofilm-mediated bioremoval of organophosphorus pesticides using an indigenous Kosakonia oryzae strain -VITPSCQ3 in a Vertical-flow Packed Bed Biofilm Bioreactor.

The widespread use of pesticides has been one of the major anthropogenic sources of environmental pollution. Organophosphorus (OP) pesticides are predominantly used in agriculture due to their broad-spectrum insecticidal activity and chemical stability. The study was focused on the biodegradation of OP pesticides, Profenofos (PF) and Quinalphos (QP) in culture media using bacterium isolated from wetland paddy rhizosphere. The strain VITPSCQ3 showed higher pesticide tolerance, efficient biofilm formation and was capable of synthesizing organophosphate degrading enzymes. Based on the 16S rRNA gene sequencing the isolate exhibited maximum sequence similarity with Kosakinia oryzae (GenBank accession number: KR149275). Biodegradation assay with various concentrations of PF and QP (200, 400, 600 and 800 mg L-1) showed maximum degradation up to 82% and 92% within 48 h. The kinetic studies revealed the biodegradation rates (k) to be 0.0844 min-1 and 0.107 min-1 with half-lives (h) of 18 h and 14.8 h for PF and QP. The degradation products were identified by GCMS and possible degradation pathways were proposed using Insilico techniques. To the best of our knowledge, this is the first report on the biodegradation of PF and QP using Kosakonia oryzae. Bioremoval of PF and QP from aqueous solution was performed using the biofilm of VITPSCQ3 developed on selected substrates in a circulating Vertical-flow packed-bed biofilm (VFPBB) bioreactor. Charcoal, gravel and mushroom (Agaricus bisporus) were used as biofilm carriers. Mushroom showed strong biofilm formation with optimum biodegradation capacity of up to 96% for PF and 92% for QP within 120 min reaction time.

Staying out or Going in? The Interplay between Type 3 and Type 5 Secretion Systems in Adhesion and Invasion of Enterobacterial Pathogens.

Enteric pathogens rely on a variety of toxins, adhesins and other virulence factors to cause infections. Some of the best studied pathogens belong to the Enterobacterales order; these include enteropathogenic and enterohemorrhagic Escherichia coli, Shigella spp., and the enteropathogenic Yersiniae. The pathogenesis of these organisms involves two different secretion systems, a type 3 secretion system (T3SS) and type 5 secretion systems (T5SSs). The T3SS forms a syringe-like structure spanning both bacterial membranes and the host cell plasma membrane that translocates toxic effector proteins into the cytoplasm of the host cell. T5SSs are also known as autotransporters, and they export part of their own polypeptide to the bacterial cell surface where it exerts its function, such as adhesion to host cell receptors. During infection with these enteropathogens, the T3SS and T5SS act in concert to bring about rearrangements of the host cell cytoskeleton, either to invade the cell, confer intracellular motility, evade phagocytosis or produce novel structures to shelter the bacteria. Thus, in these bacteria, not only the T3SS effectors but also T5SS proteins could be considered "cytoskeletoxins" that bring about profound alterations in host cell cytoskeletal dynamics and lead to pathogenic outcomes.