Single Bacterial Strain Research Articles

Engineering Microbial Consortia as Living Materials: Advances and Prospectives.

The field of Engineered Living Materials (ELMs) integrates engineered living organisms into natural biomaterials to achieve diverse objectives. Multiorganism consortia, prevalent in both naturally occurring and synthetic microbial cultures, exhibit complex functionalities and interrelationships, extending the scope of what can be achieved with individual engineered bacterial strains. However, the ELMs comprising microbial consortia are still in the developmental stage. In this Review, we introduce two strategies for designing ELMs constituted of microbial consortia: a top-down strategy, which involves characterizing microbial interactions and mimicking and reconstructing natural ecosystems, and a bottom-up strategy, which entails the rational design of synthetic consortia and their assembly with material substrates to achieve user-defined functions. Next, we summarize technologies from synthetic biology that facilitate the efficient engineering of microbial consortia for performing tasks more complex than those that can be done with single bacterial strains. Finally, we discuss essential challenges and future perspectives for microbial consortia-based ELMs.

Simulating the clinical manifestations and disease progression of human sepsis: A monobacterial injection approach for animal modeling

ABSTRACT Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, with great clinical heterogeneity, high morbidity, and high mortality. At the same time, there are many kinds of infection sources, the pathophysiology is very complex, and the pathogenesis has not been fully elucidated. An ideal animal model of sepsis can accurately simulate clinical sepsis and promote the development of sepsis-related pathogenesis, treatment methods, and prognosis. The existing sepsis model still uses the previous Sepsis 2.0 modelling standard, which has some problems, such as many kinds of infection sources, poor repeatability, inability to take into account single-factor studies, and large differences from clinical sepsis patients. To solve these problems, this study established a new animal model of sepsis. The model uses intravenous tail injection of a single bacterial strain, simplifying the complexity of multibacterial infection, and effectively solving the above problems.

First report of Pectobacterium brasiliense causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) belongs to Brassicaceae and is an important leaf vegetable widely cultivated in the Yangtze River basin and various southern provinces in China. In August 2023, the rhizome decay symptoms were observed at the stem base of leaf mustard plants (cv. Huarong) in the field of Changde City (29.05 °N; 111.59 °E), Hunan Province, China. The incidence of symptomatic leaf mustard was approximately 30% in several fields (2 ha in total). Brown and water-soaked symptoms appeared at the base of the outer leaves, and hollow rot at the base of the stem, accompanied by a fishy odor. To identify the causal agent, six infected stem samples were collected and surface sterilized by soaking in 75% ethanol for 60 seconds, rinsed three times with sterile distilled water, and finally cut into pieces (5 × 5 mm) in the sterile water. The extract was streaked on nutrient agar medium. After incubation at 28°C for 24 h, 17 strains were obtained and the colonies of all strains were creamy white, roughly circular, and convex elevation. Six single bacterial strains JC23121001-JC23121006, individually isolated from six different diseased stem samples, were selected as representative strains for further study. For preliminary identification, DNA from the six strains was extracted and identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991), and the sequences (accession nos. PP784484 to PP784489) showed 99% query coverage and 99.65% identity to Pectobacterium brasiliense type strain IBSBF1692T (Nabhan et al. 2012). In addition, five housekeeping genes acnA, mdh, mltD, pgi, and proA of the six strains were amplified with specially designed primers (Ma et al. 2007), and the resulting sequences from all six strains were 100% identical. The sequences of the representative strain JC23121001 were deposited into GenBank with accession numbers PP108247, PP066857, PP108248, PP066858, and PP066860, respectively. The maximum-likelihood phylogenetic tree clustered JC23121001 with P. brasiliense type strain IBSBF1692T (Nabhan et al. 2012). The pathogenicity test of six strains was carried out on the six-week-old leaf mustard (cv. Huarong) plants grown in the greenhouse by inoculating 10 µl of each bacterial suspension (108 CFU/ml) on needle-like wounds on the stem base of three healthy leaf mustard plants (Singh et al. 2013). Control plants were treated with sterile distilled water. After inoculation, the plants were incubated at 28°C and 90% relative humidity in a growth chamber. This trial was repeated three times. All inoculated mustard stems were slightly water-soaked after 24 hours and eventually developed into soft rot symptoms, consistent with the original symptoms observed. The control plants remained symptom-free. The strains were re-isolated from inoculated plants and re-identified as P. brasiliense by sequencing five housekeeping genes, thus fulfilling Koch's postulates. P. brasiliense has a broad host range and has been reported on other Brassica species, such as Bok choy (Brassica rapa var. chinensis) in China (Li et al. 2023). Soft rot of leaf mustard caused by Pectobacterium aroidearum has also been reported previously (Chu et al. 2023). To our knowledge, this is the first report of P. brasiliense causing soft rot on leaf mustard in China. The soft rot poses a significant threat to the local leaf mustard industry and requires further research into epidemiology and disease management options.

Điều tra bệnh hại sau thu hoạch trên giống chuối Tiêu hồng (Musa acuminata) và chuối Nam Mỹ (Musa paradisiaca) ở Việt Nam

The survey implemented in the 2021 winter season on the Tieu hong banana cultivar (Musa acuminata) grown in Phu Tho and Hung Yen provinces showed that the most severe diseases were fruit rot and fruit spot with infection rates of 79.1% and 75.5%, respectively. Peel cracking and end rot (cigar rot) were also recorded at less than 5.0%. Fruit spot was the main disease that affected the South-American banana cultivar (Musa paradisiaca) during the 2022 spring-summer season in Gia Lai province. Of the 24 fungal and 37 bacterial strains isolated from the disease samples collected during the investigation and tested for their pathogenicity via artificial infection on healthy bananas, only 17 fungal strains showed the ability of lesion formation of which 4 strains caused typical symptoms of the fruit rot disease. There was not a single bacterial strain ascertained as phytopathogen indicating that fungi were the primary agents of post-harvest diseases on bananas. These four fungal strains were taxonomically characterized based on their morphology (colony and spore), comparative analysis of the ITS sequences of the 18S rDNA gene, and were then identified as Corynespora torulosa B2B, Microdochium colombiense B6, Fusarium circinatum B13F, and Aspergillus niger GL2.1. The ITS sequences of these strains were deposited to GenBank under the accession numbers OP855524, OP855525, OP855526, and OP855527, respectively.

Immunomodulating effects of the single bacterial strain therapy EDP1815 on innate and adaptive immune challenge responses — a randomized, placebo-controlled clinical trial

The gut microbiome can modulate systemic inflammation and is therefore target for immunomodulation. Immunomodulating effects of EDP1815, a bacterial commensal strain of Prevotella histicola, were studied in healthy participants. Effects on adaptive immunity were evaluated by a neo-antigen challenge with keyhole limpet haemocyanin (KLH), while effects on innate immunity were evaluated by topical toll-like receptor 7 (TLR7) agonist imiquimod. Capsules with two enteric coating levels (EC1, EC2) were compared. Thirty-six healthy participants were included and received a daily dose of 8 × 1010 cells EDP1815-EC1, EDP1815-EC2 or placebo (randomization 1:1:1) for 60 days. They received KLH vaccinations at days 8, 24 and 36, with intradermal skin challenge at day 57. KLH challenge outcomes were antibody levels, and skin blood flow and erythema after skin challenge, measured by imaging techniques. Imiquimod administration started at day 57, for 72 h. Outcomes consisted of imaging measurements similar to the KLH challenge, and the influx of inflammatory cells and cytokines in blister fluid. There was no effect of EDP1815 treatment on the KLH challenge, neither on the imaging outcomes of the imiquimod challenge. There was a consistently lower influx of inflammatory cells in the blister fluid of EDP1815-treated participants (neutrophils, p = 0.016; granulocytes, p = 0.024), more pronounced in EC1. There was a lower influx of interleukin [IL]-1β, IL-6, IL-8, IL-10, interferon [IFN]-γ and tumour necrosis factor in blister fluid of EDP1815-treated participants. EDP1815 had immunomodulatory effects on the innate immune response driven by imiquimod, but no effect on the KLH challenge was observed. Trial registration number: NCT05682222; date: 22 July 2022.

Host-gut microbiota interactions shape parasite infections in farmed Atlantic salmon.

Animals and their associated microbiota share long evolutionary histories. However, it is not always clear how host genotype and microbiota interact to affect phenotype. We applied a hologenomic approach to explore how host-microbiota interactions shape lifetime growth and parasite infection in farmed Atlantic salmon (Salmo salar). Multi-omics data sets were generated from the guts of 460 salmon, 82% of which were naturally infected with an intestinal cestode. A single Mycoplasma bacterial strain, MAG01, dominated the gut metagenome of large, non-parasitized fish, consistent with previous studies showing high levels of Mycoplasma in the gut microbiota of healthy salmon. While small and/or parasitized salmon also had high abundance of MAG01, we observed increased alpha diversity in these individuals, driven by increased frequency of low-abundance Vibrionaceae and other Mycoplasma species that carried known virulence genes. Colonization by one of these cestode-associated Mycoplasma strains was associated with host individual genomic variation in long non-coding RNAs. Integrating the multi-omic data sets revealed coordinated changes in the salmon gut mRNA transcriptome and metabolome that correlated with shifts in the microbiota of smaller, parasitized fish. Our results suggest that the gut microbiota of small and/or parasitized fish is in a state of dysbiosis that partly depends on the host genotype, highlighting the value of using a hologenomic approach to incorporate the microbiota into the study of host-parasite dynamics.IMPORTANCEStudying host-microbiota interactions through the perspective of the hologenome is gaining interest across all life sciences. Intestinal parasite infections are a huge burden on human and animal health; however, there are few studies investigating the role of the hologenome during parasite infections. We address this gap in the largest multi-omics fish microbiota study to date using natural cestode infection of farmed Atlantic salmon. We find a clear association between cestode infection, salmon lifetime growth, and perturbation of the salmon gut microbiota. Furthermore, we provide the first evidence that the genetic background of the host may partly determine how the gut microbiota changes during parasite-associated dysbiosis. Our study therefore highlights the value of a hologenomic approach for gaining a more in-depth understanding of parasitism.

Effects of Bacillus subtilis as a single strain probiotic on growth, disease resistance and immune response of striped catfish (Pangasius hypophthalmus).

The present study investigated the potential role of Bacillus subtilis as probiotic in striped catfish (Pangasius hypophthalmus). Fish (initial weight = 150.00±2.63g n = 180) were stocked in circular tanks. Four isonitrogenous (30%) and isolipidic (3.29%) diets were formulated having supplementation of B. subtilis at four different levels (P0; 0, P1: 1×106, P2: 1×108 and P3: 1×1010 CFU/g). Each treatment had three replicates, while each replicate had fifteen fish. The trial started on second week of July and continued for eight weeks. Growth, feed conversion ratio, crude protein content, the concentration of amylase and protease, the profile of both dispensable and non-dispensable amino acids in all four dietary groups increased with a gradual increase of B. subtilis in the diet. At the end of growth experiment, fish in all four groups were exposed to Staphylococcus aureus (5×105 CFU/ml). After S. aureus challenge, fish fed with B. subtilis responded better to damage caused by reactive oxygen species and lipid peroxidation and better survival rate. The catalase and superoxide dismutase level also increased in response to bacterial challenge in B. subtilis fed groups. On the other hand, the concentration of malondialdehyde gradually decreased in these groups (+ve P0 >P1>P2>P3). It is concluded that supplementation of B. subtilis as a probiotic improved the growth, protein content, antioxidant response and immunocompetency against S. aureus in striped catfish. The optimum dosage of B. subtilis, at a concentration of 1×1010 CFU/g, resulted in the most favorable outcomes in striped catfish. This single bacterial strain can be used as an effective probiotic in large scale production of aquafeed for striped catfish. Future studies can investigate this probiotic's impact in the intensive culture of the same species.

Clinical and microbiological evaluation in patients with Fournier’s gangrene

Introduction: Necrotising fasciitis (Fournier’s gangrene) is a bacterial inflammation of the subcutaneous tissues and skin, involving the urogenital area. The treatment is complex, with a high risk of systemic infection. A key aspect of treatment is antibiotic therapy. Materials and methods: The analysis involved 30 hospitalised patients with Fournier’s gangrene. An attempt was made to determine the relationship between laboratory parameters, types of pathogens, patterns of antibiotic susceptibility/resistance, and treatment outcomes. Results: The average value of Fournier’s Gangrene Severity Index (FGSI) was 4.37 ± 2.87. In the study group, a total of 18 (60%) patients had an infection caused by a single bacterial strain, while 12 (40%) had infections caused by multiple strains. The most commonly identified bacteria was Escherichia coli (53.33%) – 50% of monocultural infections and 58.33% of polyculture infections. The majority of bacteria were sensitive to piperacillin–tazobactam – 22 (73.33%) patients, clindamycin – 20 (66.66%) patients, and metronidazole – 18 (60%) patients. The highest percentage of antibiotic resistance was observed for cefuroxime – 19 (63.33%) patients, ciprofloxacin – 19 (63.33%) patients, and ceftriaxone – 17 (56.66%) patients. The average hospitalisation length was 30.03 ± 23.01 days. The overall mortality rate was 13.33% (n = 4). No correlation was found between the type of pathogen, hospitalisation length or mortality. No differences were observed in the results of laboratory parameters, FGSI, or length of hospitalisation in the analysed subgroups. Conclusions: Effective treatment of Fournier’s gangrene requires performing a wound swab culture as quickly as possible and implementing a targeted antibiotic therapy. There is no single laboratory parameter that can predict treatment outcomes and patient prognosis. There are no differences in the clinical course and prognosis between multibacterial and monobacterial infections.

Microbe-induced drought tolerance by ABA-mediated root architecture and epigenetic reprogramming.

The use of beneficial microbes to mitigate drought stress tolerance of plants is of great potential albeit little understood. We show here that a root endophytic desert bacterium, Pseudomonas argentinensis strain SA190, enhances drought stress tolerance in Arabidopsis. Transcriptome and genetic analysis demonstrate that SA190-induced root morphogenesis and gene expression is mediated via the plant abscisic acid (ABA) pathway. Moreover, we demonstrate that SA190 primes the promoters of target genes in an epigenetic ABA-dependent manner. Application of SA190 priming on crops is demonstrated for alfalfa, showing enhanced performance under drought conditions. In summary, a single beneficial root bacterial strain can help plants to resist drought conditions.

What's the Matter with MICs: Bacterial Nutrition, Limiting Resources, and Antibiotic Pharmacodynamics.

The MIC of an antibiotic required to prevent replication is used both as a measure of the susceptibility/resistance of bacteria to that drug and as the single pharmacodynamic parameter for the rational design of antibiotic treatment regimes. MICs are experimentally estimated in vitro under conditions optimal for the action of the antibiotic. However, bacteria rarely grow in these optimal conditions. Using a mathematical model of the pharmacodynamics of antibiotics, we make predictions about the nutrient dependency of bacterial growth in the presence of antibiotics. We test these predictions with experiments in broth and a glucose-limited minimal media with Escherichia coli and eight different antibiotics. Our experiments question the sufficiency of using MICs and simple pharmacodynamic functions as measures of the pharmacodynamics of antibiotics under the nutritional conditions of infected tissues. To an extent that varies among drugs: (i) the estimated MICs obtained in rich media are greater than those estimated in minimal media; (ii) exposure to these drugs increases the time before logarithmic growth starts, their lag; and (iii) the stationary-phase density of E. coli populations declines with greater sub-MIC antibiotic concentrations. We postulate a mechanism to account for the relationship between sub-MICs of antibiotics and these growth parameters. This study is limited to a single bacterial strain and two types of culture media with different nutritive content. These limitations aside, the results of our study clearly question the use of MIC as the unique pharmacodynamic parameter to develop therapeutically oriented protocols. IMPORTANCE For studies of antibiotics and how they work, the most-often used measurement of drug efficacy is the MIC. The MIC is the concentration of an antibiotic needed to inhibit bacterial growth. This parameter is critical to the design and implementation of antibiotic therapy. We provide evidence that the use of MIC as the sole measurement for antibiotic efficacy ignores important aspects of bacterial growth dynamics. Before now, there has not been a nexus between bacteria, the conditions in which they grow, and the MIC. Most importantly, few studies have considered sub-MICs of antibiotics, despite their clinical importance. Here, we explore these concentrations in-depth, and we demonstrate MIC to be an incomplete measure of how an infection will interact with a specific antibiotic. Understanding the critiques of MIC is the first of many steps needed to improve infectious disease treatment.

Probiotic treatment (Enterococcus faecalis) improves symptoms of seasonal allergic rhinitis: A randomized controlled trial.

Probiotic treatment (Enterococcus faecalis) improves symptoms of seasonal allergic rhinitis: A randomized controlled trial.

Construction of "lock-key" biological living material based on double engineered bacteria and its application on intestinal retention in vivo

At present, the research of biological living materials mainly focuses on applications in vitro, such as using a single bacterial strain to produce biofilm and water plastics. However, due to the small volume of a single strain, it is easy to escape when used in vivo, resulting in poor retention. In order to solve this problem, this study used the surface display system (Neae) of Escherichia coli to display SpyTag and SpyCatcher on the surface of two strains, respectively, and constructed a double bacteria "lock-key" type biological living material production system. Through this force, the two strains are cross-linked in situ to form a grid-like aggregate, which can stay in the intestinal tract for a longer time. The in vitro experiment results showed that the two strains would deposit after mixing for several minutes. In addition, confocal imaging and microfluidic platform results further proved the adhesion effect of the dual bacteria system in the flow state. Finally, in order to verify the feasibility of the dual bacteria system in vivo, mice were orally administrated by bacteria A (p15A-Neae-SpyTag/sfGFP) and bacteria B (p15A-Neae-SpyCatcher/mCherry) for three consecutive days, and then intestinal tissues were collected for frozen section staining. The in vivo results showed that the two bacteria system could be more detained in the intestinal tract of mice compared with the non-combined strains, which laid a foundation for further application of biological living materials in vivo.

Amelioration of Colitis by a Gut Bacterial Consortium Producing Anti-Inflammatory Secondary Bile Acids

The Integrative Human Microbiome Project and other cohort studies have indicated that inflammatory bowel disease is accompanied by dysbiosis of gut microbiota, decreased production of secondary bile acids, and increased levels of primary bile acids. Secondary bile acids, such as ursodeoxycholic acid (UDCA) and lithocholic acid (LCA), have been reported to be anti-inflammatory, yet it remains to be studied whether introducing selected bacteria strains to restore bile acid metabolism of the gut microbiome can alleviate intestinal inflammation. In this study, we screened human gut bacterial strains for bile acid metabolism and designed a consortium of three species, including Clostridium AP sp000509125, Bacteroides ovatus, and Eubacterium limosum, and named it BAC (bile acid consortium). We showed that the three-strain gut bacterial consortium BAC is capable of converting conjugated primary bile acids taurochenodeoxycholic acid and glycochenodeoxycholic acid to secondary bile acids UDCA and LCA in vitro. Oral gavage treatment with BAC in mice resulted in protective effects against dextran sulfate sodium (DSS)-induced colitis, including reduced weight loss and increased colon length. Furthermore, BAC treatment increased the fecal level of bile acids, including UDCA and LCA. BAC treatment enhanced intestinal barrier function, which may be attributed to the increased activation of the bile acid receptor TGR5 by secondary bile acids. Finally, we examined the remodeling of gut microbiota by BAC treatment. Taken together, the three-strain gut bacterial consortium BAC restored the dysregulated bile acid metabolism and alleviated DSS-induced colitis. Our study provides a proof-of-concept demonstration that a rationally designed bacterial consortium can reshape the metabolism of the gut microbiome to treat diseases. IMPORTANCE Secondary bile acids have been reported to be anti-inflammatory, yet it remains to be studied whether introducing selected bacteria strains to restore bile acid metabolism of the gut microbiome can alleviate intestinal inflammation. To address this gap, we designed a consortium of human gut bacterial strains based on their metabolic capacity to produce secondary bile acids UDCA and LCA, and we evaluated the efficacy of single bacterial strains and the bacterial consortium in treating the murine colitis model. We found that oral gavage of the bacterial consortium to mice restored secondary bile acid metabolism to increase levels of UDCA and LCA, which induced the activation of TGR5 to improve gut-barrier integrity and reduced the inflammation in murine colitis. Overall, our study demonstrates that rationally designed bacterial consortia can reshape the metabolism of the gut microbiome and provides novel insights into the application of live biotherapeutics for treating IBD.

Effects of Phosphorus Limitation on the Bioavailability of DOM Released by Marine Heterotrophic Prokaryotes.

Heterotrophic prokaryotes (HP) contribute largely to dissolved organic matter (DOM) processing in the ocean, but they also release diverse organic substances. The bioavailability of DOM released by HP under varying environmental conditions has not been fully elucidated. In this study, we investigated the bioavailability of DOM released by a single bacterial strain (Sphingopyxis alaskensis) and 2 natural HP communities grown under P-replete and P-limited conditions. The released DOM (HP-DOM) was used as a substrate for natural HP communities at a coastal site in the Northwestern Mediterranean Sea. We followed changes in HP growth, enzymatic activity, diversity, and community composition together with the consumption of HP-DOM fluorescence (FDOM). HP-DOM produced under P-replete and P-limited conditions promoted significant growth in all incubations. No clear differences in HP-DOM lability released under P-repletion and P-limitation were evidenced based on the HP growth, and P-limitation was not demonstrated to decrease HP-DOM lability. However, HP-DOM supported the growth of diverse HP communities, and P-driven differences in HP-DOM quality were selected for different indicator taxa in the degrading communities. The humic-like fluorescence, commonly considered recalcitrant, was consumed during the incubations when this peak was initially dominating the FDOM pool, and this consumption coincided with higher alkaline phosphatase activity. Taken together, our findings emphasize that HP-DOM lability is dependent on both DOM quality, which is shaped by P availability, and the composition of the consumer community.

A8 MICROBIAL ACTIVATION OF INTESTINAL DENDRITIC CELLS IS CRITICAL FOR THE ESTABLISHMENT OF NORMAL BEHAVIOR

Abstract Background Accumulating evidence suggests that gut microbiota affects brain development and its function. It is well known that compared with conventional mice (SPF), germ-free (GF) mice display higher exploratory behavior, which normalizes after bacterial colonization. However, little is known about the underlying mechanisms and first critical steps initiating microbiota-gut-brain communication, which lead to establishment of normal behavior. Purpose To investigate the role of immune system in the establishment of normal behavior after bacterial colonization. Method We assessed behavior in GF mice before and after colonization with SPF microbiota, Altered Schaedler Flora (ASF) or the single bacterial strain E. coli JM83, and compared them to SPF mice, using the light-dark preference and tail suspension tests. Levels of brain-derived neurotrophic factor (BDNF) and c-Fos expression were measured by immunofluorescence in the hippocampus and amygdala. Colonic and brain gene expression were assessed using a NanoString technology. The immunodeficient MyD88-/- Ticam1- and SCID mice were used to study the role of the innate and adaptive immune systems. To demonstrate the role of the dendritic cells (DCs), we measured behavior before and after mono-colonization with E. coli JM83 in GF mice treated with cosalane and fingolimod, that inhibit DCs activation and migration, respectively. Brain levels of CD11b, CD11c and CD103 as DCs markers was assessed by immunofluorescence. Result(s) Compared to SPF mice, GF mice showed higher exploratory and less depressive-like behavior. The ex-germ-free mice colonized with ASF microbiota, or mono-colonized with E. coli JM83 showed similar normalization of behavior as those colonized with SPF microbiota. Mono-colonization with E. coli reduced both BDNF and c-fos levels in the hippocampus and amygdala. While colonization of GF SCID mice induced same change in behavior as in wild-type mice, GF MyD88-/-Ticam1-/- mice did not alter their behavior. Mono-colonization affected multiple genes in the colon and the brain, associated with innate immunity and neural plasticity. Treatment with both cosalane and fingolimod prevented behavioral changes after colonization, which was paralleled by absence of CD11b+CD103+CD11c+ cells in the brain, otherwise found in high numbers in control mono-colonized mice and absent in germ-free mice. Conclusion(s) The innate immune system, through activation and migration of intestinal dendritic cells into the brain, initiates the neuro-immune signaling within the gut-brain axis and leads to normalization of behavior after bacterial colonization. Our findings may impact several psychiatric conditions, in which altered innate immune signaling has been implicated. Please acknowledge all funding agencies by checking the applicable boxes below CIHR, Other Please indicate your source of funding; Balsam Family Foundation Disclosure of Interest None Declared

Direct Production of Bio-Recalcitrant Carboxyl-Rich Alicyclic Molecules Evidenced in a Bacterium-Induced Steroid Degradation Experiment.

Carboxyl-rich alicyclic molecules (CRAM) are highly unsaturated compounds extensively distributed throughout aquatic environments and sediments. This molecular group is widely referred to as a major proxy of recalcitrant organic materials, but its direct biosynthesis remains unclear. Steroids are a typical anthropogenic contaminant and have been previously suggested to be precursors of CRAM; however, experimental evidence to support this hypothesis is lacking. Here, a steroid-degrading bacterium, Comamonas testosteroni ATCC 11996, was incubated in a liquid medium supplemented with testosterone (a typical steroid) as the sole carbon source for 90 days. Testosterone-induced metabolites (TIM) were extracted for molecular characterization and to examine the bioavailability during an additional 90-day incubation after inoculation with a natural coastal microbial assemblage. The results showed that 1,775 molecular formulas (MFs) were assigned to TIM using ultrahigh-resolution mass spectrometry, with 66.99% categorized as CRAM-like constituents. A large fraction of TIM was respired or transformed during the additional 90-day seawater incubation; nevertheless, 638 MFs of the TIM persisted or increased during incubation. Among the 638 MFs, 394 were commonly assigned in natural deep seawater samples (depths of 500 to 2,000 m) from the South China Sea. Compared to the catabolites of the well-established testosterone degradation pathway, we compiled a list of bio-refractory MFs and potential chemical structures, some of which shared structural homology with CRAM. These results demonstrated direct microbial production of bio-refractory CRAM from steroid hormones and indicated that some of the biogenic CRAM resisted microbial decomposition, potentially contributing to the aquatic refractory dissolved organic matter (DOM) pool. IMPORTANCE CRAM are an operationally defined DOM group comprising a complex mixture of carboxylated and fused alicyclic structures. This DOM group is majorly characterized as refractory DOM in the marine environment. However, the origins of the complex CRAM remain unclear. In this study, we demonstrated that testosterone (a typical steroid) could be transformed into bio-refractory CRAM by a single bacterial strain and observed that some of the CRAM highly resisted microbial degradation. Through molecular comparison and screening, potential chemical structures of steroid-induced CRAM were suggested. This study established the biological connection between steroids and bio-refractory CRAM, and it provides a novel perspective explaining the fate of terrestrial contaminants in aquatic environments.

Rapid Identification of Infectious Pathogens at the Single-Cell Level via Combining Hyperspectral Microscopic Images and Deep Learning.

Identifying infectious pathogens quickly and accurately is significant for patients and doctors. Identifying single bacterial strains is significant in eliminating culture and speeding up diagnosis. We present an advanced optical method for the rapid detection of infectious (including common and uncommon) pathogens by combining hyperspectral microscopic imaging and deep learning. To acquire more information regarding the pathogens, we developed a hyperspectral microscopic imaging system with a wide wavelength range and fine spectral resolution. Furthermore, an end-to-end deep learning network based on feature fusion, called BI-Net, was designed to extract the species-dependent features encoded in cell-level hyperspectral images as the fingerprints for species differentiation. After being trained based on a large-scale dataset that we built to identify common pathogens, BI-Net was used to classify uncommon pathogens via transfer learning. An extensive analysis demonstrated that BI-Net was able to learn species-dependent characteristics, with the classification accuracy and Kappa coefficients being 92% and 0.92, respectively, for both common and uncommon species. Our method outperformed state-of-the-art methods by a large margin and its excellent performance demonstrates its excellent potential in clinical practice.

Effects of erythromycin and roxithromycin on river periphyton: Structure, functions and metabolic pathways

Macrolides have been frequently detected in the surface waters worldwide, posing a threat to the aquatic microbes. Several studies have evaluated the ecotoxicological effects of macrolides on single algal and bacterial strains. However, without considering the species interaction in the aquatic microbial community, these results cannot be extrapolated to the field. Thus, the present study aimed to evaluate the effects of two macrolides (erythromycin and roxithromycin) on the structure, photosynthetic process, carbon utilization capacity, and the antibiotic metabolic pathways in river periphyton. The colonized periphyton was exposed to the graded concentration (0 μg/L (control), 0.5 μg/L (low), 5 μg/L (medium), 50 μg/L (high)) of ERY and ROX, respectively, for 7 days. Herein, high levels of ERY and ROX altered the community composition by reducing the relative abundance of Chlorophyta in the eukaryotic community. Also, the Shannon and Simpson diversity indexes of prokaryotes were reduced, although similar effects were seldomly detected in the low and medium groups. In contrast to the unchanged carbon utilization capacity, the PSII reaction center involved in the periphytic photosynthesis was significantly inhibited by macrolides at high levels. In addition, both antibiotics had been degraded by periphyton, with the removal rate of 51.63–66.87% and 41.85–48.27% for ERY and ROX, respectively, wherein the side chain and ring cleavage were the main degradation pathways. Overall, this study provides an insight into the structural and functional toxicity and degradation processes of macrolides in river periphyton.–

Divergent taxonomic responses of below-ground microbial communities to silicate fertilizer and biofertilizer amendments in two rice ecotypes

Using silicate fertilizer and bacterial inoculum as biofertilizer is significant for increasing soil silicon (Si) availability and rice agronomic performance. To use microbial technology for sustainable agriculture, it is crucial to have a deeper knowledge of how microbial populations shift among the plant hosts and related compartments, as well as how they respond to various fertilization models. In this study, the effects of silicate fertilizer, a single bacterial strain Bacillus mucilagniosis as biofertilizer, and their integrated application on soil physiochemical properties and soil microbiota structure, composition, and diversity in two eco-geographically diverse races (Indica and Japonica rice) were evaluated. Plant compartment, cultivar type, and fertilizer treatments contributed to microbiome variation. Indica and Japonica harbor different root microbiota; notably, taxa enriched in the rhizosphere soil were more diverse than in the root. Bacterial genera Leptonema, Azospira, Aquabacterium, Fluviicola, Aquabacterium, Leptonema, and fungal genera Metarhizium, Malassezia, and Cladosporium all were found in the rice core microbiome. Both silicate and biofertilizer applications increase the relative abundance of Betaproteobacteria, Deltaproteobacteria, and Actinobacteria, while suppressing fungal pathogens Alternaria and Fusarium. Silicate and bacterial inoculum applications increased the soil pH, available silicon content (ASi), available phosphorous (AP), available potassium (AK), and organic carbon (OC), while reduced the total nitrogen (N). These changes were also associated with major bacterial phyla Spirochaetes, Bacteroidetes, Actinobacteria, and Proteobacteria, except for Acidobacteria, and fungal phyla Ascomycota, Mortierellomycota and unassigned fungi. Several treatment-specific biomarkers were revealed through Linear discriminant analysis effect size (LEfSe) analysis. In conclusion, the change in the structure of root-associated communities driven by plant compartment and genetics suggests dynamic interactions in the host plant microbiome. Short-term silicate and biofertilizer amendments improved soil physiochemical status and altered bacterial and saprotrophic fungal communities, which have important implications for sustainable rice production.

Within-Host Genotypic and Phenotypic Diversity of Contemporaneous Carbapenem-Resistant Klebsiella pneumoniae from Blood Cultures of Patients with Bacteremia.

It is unknown whether bacterial bloodstream infections (BSIs) are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole-genome sequences of 10 sequence type 258 (ST258) CRKP strains from blood cultures in each of 6 patients (Illumina HiSeq). Strains clustered by patient by core genome and pan-genome phylogeny. In 5 patients, there was within-host strain diversity by gene mutations, presence/absence of antibiotic resistance or virulence genes, and/or plasmid content. Accessory gene phylogeny revealed strain diversity in all 6 patients. Strains from 3 patients underwent long-read sequencing for genome completion (Oxford Nanopore) and phenotypic testing. Genetically distinct strains within individuals exhibited significant differences in carbapenem and other antibiotic responses, capsular polysaccharide (CPS) production, mucoviscosity, and/or serum killing. In 2 patients, strains differed significantly in virulence during mouse BSIs. Genetic or phenotypic diversity was not observed among strains recovered from blood culture bottles seeded with index strains from the 3 patients and incubated in vitro at 37°C. In conclusion, we identified genotypic and phenotypic variant ST258 CRKP strains from blood cultures of individual patients with BSIs, which were not detected by the clinical laboratory or in seeded blood cultures. The data suggest a new paradigm of CRKP population diversity during BSIs, at least in some patients. If validated for BSIs caused by other bacteria, within-host microbial diversity may have implications for medical, microbiology, and infection prevention practices and for understanding antibiotic resistance and pathogenesis. IMPORTANCE The long-standing paradigm for pathogenesis of bacteremia is that, in most cases, a single organism passes through a bottleneck and establishes itself in the bloodstream (single-organism hypothesis). In keeping with this paradigm, standard practice in processing positive microbiologic cultures is to test single bacterial strains from morphologically distinct colonies. This study is the first genome-wide analysis of within-host diversity of Klebsiella pneumoniae strains recovered from individual patients with bloodstream infections (BSIs). Our finding that positive blood cultures comprised genetically and phenotypically heterogeneous carbapenem-resistant K. pneumoniae strains challenges the single-organism hypothesis and suggests that at least some BSIs are caused by mixed bacterial populations that are unrecognized by the clinical laboratory. The data support a model of pathogenesis in which pressures in vivo select for strain variants with particular antibiotic resistance or virulence attributes and raise questions about laboratory protocols and treatment decisions directed against single strains.