Bacterial Gene Research Articles

Development of a bacterial gene transcription activating strategy based on transcriptional activator positive feedback

IntroductionTranscription of biological nitrogen fixation (nif) genes is activated by the NifA protein which recognizes specific activating sequences upstream of σ54-dependent nif promoters. The large quantities of nitrogenase which can make up 20% of the total proteins in the cell indicates high transcription activating efficiency of NifA and high transcription level of nifHDK nitrogenase genes. ObjectivesDevelopment of an efficient gene transcription activating strategy in bacteria based on positive transcription regulatory proteins and their regulating DNA sequences. MethodsWe designed a highly efficient gene transcription activating strategy in which the nifA gene was placed directly downstream of its regulating sequences. The NifA protein binds its regulating sequences and stimulates transcription of itself and downstream genes. Overexpressed NifA causes transcription activation by positive reinforcement. ResultsWhen this gene transcription activating strategy was used to overexpress NifA in Pseudomonas stutzeri DSM4166 containing the nif gene cluster, the nitrogenase activity was increased by 368 folds which was 16 times higher than that obtained by nifA driven by the strongest endogenous constitutive promoter. When this strategy was used to activate transcription of exogenous biosynthetic genes for the plant auxin indole-3-acetic acid and the antitumor alkaloid pigment prodigiosin in DSM4166, both of them resulted in better performance than the strongest endogenous constitutive promoter and the highest reported productions in heterologous hosts to date. Finally, we demonstrated the universality of this strategy using the positive transcriptional regulator of the psp operon, PspF, in E. coli and the pathway-specific positive transcription regulator of the polyene antibiotic salinomycin biosynthesis, SlnR, in Streptomyces albus. ConclusionMany positive transcription regulatory proteins and their regulating DNA sequences have been identified in bacteria. The gene transcription activating strategy developed in this study will have broad applications in molecular biology and biotechnology.

EE168 A Markov Model to Determine the Cost-Effectiveness of a Multi-Targeting Bacterial Gene Therapy (AUP-16) at Healing a Diabetic Foot Ulcer When Compared to the Current Standard of Care

EE168 A Markov Model to Determine the Cost-Effectiveness of a Multi-Targeting Bacterial Gene Therapy (AUP-16) at Healing a Diabetic Foot Ulcer When Compared to the Current Standard of Care

Tributyrin oral administration significantly attenuates the loss of intestinal butyrate‐producing bacteria and the ensuing cognitive dysfunction associated with Alzheimer’s Disease pathogenesis in 3xTg‐AD mice

AbstractBackgroundAlzheimer’s Disease (AD) development is linked to the loss of overall gut microbial diversity. However, the specific alterations are only beginning to be examined. In this regard, decreases in intestinal butyrate‐producing bacteria have been indicated by pre‐clinical and clinical studies in AD. The current work, employing the 3xTg‐AD mouse model examined the causal association of loss of butyrate‐producing bacterial communities with cognitive dysfunction associated with AD pathogenesis.MethodCecal samples from tributyrin (TB) treated and untreated female 3xTg‐AD mice were used to perform metataxonomic analysis by sequencing the 16S rRNA gene (V4 region) on a large‐scale Illumina MiSeq platform. Inferred metagenomics analysis was performed using the PICRUSt2 pipeline. TB treatment (oral gavage, 2mg/kg, twice a week) was initiated at 6‐months of age until 17‐months. Spatial and short‐term memory functions were tested using the Y‐maze and Novel Object Recognition Test (NORT).ResultMetataxonomic analysis showed an age‐dependent, significant decline in alpha diversity represented by measures of OTUs, Chao‐1 and Shannon Index from 2‐ to 18‐months. Additionally, age‐dependent changes in gut microbial composition were demonstrated by a significant decrease in the Firmicutes‐to‐Bacteroidota (F/B) ratio. Further analysis of specific features of the gut microbiome demonstrated an age‐dependent decrease in total butyrate‐producing families within the Firmicutes phylum; particularly, a significant loss of critical butyrate producing family Ruminococcacea was identified. Predictive functional analysis using PICRUSt2 also showed a significant reduction in the abundance of specific bacterial genes involved in butyrate synthesis. Importantly, these age‐dependent markers of gut microbial dysbiosis coincided with diminished measures of spatial and short‐term memory. Notably, administration of TB, a butyrate pro‐drug, significantly attenuated the age‐dependent (i) decline in gut microbial diversity & butyrate‐producing bacteria and (ii) loss of spatial and short‐term memory.ConclusionThe findings from this study identify that decline in total butyrate‐producing families is a key pathogenic feature of AD‐associated microbial dysbiosis and cognitive dysfunction. Importantly, these findings have the potential to inform the development of treatment strategies targeting gut microbial dysbiosis using TB, to prevent onset and development of cognitive decline in AD.

High risk HPV-positive women cervicovaginal microbial profiles in a Greek cohort: a retrospective analysis of the GRECOSELF study

Increasing evidence supports a role for the vaginal microbiome (VM) in the severity of HPV infection and its potential link to cervical intraepithelial neoplasia. However, a lot remains unclear regarding the precise role of certain bacteria in the context of HPV positivity and persistence of infection. Here, using next generation sequencing (NGS), we comprehensively profiled the VM in a series of 877 women who tested positive for at least one high risk HPV (hrHPV) type with the COBAS® 4,800 assay, after self-collection of a cervico-vaginal sample. Starting from gDNA, we PCR amplified the V3–V4 region of the bacterial 16S rRNA gene and applied a paired-end NGS protocol (Illumina). We report significant differences in the abundance of certain bacteria compared among different HPV-types, more particularly concerning species assigned to Lacticaseibacillus, Megasphaera and Sneathia genera. Especially for Lacticaseibacillus, we observed significant depletion in the case of HPV16, HPV18 versus hrHPVother. Overall, our results suggest that the presence or absence of specific cervicovaginal microbial genera may be linked to the observed severity in hrHPV infection, particularly in the case of HPV16, 18 types.

Spatial distribution characteristics of denitrification functional genes and the environmental drivers in Liaohe estuary wetland.

Genes nirS, nirK, and nosZ are specific for the denitrification process, which is associated with greenhouse gas N2O emission. The abundances and diversities of community containing these three genes are usually used as a common index to reflect the denitrification process, and they would be affected by differences in environmental factors caused by changes from warm to cold conditions. The quantification of denitrification in natural wetlands is complex, and straightforward identification of spatial distribution and drivers affecting the process is still developing. In this study, the bacterial communities, gene diversities, and relative abundances involved in denitrification were investigated in Liaohe Estuary Wetland. We analyzed the relative abundances, diversities, and communities of bacteria containing the three genes at warm and cold conditions using Illumina MiSeq sequencing and detected the potential environmental factors influencing their distribution by using a random forest algorithm. There are great differences in the community composition of the bacteria containing genes nirS, nirK, and nosZ. All the abundant taxa of nirS and nirK communities belonged to phylum Proteobacteria. Compared with the community composition of bacteria containing nirS and nirK, the community of bacteria containing nosZ is more diverse, and the subdivision taxa of phylum Euryarchaeota was also abundant in the community containing nosZ. The distribution characteristics of the relative abundance of nirS and nirK showed obvious differences both at warm and cold climate conditions. The oxidation-reduction potential, nitrite nitrogen, and salinity were detected as potential variables that might explain the diversity of nirS. The total nitrogen and nitrite nitrogen were the important variables for predicting the relative abundance of nirS at warm climate condition, while oxidation-reduction potential and pH contributed to the diversity of nirS at cold condition. The bulk density of sediment was detected as a potential variable affecting the relative abundance of nirK at warm and cold conditions, and diversity of nirK at warm condition, while nitrite nitrogen was detected as an important environmental factor for predicting the diversity of nirK at cold condition. Overall, our results show that the key environmental factors, which affect the relative abundance, diversity, and community of bacteria containing the functional denitrification genes, are not exactly the same, and the diversities of nirS, nirK, and nosZ have a higher environmental sensitivity than their relative abundances.

Bacterial amidohydrolases and modified 5-fluorocytidine compounds: Novel enzyme-prodrug pairs.

Gene-directed enzyme prodrug therapy is an emerging strategy for cancer treatment based on the delivery of a gene that encodes an enzyme that is able to convert a prodrug into a potent cytotoxin exclusively in target cancer cells. However, it is limited by the lack of suitable enzyme variants and a scarce choice of chemical bonds that could be activated. Therefore, this study is aimed to determine the capability of bacterial amidohydrolases YqfB and D8_RL to activate novel prodrugs and the effect such system has on the viability of eukaryotic cancer cells. We have established cancer cell lines that stably express the bacterial amidohydrolase genes and selected several N4-acylated cytidine derivatives as potential prodrugs. A significant decrease in the viability of HCT116 human colon cancer cell lines expressing either the YqfB or the D8_RL was observed after exposure to the novel prodrugs. The data we acquired suggests that bacterial YqfB and D8_RL amidohydrolases, together with the modified cytidine-based prodrugs, may serve as a promising enzyme-prodrug system for gene-directed enzyme prodrug therapy.

Prebiotics increase iron absorption and reduce the adverse effects of iron on the gut microbiome and inflammation: a randomized controlled trial using iron stable isotopes in Kenyan infants

BackgroundIron fortificants tend to be poorly absorbed and may adversely affect the gut, especially in African children. ObjectiveWe assessed the effects of prebiotic galacto-oligosaccharides/fructo-oligosaccharides (GOS/FOS) on iron absorption and gut health when added to iron-fortified infant cereal. MethodsWe randomly assigned Kenyan infants (n = 191) to receive daily for 3 wk a cereal containing iron and 7.5 g GOS/FOS (7.5 g+iron group), 3 g (3-g+iron group) GOS/FOS, or no prebiotics (iron group). A subset of infants in the 2 prebiotic+iron groups (n = 66) consumed 4 stable iron isotope–labeled test meals without and with prebiotics, both before and after the intervention. Primary outcome was fractional iron absorption (FIA) from the cereal with or without prebiotics regardless of dose, before and after 3 wk of consumption. Secondary outcomes included fecal gut microbiota, iron and inflammation status, and effects of prebiotic dose. ResultsMedian (25th–75th percentiles) FIAs from meals before intervention were as follows: 16.3% (8.0%–27.6%) without prebiotics compared with 20.5% (10.4%–33.4%) with prebiotics (Cohen d = 0.53; P < 0.001). FIA from the meal consumed without prebiotics after intervention was 22.9% (8.5%–32.4%), 41% higher than from the meal without prebiotics before intervention (Cohen d = 0.36; P = 0.002). FIA from the meal consumed with prebiotics after intervention was 26.0% (12.2%–36.1%), 60% higher than from the meal without prebiotics before intervention (Cohen d = 0.45; P = 0.007). After 3 wk, compared with the iron group, the following results were observed: 1) Lactobacillus sp. abundances were higher in both prebiotic+iron groups (P < 0.05); 2) Enterobacteriaceae sp. abundances (P = 0.022) and the sum of pathogens (P < 0.001) were lower in the 7.5-g+iron group; 3) the abundance of bacterial toxin-encoding genes was lower in the 3-g+iron group (false discovery rate < 0.05); 4) fecal pH (P < 0.001) and calprotectin (P = 0.033) were lower in the 7.5-g+iron group. ConclusionsAdding prebiotics to iron-fortified infant cereal increases iron absorption and reduces the adverse effects of iron on the gut microbiome and inflammation in Kenyan infants.This trial was registered at clinicaltrials.gov as NCT03894358.

Biological characteristics of the enteroaggregative &lt;i&gt;escherichia coli &lt;/i&gt;ont:h30 18-726 (no. B-8857) strain isolated from a patient with ulcerative colitis and a new method of pcr identification

The aim of the work is to assess biological properties of the enteroaggregativeEscherichia coli(EAgEC) strain isolated from the colon mucosa biopsy material from a patient with ulcerative colitis, and to develop a new method for PCR identification ofE. colistrains.&#x0D; Materials and methods.The study involved 46 patients with a verified ulcerative colitis. Isolation of 87E. colistrains was carried out by using colon biopsy material obtained during a standard endoscopic procedure. The description of the biochemical bacterial properties was combined with the molecular genetic detection of virulence determinants and presence of antibiotic resistance mechanisms. Using the “AmpliSense®Escherichiose — FL” reagent kit, a screening for the presence of diarrheagenicE. colistrains was performed, which revealed the presence of a single strain of EAgEC. The methodological approach to creating a new method for strain identification was based on the search for a unique genetic sequence within the glutamate decarboxylase(gad)gene.&#x0D; Results.E. coli18-726 can be described biochemically typical to its species. The studied strain was characterized by a multiple resistance phenotype (MDR) to antibiotics, as well as a lack of sensitivity to five bacteriophages. TheE. coli18-726 strain had a unique sequence of the gene encoding the O-antigen, which differed from 188 known O-antigens (ONT) and, by the identity of the nucleotide sequence of the gene encoding the synthesis of the H-antigen, the strain belonged to the H30 serovar. Thus, the antigenic formula of strain EAgEC 18-726 was expressed as ONT:H30. TheE. coli18-726 strain contained a significant spectrum of genes that enable properties of virulence(iss, capU, aggA, aggB, aggC, aggD, aap, aar)and resistance to antibiotics(blaCTX-M-15, blaTEM-1B, aadA1, aadA5, mph(A), catB3). A new method for identifying EAgEC in chronic bowel disease is based on the polymerase chain reaction method using primers for a specific region of bacterial glutamate decarboxylase(gad)gene.Conclusion.1) The biochemical, antigenic and molecular genetic properties ofE. coliONT:H30 18-726 strain (No. B-8857) isolated from a patient with histomorphologically diagnosed ulcerative colitis were analyzed. 2) A method for PCR identification of EAgEC strains based on the detection of a specific region within the bacterial genomic glutamate decarboxylase gene has been created and patented.

ArdA genes from pKM101 and from B. bifidum chromosome have a different range of regulated genes

The ardA genes are present in a wide variety of conjugative plasmids and play an important role in overcoming the restriction barrier. To date, there is no information on the chromosomal ardA genes. It is still unclear whether they keep their antirestriction activity and why bacterial chromosomes contain these genes. In the present study, we confirmed the antirestriction function of the ardA gene from the Bifidobacterium bifidum chromosome. Transcriptome analysis in Escherichia coli showed that the range of regulated genes varies significantly for ardA from conjugative plasmid pKM101 and from the B. bifidum chromosome. Moreover, if the targets for both ardA genes match, they often show an opposite effect on regulated gene expression. The results obtained indicate two seemingly mutually exclusive conclusions. On the one hand, the pleiotropic effect of ardA genes was shown not only on restriction-modification system, but also on expression of a number of other genes. On the other hand, the range of affected genes varies significally for ardA genes from different sources, which indicates the specificity of ardA to inhibited targets.Author Summary. Conjugative plasmids, bacteriophages, as well as transposons, are capable to transfer various genes, including antibiotic resistance genes, among bacterial cells. However, many of those genes pose a threat to the bacterial cells, therefore bacterial cells have special restriction systems that limit such transfer.Antirestriction genes have previously been described as a part of conjugative plasmids, and bacteriophages and transposons. Those plasmids are able to overcome bacterial cell protection in the presence of antirestriction genes, which inhibit bacterial restriction systems.This work unveils the antirestriction mechanisms, which play an important role in the bacterial life cycle. Here, we clearly show that antirestriction genes, which are able to inhibit cell protection, exist not only in plasmids but also in the bacterial chromosomes themselves.Moreover, antirestrictases have not only an inhibitory function but also participate in the regulation of other bacterial genes. The regulatory function of plasmid antirestriction genes also helps them to overcome the bacterial cell protection against gene transfer, whereas the regulatory function of genomic antirestrictases has no such effect.

Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs.

Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of Moraxella: M. bovis and M. bovoculi. A third novel Moraxella spp., designated tentatively as M. oculi, has been identified from the eyes of cattle with and without pinkeye. These 3 Moraxella spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessing Moraxella diversity found in IBK- and non-IBK-affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3 Moraxella spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3 Moraxella spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3 Moraxella spp. Targeted NGS with sequencing of Moraxella spp. housekeeping genes appears to be a suitable method for speciation of Moraxella directly from ocular swabs.

Microbiome and plant cell transformation trigger insect gall induction in cassava.

Several specialised insects can manipulate normal plant development to induce a highly organised structure known as a gall, which represents one of the most complex interactions between insects and plants. Thus far, the mechanism for insect-induced plant galls has remained elusive. To study the induction mechanism of insect galls, we selected the gall induced by Iatrophobia brasiliensis (Diptera: Cecidomyiidae) in cassava (Euphorbiaceae: Manihot esculenta Crantz) as our model. PCR-based molecular markers and deep metagenomic sequencing data were employed to analyse the gall microbiome and to test the hypothesis that gall cells are genetically transformed by insect vectored bacteria. A shotgun sequencing discrimination approach was implemented to selectively discriminate between foreign DNA and the reference host plant genome. Several known candidate insertion sequences were identified, the most significant being DNA sequences found in bacterial genes related to the transcription regulatory factor CadR, cadmium-transporting ATPase encoded by the cadA gene, nitrate transport permease protein (nrtB gene), and arsenical pump ATPase (arsA gene). In addition, a DNA fragment associated with ubiquitin-like gene E2 was identified as a potential accessory genetic element involved in gall induction mechanism. Furthermore, our results suggest that the increased quality and rapid development of gall tissue are mostly driven by microbiome enrichment and the acquisition of critical endophytes. An initial gall-like structure was experimentally obtained in M. esculenta cultured tissues through inoculation assays using a Rhodococcus bacterial strain that originated from the inducing insect, which we related to the gall induction process. We provide evidence that the modification of the endophytic microbiome and the genetic transformation of plant cells in M. esculenta are two essential requirements for insect-induced gall formation. Based on these findings and having observed the same potential DNA marker in galls from other plant species (ubiquitin-like gene E2), we speculate that bacterially mediated genetic transformation of plant cells may represent a more widespread gall induction mechanism found in nature.

Bacterial dynamic of flue-cured tobacco leaf surface caused by change of environmental conditions

Microorganisms present on the surface of tobacco leaves play a significant role in shaping the composition of the tobacco microbial ecosystem, which undergoes continuous changes throughout the curing process. In the present study, a total of four distinct tobacco curing periods were selected for sampling, namely the fresh, yellowing, leaf-drying, and stem-drying stages. The bacterial 16S rRNA gene sequences of the collected samples were subsequently analyzed to identify operational taxonomic units (OTUs). The findings indicated that the complete dataset of leaf microbial samples was clustered, resulting in the identification of 1,783 operational taxonomic units (OTUs). Furthermore, the analysis of diversity revealed a pattern of initially increasing and subsequently decreasing community diversity. Redundancy Analysis (RDA) and weighted gene correlation networks for analysis (WGCNA) were employed in conjunction with environmental factors to assign OTUs to 22 modules for functional analysis. Additionally, a classification model utilizing the random forest algorithm was utilized to identify seven marker microorganisms (Escherichia coli, Faecalibacterium prausnitzii, Faecalibacterium, Escherichia-Shigella, Peptostreptococcaceae, Peptostreptococcales-Tissierellales, and Proteobacteria) that exhibited discriminative characteristics across different time periods. This study aimed to investigate the dynamic changes in the bacterial community throughout the curing process and their impact on the community’s function. Additionally, certain bacteria were identified as potential markers for detecting changes in the curing stage. These findings offer a novel opportunity to accurately regulate the curing environment, thereby enhancing the overall quality of tobacco leaf curing.

Long‐term fertilization regulates dissimilatory nitrate reduction processes by altering paddy soil organic carbon components

AbstractTo enhance rice yields, synthetic nitrogen (N) fertilizers are often applied in rice paddy fields. Under waterlogged conditions, denitrification, anaerobic ammonium oxidation (anammox) and dissimilatory nitrate reduction to ammonium (DNRA) are the main N transformation processes. However, the effects of long‐term fertilization on the dissimilatory nitrate reduction processes remain largely unclear. Here, we investigated rates of denitrification, anammox and DNRA and the abundance of bacterial and nitrate reduction‐related genes (16S rRNA, narG, nirK, nirS, nosZ and hzsB) in rice paddy fields under different fertilization regimes [i.e., non‐fertilization (CK), NPK fertilization (NPK) and NPK fertilization plus straw returning (NPKS)]. The rates of denitrification, anammox and DNRA in the CK treatment were 46.18, 0.35 and 1.29 nmoL N g−1 h−1, respectively, which were significantly (p &lt; .05) increased by 7.32%–27.78%, 2.45–4.12 folds and 1.01–13.23 folds by NPK and NPKS treatments. This was largely consistent with the increased bacterial and nitrate reduction‐related gene abundances in the NPK and NPKS treatments. In addition, NPKS also significantly increased the relative contribution of DNRA to the total nitrate reduction relative to NPK and CK treatments, indicating that NPKS was more beneficial to N retention in the paddy field. Changes in rates of dissimilatory nitrate reduction processes in response to NPK and NPKS treatments were correlated to variations of soil total N, ammonium contents, pH, soil organic carbon (SOC) contents and nitrate reduction‐related genes, among which SOC contents had the highest explanation. Specifically, fulvic‐like compounds, alkyl C and methoxyl and nitrogen‐alkyl C were the key components determining denitrification, anammox and DNRA rates, respectively. Overall, our study suggests that long‐term NPK and NPKS fertilization stimulate rates of dissimilatory nitrate reduction processes mainly by altering soil C components and NPKS is more effective in maintaining soil fertility in the paddy field.

Bacterial immunotherapy: is it a weapon in our arsenal in the fight against cancer?

Advances in understanding the genetic basis of cancer have driven alternative treatment approaches. Recent findings have demonstrated the potential of bacteria and it's components to serve as robust theranostic agents for cancer eradication. Compared to traditional cancer therapies like surgery, chemotherapy, radiotherapy, bacteria mediated tumor therapy has exhibited superior cancer suppressing property which is attributed a lot to it's tumor proliferating and accumulating characteristics. Genetically modified bacteria hasreduced inherent toxicity and enhanced specificity towards tumor microenvironment. This anti- tumor activity of bacteria is attributed to its toxins and other active components from the cell membrane, cell wall and spores. Furthermore, bacterial genes can be regulated to express and deliver cytokines, antibodies and cancer therapeutics. Although there is less clinical data available, the pre- clinical research clearly indicates the feasibility and potential of bacteria- mediated cancer therapy.

875. Development of a Cheap, Portable Assay to Rapidly Profile Resistant Bacterial Infections

Abstract Background Antimicrobial resistant (AMR) bacterial infections pose a significant and growing health threat worldwide. The burden of these resistant infections is often most severe in lower income areas, where the medical infrastructure necessary for their diagnosis is lacking. The development of cost-effective and accessible diagnostic tools for the rapid identification of resistant infections in low-resource settings is crucial. Specific High-sensitivity Enzymatic Reporter Unlocking, or SHERLOCK, is a promising CRISPR-based system that can aid in the diagnosis of infections and support clinical decision making. Here, we present an assay termed BADLOCK (Bacteria and AMR Detection by SHERLOCK) that uses SHERLOCK technology to rapidly identify bacterial species and AMR genes on blood cultures. SHERLOCK Schematic Enzymatic activity of the Cas protein is turned on when the target gene is bound, which then activates reporter probes. Methods We combine isothermal amplification of gene targets with SHERLOCK-based detection into a single step, allowing for a streamlined workflow and a total time-to-detection of less than two hours. The assay targets the topA gene for species identification, which is highly conserved within species but has sufficient variability between them to allow for discrimination between even closely related pathogens. A panel of clinically important AMR genes, including CTX-M-15, mecA, and five canonical carbapenemases, is also targeted to provide information on resistance profiles. BADLOCK Targets Gene targets included in the BADLOCK assay. Results BADLOCK has high sensitivity and specificity for both AMR genes and species identification and leverages a lateral-flow platform to provide a simple visual readout. We are currently validating BADLOCK on a library of several hundred clinical isolates with known genetic backgrounds, including a large collection of carbapenem-resistant Enterobacterales, which are major contributors to the global burden of resistant infections. Lateral Flow Assay Results Lateral flow results. Strip 1 is the topA gene specific to K. pneumoniae. Strips 2-4 are AMR genes KPC, CTX-M-15, and MecA. Lane 5 is a positive control; lane 6 is a NTC with bottom banding. “+” and “-” symbols mark representative readouts. Conclusion We have developed an assay for the cheap, rapid detection of bacterial infections of the bloodstream capable of detecting common pathogens as well as a panel of AMR genes. Our work has the potential to improve the diagnostic landscape for resistant infections in low-and-middle-income countries, which would represent a major step forward in infectious disease treatment in these underserved areas. Disclosures All Authors: No reported disclosures

Active soil microbial composition and proliferation are directly affected by the presence of biocides from building materials

Combinations of biocides are commonly added to building materials to prevent microbial growth and thereby cause degradation of the façades. These biocides reach the environment by leaching from façades posing an environmental risk. Although ecotoxicity to the aquatic habitat is well established, there is hardly any data on the ecotoxicological effects of biocides on the soil habitat. This study aimed to characterize the effect of the biocides terbutryn, isoproturon, octhilinone, and combinations thereof on the total and metabolically active soil microbial community composition and functions. Total soil microbial community was retrieved directly from the nucleic acid extracts, while the DNA of the active soil microbial community was separated after bromodeoxyuridine labeling. Bacterial 16S rRNA gene and fungal internal transcribed spacer region gene-based amplicon sequencing was carried out for both active and total, while gene copy numbers were quantified only for the total soil microbial community. Additionally, soil respiration and physico-chemical parameters were analyzed to investigate overall soil microbial activity. The bacterial and fungal gene copy numbers were significantly affected by single biocides and combined biocide soil treatment but not soil respiration and physico-chemical parameters. While the total soil microbiome experienced only minor effects from single and combined biocide treatment, the active soil microbiome was significantly impacted in its diversity, richness, composition, and functional patterns. The active bacterial richness was more sensitive than fungal richness. However, the adverse effects of the biocide combination treatments on soil bacterial richness were highly dependent on the identities of the biocide combination. Our results demonstrate that the presence of biocides frequently used in building materials affects the active soil microbiome. Thereby, the approach described herein can be used as an ecotoxicological measure for the effect on complex soil environments in future studies.

A Mendelian Randomization-Based Causal Investigation on Bacterial Infection-Related Genes and Neurodegenerative Diseases.

Neurodegenerative diseases remain the most prevalent and unsolved health problems in human society, especially Alzheimer's disease (AD) and Parkinson's disease (PD). The pathogenesis, pathology, and potential clinical treatments of neurodegenerative diseases still require in-depth research. In the wake of the association between pandemics and a growing number of neurodegeneration patients, there has been growing speculation that infections are linked to AD and PD. The Aβ peptide is an important causal-related biomarker of AD and is reported to share structural and functional similarities with certain antimicrobial peptides, suggesting that it has a role in eliciting an immune response against microbes. But how neurodegeneration is related to bacterial chronic infection has not been thoroughly investigated. Using the data from genome-wide association studies (GWAS), we performed Mendelian Randomization (MR) and map 7 genes in multiple bacterial infection pathways as exposure, which show a significant association with the outcome of AD or PD. As co-verification, we perform Gene Set Enrichment Analysis (GSEA) on selected genetic variants incorporating their perturb-seq gene list (combining single-cell RNA-seq and CRISPR-based perturbations). We observed clustering of the differentially expressed genes (DEGs) in the upstream and downstream of AD and PD-related KEGG pathways, hence confirming their causal association with AD and PD and providing new perspectives on the true cause of neurodegeneration.

The Microbiome in Quiescent Crohn’s Disease With Persistent Symptoms Show Disruptions in Microbial Sulfur and Tryptophan Pathways

Even in the absence of inflammation, persistent symptoms in Crohn's disease (CD) are prevalent and worsen quality of life. Amongst patients without inflammation (quiescent CD), we hypothesized that microbial community structure and function, including tryptophan metabolism, would differ between patients with persistent symptoms (qCD+ S) and without persistent symptoms (qCD-S). We performed a multicenter observational study nested within the Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease. Quiescent inflammation was defined by fecal calprotectin level <150 mcg/g. Persistent symptoms were defined by Crohn's Disease Patient-Reported Outcome-2. Active CD, diarrhea-predominant irritable bowel syndrome, and healthy controls were included as controls. Stool samples underwent whole-genome shotgun metagenomic sequencing. Thirty-nine patients with qCD+ S, 274 qCD-S, 21 active CD, 40 diarrhea-predominant irritable bowel syndrome, and 50 healthy controls were included for analysis. Patients with qCD+ S had a less-diverse microbiome. Furthermore, patients with qCD+ S showed significant enrichment of bacterial species that are normal inhabitants of the oral microbiome (eg Rothia dentocariosa, Fusobacterium nucleatum) and sulfidogenic microbes (eg Prevotella copri, Bilophila spp.). Depletion of important butyrate and indole producers (eg Eubacterium rectale, Faecalibacterium prausnitzii) was also noted in qCD+ S. Potential metagenome-related functional changes in cysteine and methionine metabolism, ATP transport, and redox reactions were disturbed in qCD+ S, also suggestive of altered sulfur metabolism. Finally, qCD+ S showed significant reductions in bacterial tnaA genes, which mediate tryptophan metabolism to indole, and significant tnaA allelic variation compared with qCD-S. The microbiome in qCD+ S showed significant differences in sulfidogenesis, butyrate producers, and typically oral microbes compared to qCD-S and active CD. These results suggest that inflammation may lead to durable microbiome alterations which may mediate persistent symptoms through testable mechanisms.

Targeted amplicon sequencing facilitated a novel risk assessment framework for assessing the prevalence of broad spectrum bacterial and coronavirus diseases

How to effectively leverage wastewater data to estimate the risk of various infectious diseases remains a great challenge. To address this issue, we conducted continuous wastewater surveillance in Dalian city during the summer-autumn seasons of 2022, targeting coronavirus and bacterial diseases. The surveillance included daily sampling at a wastewater treatment plant (WWTP) and weekly sampling in three sewersheds. Targeting the bacteria's 16S rRNA gene and the coronavirus's RNA-dependent RNA polymerase (RdRp) gene, we first employed RT–PCR and amplicon sequencing techniques to analyze the presence and phylogenetic relationship of detected coronavirus and bacterial pathogens. Next, qPCR was used to quantify the abundances of detected coronavirus and bacterial species. Based on the daily shedding dynamics of SARS-CoV-2, a novel model was developed to predict daily new cases. Based on the medium shedding density of 12 pathogens, two thresholds of sewage pathogen load (indicating 0.1 % and 1 % infection rates) were proposed. Our PanCoV RT–PCR detected coronavirus on 12th August and from 26th August to 12th September 2022. Targeted amplicon sequencing further identified human coronavirus OC43 (hCoV-OC43) on 12th August and the SARS-CoV-2 Omicron variant since 26th August in samples from WWTPs and sewersheds. Phylogenetic analysis revealed that hCoV-OC43 from this study belonged to genotype K and suggested a close relationship between the amplified coronavirus sequences from wastewater and clinical samples in a local COVID-19 outbreak on 26th August. Amplicon sequencing targeting the bacterial 16S rRNA gene also revealed the presence of several bacterial pathogens. Finally, we assessed the microbial risk of specific pathogens in sewersheds and identified a number of pathogens that reached high (>1 % prevalence) and medium risk levels (>0.1 % prevalence) at sewershed B. Our findings underline wastewater surveillance as a valuable early warning system for coronavirus and other waterborne bacterial diseases, complementing public health response measures.

Effect of RpoS on the survival, induction, resuscitation, morphology, and gene expression of viable but non-culturable Salmonella Enteritidis in powdered infant formula

Involvement of the transcriptional regulator RpoS in the persistence of viable but non-culturable (VBNC) state has been demonstrated in several species of bacteria. This study investigated the role of the RpoS in the formation and resuscitation of VBNC state in Salmonella enterica serovar Enteritidis CICC 21482 by measuring bacterial survival, morphology, physiological characteristics, and gene expression in wild-type (WT) and rpoS-deletion (ΔrpoS) strains during long-term storage in powdered infant formula (PIF). The ΔrpoS strain was produced by allelic exchange using a suicide plasmid. Bacteria were inoculated into PIF for 635-day storage. Survival, morphology, intracellular reactive oxygen species (ROS) levels and intercellular quorum sensing autoinducer-2 (AI-2) contents were regularly measured. Resuscitation assays were conducted after obtaining VBNC cells. Gene expression was measured using real-time quantitative polymerase chain reaction (qPCR). The results showed that RpoS and low temperature conditions were associated with enhanced culturability and recoverability of Salmonella Enteritidis after desiccation storage in low water activity (aw) PIF. In addition, the synthesis of intracellular ROS and intercellular quorum sensing AI-2 was regulated by RpoS, inducing the formation and resuscitation of VBNC cells. Gene expression of soxS, katG and relA was found strongly associated with RpoS. Due to the lack of RpoS factor, the ΔrpoS strain could not normally synthesize SoxS, catalase and (p)ppGpp, resulting in its early shift to the VBNC state. This study elucidates the role of rpoS in desiccation stress and the formation and resuscitation mechanism of VBNC cells under desiccation stress. It serves as the basis for preventing and controlling the recovery of pathogenic bacteria in VBNC state in low aw foods.