Project description:Is there a universal genetically programmed defense providing tolerance to antibiotics when bacteria grow as biofilms? A comparison between biofilms of three different bacterial species by transcriptomic and metabolomic approaches uncovered no evidence of one. Single-species biofilms of three bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii) were grown in vitro for three days then challenged with respective antibiotics (ciprofloxacin, daptomycin, tigecycline) for an additional 24 h. All three microorganisms displayed reduced susceptibility in biofilms compared to planktonic cultures. Global transcriptomic profiling of gene expression comparing biofilm to planktonic and antibiotic-treated biofilm to untreated biofilm was performed. Extracellular metabolites including 18 amino acids, glucose, lactate, acetate, formate, and ethanol were measured to characterize the utilization of carbon sources between biofilms, treated biofilms, and planktonic cells. While all three bacteria exhibited a species-specific signature of stationary phase, no conserved gene, gene set, or common functional pathway could be identified that changed consistently across the three microorganisms. Across the three species, glucose consumption was increased in biofilms compared to planktonic cells and alanine and aspartic acid utilization were decreased in biofilms compared to planktonic cells. The reasons for these changes were not readily apparent in the transcriptomes. No common shift in the utilization pattern of carbon sources was discerned when comparing untreated to antibiotic-exposed biofilms. Overall, our measurements do not support the existence of a common genetic or biochemical basis for biofilm tolerance against antibiotics. Rather, there are likely myriad genes, proteins, and metabolic pathways that influence the physiological state of microorganisms in biofilms contributing to antibiotic tolerance. The Acinetobacter baumannii microarray data from the study described above is deposited here.
Project description:Is there a universal genetically programmed defense providing tolerance to antibiotics when bacteria grow as biofilms? A comparison between biofilms of three different bacterial species by transcriptomic and metabolomic approaches uncovered no evidence of one. Single-species biofilms of three bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii) were grown in vitro for three days then challenged with respective antibiotics (ciprofloxacin, daptomycin, tigecycline) for an additional 24 h. All three microorganisms displayed reduced susceptibility in biofilms compared to planktonic cultures. Global transcriptomic profiling of gene expression comparing biofilm to planktonic and antibiotic-treated biofilm to untreated biofilm was performed. Extracellular metabolites including 18 amino acids, glucose, lactate, acetate, formate, and ethanol were measured to characterize the utilization of carbon sources between biofilms, treated biofilms, and planktonic cells. While all three bacteria exhibited a species-specific signature of stationary phase, no conserved gene, gene set, or common functional pathway could be identified that changed consistently across the three microorganisms. Across the three species, glucose consumption was increased in biofilms compared to planktonic cells and alanine and aspartic acid utilization were decreased in biofilms compared to planktonic cells. The reasons for these changes were not readily apparent in the transcriptomes. No common shift in the utilization pattern of carbon sources was discerned when comparing untreated to antibiotic-exposed biofilms. Overall, our measurements do not support the existence of a common genetic or biochemical basis for biofilm tolerance against antibiotics. Rather, there are likely myriad genes, proteins, and metabolic pathways that influence the physiological state of microorganisms in biofilms contributing to antibiotic tolerance. The Staphylococcus aureus microarray data from the study described above is deposited here.
Project description:The heterogeneity of bacterial cells in biofilms is an important property. We performed single-cell sequencing to profile heterogeneous cell populations within Vibrio alginolyticus biofilm. Our analysis has revealed the key functional compartments for the composition and development of biofilms
Project description:Campylobacter jejuni, an important pathogen of bacterial gastrointestinal infections, forms biofilms that are crucial for its survival in different environments. We used RNA sequencing to investigate the changes in gene expression at different stages of biofilm formation (16 h, 24 h, 48 h and 72 h). The results showed that early biofilms (16 h and 24 h biofilms) show increased expression of genes involved in cysteine and methionine amino acid metabolism, while mature biofilms (48 h and 72 h biofilms) show decreased expression of capsular polysaccharides and lipooligosaccharides. Both early and mature biofilms are characterized by increased expression of genes involved in flagella formation, leucine amino acid metabolism and oxidative stress response, and decreased expression of genes involved in energy metabolism, iron acquisition and transmembrane transport. Thus, we provide a deeper understanding of the molecular mechanisms of C. jejuni biofilm maturation, environmental resistance and the dynamic nature of gene expression that facilitates adaptation to biofilm-associated lifestyles
Project description:Biofilms are heterogeneous bacterial communities featured by high persister prevalence, responsible for antibiotic tolerance. However, the mechanisms underlying persister formation within biofilms remained ambiguous. Here, by developing and utilizing a ribosomal RNA depleted bacterial single-cell RNA-seq method, RiboD-mSPLiT, we resolved biofilm heterogeneity and discovered pdeI as a marker gene for persister subgroup within biofilms. Remarkably, our findings elucidated that PdeI upregulates cellular levels of c-di-GMP, which acts as an antitoxin to modulate the toxicity of toxin protein HipH. HipH localizes on nucleoid and functions as a potent DNase, inducing cells into a viable but non-culturable state. Conversely, c-di-GMP interacts with HipH, reducing its genotoxic effects and enabling cells to enter a persister state, resulting in drug tolerance. Importantly, by targeting this toxin-antitoxin system, we repressed drug tolerance in Uropathogenic Escherichia coli infections, offering promising therapeutic strategies against chronic and relapsing infections.
Project description:Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms, as well as responding leukocytes, may impede wound healing. In this study, we used oxygen microsensors to measure oxygen transects through in vitro-cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse model, and ex vivo human chronic wound specimens. The results show that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17-72 mmHg on live mice and 6.4-1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. No oxygen gradients were observed for heat-killed mouse scabs, suggesting that active metabolism by the viable bacteria and host cells contributed to the reduced oxygen partial pressure of the scabs. To characterize the metabolic activities of the bacteria in the mouse scabs, we performed transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds using Affymetrix microarrays. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results indicated that the bacteria within the wounds also experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results support the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions.
Project description:In this study, we probed factors that could influence Shigella pathogenesis. We show that in basic pH conditions, deoxycholate-induced biofilm formation and virulence of Shigella are attenuated. We utilized RNA-sequencing to investigate pathways enriched in bacterial cells in biofilms.
Project description:In contrast to mammalian cells, bacterial cells lack mRNA polyadenylated tails, presenting a hurdle in isolating mRNA amidst the prevalent rRNA during single-cell RNA-seq. This study introduces a novel method, Ribosomal RNA-derived cDNA Depletion (RiboD), seamlessly integrated into the PETRI-seq technique, yielding RiboD-PETRI. This innovative approach offers a cost-effective, equipment-free, and high-throughput solution for bacterial single-cell RNA sequencing (scRNA-seq). By efficiently eliminating rRNA reads and substantially enhancing mRNA detection rates (up to 92%), our method enables precise exploration of bacterial population heterogeneity. Applying RiboD-PETRI to investigate biofilm heterogeneity, distinctive subpopulations marked by unique genes within biofilms were successfully identified. Notably, PdeI, a marker for the cell-surface attachment subpopulation, was observed to elevate cyclic diguanylate (c-di-GMP) levels, promoting persister cell formation. Thus, we address a persistent challenge in bacterial single-cell RNA-seq regarding rRNA abundance, exemplifying the utility of this method in exploring biofilm heterogeneity. Our method effectively tackles a long-standing issue in bacterial scRNA-seq: the overwhelming abundance of rRNA. This advancement significantly enhances our ability to investigate the intricate heterogeneity within biofilms at unprecedented resolution.
Project description:In contrast to mammalian cells, bacterial cells lack mRNA polyadenylated tails, presenting a hurdle in isolating mRNA amidst the prevalent rRNA during single-cell RNA-seq. This study introduces a novel method, Ribosomal RNA-derived cDNA Depletion (RiboD), seamlessly integrated into the PETRI-seq technique, yielding RiboD-PETRI. This innovative approach offers a cost-effective, equipment-free, and high-throughput solution for bacterial single-cell RNA sequencing (scRNA-seq). By efficiently eliminating rRNA reads and substantially enhancing mRNA detection rates (up to 92%), our method enables precise exploration of bacterial population heterogeneity. Applying RiboD-PETRI to investigate biofilm heterogeneity, distinctive subpopulations marked by unique genes within biofilms were successfully identified. Notably, PdeI, a marker for the cell-surface attachment subpopulation, was observed to elevate cyclic diguanylate (c-di-GMP) levels, promoting persister cell formation. Thus, we address a persistent challenge in bacterial single-cell RNA-seq regarding rRNA abundance, exemplifying the utility of this method in exploring biofilm heterogeneity. Our method effectively tackles a long-standing issue in bacterial scRNA-seq: the overwhelming abundance of rRNA. This advancement significantly enhances our ability to investigate the intricate heterogeneity within biofilms at unprecedented resolution.