New strategies to treat diseases wherein biofilms contribute significantly to pathogenesis are needed as biofilm-resident bacteria are highly recalcitrant to antibiotics due to physical biofilm architecture and a canonically quiescent metabolism, among many additional attributes. We, and others, have shown that when biofilms are dispersed or disrupted, bacteria released from biofilm residence are in a distinct physiologic state that, in part, renders these bacteria highly sensitive to killing by specific antibiotics. We sought to demonstrate the breadth of ability of a recently humanized monoclonal antibody against an essential biofilm structural element (DNABII protein) to disrupt biofilms formed by respiratory tract pathogens and potentiate antibiotic-mediated killing of bacteria released from biofilm residence. Biofilms formed by six respiratory tract pathogens were significantly disrupted by the humanized monoclonal antibody in a dose- and time-dependent manner, as corroborated by CLSM imaging. Bacteria newly released from the biofilms of 3 of 6 species were significantly more sensitive than their planktonic counterparts to killing by 2 of 3 antibiotics currently used clinically and were now also equally as sensitive to killing by the 3 rd antibiotic. The remaining 3 pathogens were significantly more susceptible to killing by all 3 antibiotics. A humanized monoclonal antibody directed against protective epitopes of a DNABII protein effectively released six diverse respiratory tract pathogens from biofilm residence in a phenotypic state that was now as, or significantly more, sensitive to killing by three antibiotics currently indicated for use clinically. These data support this targeted, combinatorial, species-agnostic therapy to mitigate chronic bacterial diseases.
The microbiome has been recognized as being highly impactful for cancer outcomes, both with respect to the diversity and composition of the microbial population, as well as overall abundance. Presence or absence of certain microbial species has been correlated to improved cancer outcomes, in concert with specific treatments. However, little research has been performed to consider the reciprocal of this relationship: how different treatments, or the progression of disease, impact the microbiome. Further, though correlations have been demonstrated between dietary intake of both fats and carbohydrates and the composition and breadth of the microbiome, no studies have investigated the microbiome population as it is impacted by cancer and therapeutic treatments under different dietary regiments. This study aims to investigate how dietary patterns, in concert with progression of cancer and both surgical and therapeutic interventions, impact the diversity and abundance of the murine microbiome. A total of 116 c57BI/6 female mice (7‐8 weeks old) were randomized into four groups (n=29/group, each) one week post ovariectomy. Mice received a specific, semi‐purified diet consisting of A) low sucrose, high omega 3 fatty acid; B) low sucrose, low omega 3 fatty acid; C) high sucrose, high omega 3 fatty acid; or D) high sucrose, high omega 3 fatty acid. For high omega 3 fatty acid groups, 2% of daily calories were supplied to the mice as EPA and DHA in a 1.5:1 ratio. One week after implementation of diets all mice were inoculated with E0771 breast cancer cells. Twelve days following inoculation all mice were subject to lumpectomy. Eight days following, measurement procedures were repeated, as at baseline. Ten days following lumpectomy, half of the mice from each dietary cohort were treated with saline, while the other half were treated with a chemotherapeutic agent (9 mg/kg body weight doxorubicin and 90 mg/kg body weight cyclophosphamide). Six days later, the experiment ended, and mice were sacrificed following IACUC guidelines. Fecal samples, which had been collected at each time point noted above, were processed using QIAmp® PowerFecal® DNA isolation kit and were sent to MCIC (OSU Wooster Campus) for 16S ribosomal genetic profiling. A genomic library was generated, using previously validated primers for the V4 region. FASTQ files were filtered, trimmed, dereplicated, and denoised. Cleaned FASTQ files were then used to infer ASVs. Over the progression of cancer, there has been a consistent decrease in the alpha diversity of the microbiome. The effect of diet was mitigated by introduction of chemotherapeutic agents, though the significance of this effect is yet to be determined. While the analysis is still ongoing, it has been determined that there is a clear dietary effect which interacts with abundance. Multiple ASVs have been shown to be positively associated with high fat consumption, though inverse effects, as well as timepoint and treatment effects, have also been noted. Akkermansia muciniphila was a highly present bacter...
The intestinal microbial population is recognized for its impact on cancer treatment outcomes. Little research has reported microbiome changes during cancer progression or the interplay of disease progression, dietary sugar/fat intake, and the microbiome through surgery and chemotherapy. In this study, the murine gut microbiome was used as a model system, and changes in microbiome diversity, richness, and evenness over the progression of the cancer and treatment were analyzed. Mice were categorized into four diet cohorts, combinations of either high or low sucrose and high or low omega-3 fatty acids, and two treatment cohorts, saline vehicle or chemotherapy, for a total of eight groups. Fecal samples were collected at specific timepoints to assess changes due to diet implementation, onset of cancer, lumpectomy, and chemotherapy. Akkermansia muciniphila abundance was very high in some samples and negatively correlated with overall Amplicon Sequence Variant (ASV) richness (r(64) = −0.55, p = 3 × 10−8). Throughout the disease progression, ASV richness significantly decreased and was impacted by diet and treatment. Alpha-diversity and differential microbial abundance were significantly affected by disease progression, diet, treatment, and their interactions. These findings help establish a baseline for understanding how cancer progression, dietary macronutrients, and specific treatments impact the murine microbiome, which may influence outcomes.
Objectives This study aims to investigate how dietary patterns, in concert with progression of cancer and both surgical and therapeutic interventions, impact the diversity and abundance of the murine microbiome. Methods 116 c57BI/6 female mice (7–8 weeks old) post-ovariectomy, were placed into diet groups A) low-sucrose/high-omega-3; B) low-sucrose/low-omega-3; C) high-sucrose/high-omega-3; or D) high-sucrose/high-omega-3. Mice were inoculated with breast cancer cells, subjected to lumpectomy, and treated with chemotherapy (9 mg/kg doxorubicin/90 mg/kg cyclophosphamide; and half received saline). Fecal samples were collected for gut microbiome analysis over the course of experiment and processed for 16S ribosomal genetic profiling using QIAmp® PowerFecal® DNA kit. A genomic library was generated, using previously validated primers for the V4 region. Cleaned FASTQ files were then used to infer ASVs (Amplicon Sequence Variants). Results There is a clear dietary effect which interacts with microbial abundance. Multiple ASVs have shown to be positively associated with high fat consumption, though inverse effects, as well as timepoint and treatment effects, have also been noted. Although Akkermansia muciniphila is usually seen as a minor gut resident, the species was abundant in all samples. Over the progression of cancer, a consistent decrease in the alpha diversity of the microbiome was observed. Notably, this effect was somewhat mitigated by the introduction of chemotherapeutic agents. Conclusions The findings of this study provide the foundation for future investigation of cancer patients specimens, with the overall goal of treatment improvement, discomfort mitigation, and better overall quality of life. Funding Sources This study was completed using The Ohio State University Ohio Agricultural Research and Development Center Hatch Funds.
IntroductionThe “silent” antimicrobial resistance (AMR) pandemic is responsible for nearly five million deaths annually, with a group of seven biofilm-forming pathogens, known as the ESKAPEE pathogens, responsible for 70% of these fatalities. Biofilm-resident bacteria, as they exist within the disease site, are canonically highly resistant to antibiotics. One strategy to counter AMR and improve disease resolution involves developing methods to disrupt biofilms. These methods aim to release bacteria from the protective biofilm matrix to facilitate their killing by antibiotics or immune effectors. Several laboratories working on such strategies have demonstrated that bacteria newly released from a biofilm display a transient phenotype of significantly increased susceptibility to antibiotics. Similarly, we developed an antibody-based approach for biofilm disruption directed against the two-membered DNABII family of bacterial DNA-binding proteins, which serve as linchpins to stabilize the biofilm matrix. The incubation of biofilms with α-DNABII antibodies rapidly collapses them to induce a population of newly released bacteria (NRel).MethodsIn this study, we used a humanized monoclonal antibody (HuTipMab) directed against protective epitopes of a DNABII protein to determine if we could disrupt biofilms formed by the high-priority ESKAPEE pathogens as visualized by confocal laser scanning microscopy (CLSM) and COMSTAT2 analysis. Then, we demonstrated the potentiated killing of the induced NRel by seven diverse classes of traditional antibiotics by comparative plate count.ResultsTo this end, ESKAPEE biofilms were disrupted by 50%−79% using a single tested dose and treatment period with HuTipMab. The NRel of each biofilm were significantly more sensitive to killing than their planktonically grown counterparts (heretofore, considered to be the most sensitive to antibiotic-mediated killing), even when tested at a fraction of the MIC (1/250–1/2 MIC). Moreover, the bacteria that remained within the biofilms of two representative ESKAPEE pathogens after HuTipMab disruption were also significantly more susceptible to killing by antibiotics.DiscussionNew data presented in this study support our continued development of a combinatorial therapy wherein HuTipMab is delivered to a patient with recalcitrant disease due to an ESKAPEE pathogen to disrupt a pathogenic biofilm, along with a co-delivered dose of an antibiotic whose ability to rapidly kill the induced NRel has been demonstrated. This novel regimen could provide a more successful clinical outcome to those with chronic, recurrent, or recalcitrant diseases, while limiting further contribution to AMR.
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