Ophthalmic viscoelastics commonly used in cataract surgery: A microbiota investigation.

PURPOSE: To survey commonly used, sterile ophthalmic viscoelastic materials used during routine cataract surgery for the presence of bacterial DNA and/or viable bacteria and endotoxin quantification. METHODS: Samples from three different ophthalmic viscoelastic manufacturers and three different production lots per manufacturer were collected for 16 S ribosomal ribonucleic acid (rRNA) sequencing and conventional aerobic and capnophilic bacterial culture. Other samples of viscoelastic material from the same three manufacturers were collected for endotoxin quantification using a commercially available Limulus amebocyte lysate (LAL) assay. Statistical analysis was performed using Sigma Plot 14.0, and R v4.0.2.0. Differences (p ≤ .05) between sample collection sites in total DNA concentration, microbial richness, mean intra-group distances, and endotoxin quantification alongside reagent controls were evaluated. RESULTS: Culture yielded two isolates, identified as Staphylococcus epidermidis and Bacillus megaterium. 16 S rRNA sequencing revealed no differences between brands in richness or overall composition. The most common bacterial DNA detected across all brands was Staphylococcus sp., Cutibacterium sp., Flavobacterium sp., and Lactobacillus sp. A significant difference was found between the median endotoxin concentration between Anvision and Hyvisc® viscoelastic (Anvision: 0.171 EU/mL, Hyvisc®: 0.03 EU/mL; p < .001). CONCLUSIONS: No brand-specific differences in bacterial DNA were detected in the viscoelastic materials. Staphylococcus, Cutibacterium, Flavobacterium, and Lactobacillus were the dominant contributors to the bacterial DNA detected. Although Anvision viscoelastic samples contained significantly more endotoxin than Hyvisc® viscoelastic samples, endotoxin concentrations were below the FDA limit of 0.2 EU/mL for both manufacturers. These data further the understanding of inflammatory outcomes following cataract surgery.

Acute and long-term effects of antibiotics commonly used in laboratory animal medicine on the fecal microbiota.

Biomedical research relies on the use of animal models, and the animals used in those models receive medical care, including antibiotics for brief periods of time to treat conditions such as dermatitis, fight wounds, and suspected bacterial pathogens of unknown etiology. As many mouse model phenotypes are sensitive to changes in the gut microbiota, our goal was to examine the effect of antibiotics commonly administered to mice. Therefore, four treatment groups (subcutaneous enrofloxacin for 7 days, oral enrofloxacin for 14 days, oral trimethoprim-sulfamethoxazole for 14 days, and topical triple antibiotic ointment for 14 days) alongside a fifth control group receiving no treatment (n = 12/group) were included in our study. Fecal samples were collected prior to treatment, immediately after two weeks of exposure, and four weeks after cessation of treatment, and subjected to 16S rRNA library sequencing. The entire experimental design was replicated in mice from two different suppliers. As expected, several treatments including enrofloxacin and triple antibiotic ointment substantially decreased the amount of DNA recovered from fecal material, as well as the microbial richness. Notably, many of these effects were long-lasting with diminished gut microbiota (GM) richness four weeks following exposure, in both substrains of mice. Trimethoprim-sulfamethoxazole induced minimal to no discernible changes in the taxonomic composition beyond that seen in control mice. Collectively, these data highlight the need to consider the impact on GM of brief and seemingly routine use of antibiotics in the clinical care of research animals.

Effect size of delayed freezing, diurnal variation, and hindgut location on the mouse fecal microbiome.

Practical considerations in fecal sample collection for microbiome research include time to sample storage, time of collection, and hindgut position during terminal collections. Here, parallel experiments were performed to investigate the relative effect of these factors on microbiome composition in mice colonized with two different vendor-origin microbiomes. 16S rRNA amplicon sequencing of immediately flash-frozen feces showed no difference in alpha or beta diversity compared to samples incubated up to 9 h at room temperature. Samples collected in the morning showed greater alpha diversity compared to samples collected in the afternoon. While a significant effect of time was detected in all hindgut regions, the effect increased from cecum to distal colon. This study highlights common scenarios in microbiome research that may affect outcome measures of microbial community analysis. However, we demonstrate a relatively low effect size of these technical factors when compared to a primary experimental factor with large intergroup variability.

Standardized Complex Gut Microbiomes Influence Fetal Growth, Food Intake, and Adult Body Weight in Outbred Mice.

Obesity places a tremendous burden on individual health and the healthcare system. The gut microbiome (GM) influences host metabolism and behaviors affecting body weight (BW) such as feeding. The GM of mice varies between suppliers and significantly influences BW. We sought to determine whether GM-associated differences in BW are associated with differences in intake, fecal energy loss, or fetal growth. Pair-housed mice colonized with a low or high microbial richness GM were weighed, and the total and BW-adjusted intake were measured at weaning and adulthood. Pups were weighed at birth to determine the effects of the maternal microbiome on fetal growth. Fecal samples were collected to assess the fecal energy loss and to characterize differences in the microbiome. The results showed that supplier-origin microbiomes were associated with profound differences in fetal growth and excessive BW-adjusted differences in intake during adulthood, with no detected difference in fecal energy loss. Agreement between the features of the maternal microbiome associated with increased birth weight here and in recent human studies supports the value of this model to investigate the mechanisms by which the maternal microbiome regulates offspring growth and food intake.

Molecular and culture-based assessment of the microbiome in a zebrafish (Danio rerio) housing system during set-up and equilibration.

BACKGROUND: Zebrafish used in research settings are often housed in recirculating aquaculture systems (RAS) which rely on the system microbiome, typically enriched in a biofiltration substrate, to remove the harmful ammonia generated by fish via oxidation. Commercial RAS must be allowed to equilibrate following installation, before fish can be introduced. There is little information available regarding the bacterial community structure in commercial zebrafish housing systems, or the time-point at which the system or biofilter reaches a microbiological equilibrium in RAS in general. METHODS: A zebrafish housing system was monitored at multiple different system sites including tank water in six different tanks, pre- and post-particulate filter water, the fluidized bed biofilter substrate, post-carbon filter water, and water leaving the ultra-violet (UV) disinfection unit and entering the tanks. All of these samples were collected in quadruplicate, from prior to population of the system with zebrafish through 18 weeks post-population, and analyzed using both 16S rRNA amplicon sequencing and culture using multiple agars and annotation of isolates via matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. Sequencing data were analyzed using traditional methods, network analyses of longitudinal data, and integration of culture and sequence data. RESULTS: The water microbiome, dominated by Cutibacterium and Staphylococcus spp., reached a relatively stable richness and composition by approximately three to four weeks post-population, but continued to evolve in composition throughout the study duration. The microbiomes of the fluidized bed biofilter and water leaving the UV disinfection unit were distinct from water at all other sites. Core taxa detected using molecular methods comprised 36 amplicon sequence variants, 15 of which represented Proteobacteria including multiple members of the families Burkholderiaceae and Sphingomonadaceae. Culture-based screening yielded 36 distinct isolates, and showed moderate agreement with sequencing data. CONCLUSIONS: The microbiome of commercial RAS used for research zebrafish reaches a relatively stable state by four weeks post-population and would be expected to be suitable for experimental use following that time-point.

Effects of Fenbendazole-impregnated Feed and Topical Moxidectin during Quarantine on the Gut Microbiota of C57BL/6 Mice.

To protect the biosecurity of research rodent colonies, research institutions frequently require a quarantine period for live animals transferred into their facilities. Quarantine practices often include antibiotic and antiparasitic treatment with drugs such as fenbendazole and macrolide lactones. The influence of these compounds on the resident gut microbiota of mice is unknown, and any effects might subsequently affect model reproducibility. To test the influence of standard quarantine procedures on the composition of the microbiota, C57BL/6 mice, purchased from 2 different commercial suppliers, were randomly assigned to treatment groups (n = 12) by vendor and treated with fenbendazole-supplemented feed, topical moxidectin, both treatments, or no treatment (control), according to our institution's standard treatment regimen and duration. Feces were collected on arrival, immediately after completing the 8-wk treatment, and at 2 and 4 wk after treatment. Fecal DNA was extracted, sequenced, and analyzed to compare the changes in the microbiota of treated and control groups. Although significant main effects of time and treatment and interactions between those variables were detected in comparisons of richness, α-diversity, and ß-diversity, the effect sizes associated with any particular treatment were consistently much smaller than that associated with acclimation to a new facility in the absence of any quarantine treatments. This outcome, along with the visual evaluation of principal coordinate analysis based on multiple similarity indices, suggests that time or institution plays a larger role in alterations of the murine gut microbiota than do quarantine treatments on its composition.

Variable Colonization after Reciprocal Fecal Microbiota Transfer between Mice with Low and High Richness Microbiota.

Several associations have been made between characteristics of the resident gut microbiota and human health and disease susceptibility. Animal models provide the means to test these correlations prospectively and evaluate causality. Experimental fecal microbiota transfer (FMT), or the intentional transplantation of gut microbes into recipient mice depleted of their autochthonous microbes with antibiotics, is a commonly used method of testing these relationships. The true completeness of microbial transfer through such procedures is poorly documented in the literature, particularly in the context of reciprocal transfer of microbes between recipient and donor mice harboring microbial populations of differing richness and diversity. Moreover, it is unclear whether the use of frozen fecal contents or cecal contents would confer any difference in the outcomes of transfer. Herein, groups of mice colonized with distinct gut microbiota of differing richness and composition were used in a reciprocal FMT study, with different groups receiving transfer of material prepared from fresh cecal contents, fresh feces, or frozen feces. Targeted 16S rRNA gene amplicon sequencing was used at intervals throughout the study to characterize the microbiota. Notably, despite comparable depletion of the microbiota in recipient mice prior to transfer, donor-specific taxa reliably colonized recipients only when relatively rich donor material was transferred to mice originally colonized with a simpler microbiota. It is unclear whether these differences were due to differences in the endogenous recipient microbiota or host factors induced in early life by microbial factors. These findings are of practical import for researchers using FMT to prospectively assess the influence of the gut microbiota in mouse models, and to those studying host-microbial interactions and their influence on gut barrier function.