| Metadata |
| datasetIdentifier | PASS01632 |
| datasetType | SRM |
| submitter | Ulrike Kusebauch <ukusebauch@systemsbiology.org> |
| submitter_organization | Institute f. Systems Biology |
| lab_head_full_name | Robert L. Moritz |
| lab_head_email | Robert.Moritz@systemsbiology.org |
| lab_head_organization | Institute f. Systems Biology |
| lab_head_country | United States |
| datasetTag | cefoperazone |
| datasetTitle | Quantification of cefoperazone in fecal and mouse samples of mice |
| publicReleaseDate | 2020-10-02 00:00:00 |
| finalizedDate | |
| summary | Broad spectrum antibiotics can cause both transient and lasting damage to the ecology of the gut microbiome. Antibiotic-induced loss of gut bacterial diversity has been linked to susceptibility to enteric infections. Antibiotic-tolerant populations of cells are known to arise spontaneously in single-strain systems. Furthermore, prior work on subtherapeutic antibiotic treatment in humans and therapeutic treatments in non-human animals have suggested that entire gut communities may exhibit tolerance phenotypes. In this study, we validate the existence of these community tolerance phenotypes in the murine gut and explore how antibiotic treatment duration or a diet enriched in antimicrobial phytochemicals might influence the frequency of this phenotype. We find that almost a third of mice exhibit whole-community tolerance to a high dose of the β-lactam antibiotic cefoperazone, independent of antibiotic treatment duration or dietary phytochemical amendment. These non-responder (i.e. antibiotic tolerant) microbiota did not exhibit the biomass depletion, transient ecological community collapse, and persistent species losses seen in the responder (i.e. susceptible) microbiota. We observed few compositional differences between non-responder microbiota during antibiotic treatment and the untreated control microbiota at the phylum level. However, gene expression was vastly different between non-responder microbiota and controls during treatment, with non-responder communities showing an upregulation of antimicrobial tolerance genes, like efflux transporters, and a down-regulation of central metabolism. Finally, we observed slightly lower average cefoperazone concentrations in a subset of non-responder fecal samples two days following antibiotic treatment, when compared to a subset of responder fecal samples. Thus, most (but not all) non-responder communities show slightly lower in situ antibiotic concentrations, either due to host- or microbial-associated heterogeneity, and appear to avoid antibiotic-induced ecological damage through a combination of efflux transporter upregulation and a reduced growth rate across the entire gut community. Future work should focus on what specific host- or microbiome-associated factors are responsible for tipping entire communities from responder and non-responder phenotypes so that we might learn to harness this phenomenon to protect our microbiota from exposure to routine antibiotic treatment. |
| contributors | Christian Diener, Anna C. H. Hoge, Sean M. Kearney, Ulrike Kusebauch, Sushmita Patwardhan, Robert L. Moritz, Susan E. Erdman, and Sean M. Gibbons |
| publication | Diener, C, Hoge, ACH, Kearney, SM, Kusebauch, U, Patwardhan, S, Moritz, RL, Erdman, SE, and Gibbons, SM, Non-responder phenotype reveals apparent microbiome-wide antibiotic tolerance in the murine gut, submitted |
| growth | |
| treatment | A cohort of 28 mice were split randomly into two diet treatment groups and were fed with either a custom chow diet (Bio-Serv, Flemington NJ) containing 1% raw seaweed nori (Izumi Brand) or a standard control diet (product no. F3156; AN-93G; Bio-Serv, Flemington NJ). Prior to the experiment, animals were co-housed for 10 days and then singly housed for 7 days prior to separation into the seaweed treatment and control groups. After 20 days of dietary treatment, all mice resumed the standard diet. From day 26 to day 31, 8 mice from each diet group were administered 0.5 mg/mL cefoperazone in their drinking water as in the duration experiment. All mice were weighed and assessed daily. As per our IACUC protocol, any mouse showing significant signs of morbidity/suffering would be humanely euthanized. No mice showed signs of morbidity or distress during the course of both experiments. Fresh fecal samples were obtained within an hour of one another each day from all animals in all groups. Fecal samples were collected into anaerobic 40% glycerol containing 0.1% cysteine and transferred immediately to dry ice before being stored at -80° C prior to nucleic acid extraction. |
| extraction | Mice fecal sample preparation
To extract cefoperazone from fecal samples of mice (collected on day 15, 27, 32 and 33), 750 µL of 90% acetonitrile/10% water and 50 µL internal standard (IS) ceftiofur at 50 µg/mL (dissolved in water) were added to each sample in a SK38 Soil Kit 2mL tube (containing 0.1mm glass beads, 1.4mm ceramic (zirconium oxide) beads and a 3mm glass bead, Bertin Corp). Samples were disrupted at 4°C using a Precellys 24 homogenizer (Bertin Corp) at 6500 rpm for 3 x 30 s. Samples were centrifuged for 8 min at 16,000 rpm at 4°C. The supernatant was removed, a 5 µL aliquot diluted 1:250 with 60% acetonitrile/40% water, and 5 µL injected for SRM analysis.
Mice plasma sample preparation
Extraction of cefoperazone from plasma was guided by Wu et al. 61. 500 µL acetonitrile, 12 µL water and 38 µL IS ceftiofur at 0.4 µg/mL (dissolved in water) were added to 50 µL of plasma (collected on day 31). Samples were vortexed for 5 min, then centrifuged for 8 min at 16,000 rpm at 4°C. The supernatant was removed, an aliquot diluted 1:1 with Millipore water, and 5 µL injected for SRM analysis.
Dilution curve for cefoperazone
A 10-step dilution series of cefoperazone covering more than 5 orders of magnitude (262,144 fold range) with 8 µg/mL as the highest and 3.05*10-5 µg/mL as the lowest concentration on column was prepared in 60% water/40% acetonitrile. Each concentration was measured in technical triplicates. |
| separation | |
| digestion | |
| acquisition | Selected Reaction Monitoring (SRM) analysis
Samples were analyzed with a 5500 QTRAP equipped with a Turbo Spray Ion Source (Sciex, Foster City, CA) and an 1290 Infinity HPLC system including a G4220A binary pump and column heater (Agilent Technologies Inc., Santa Clara, CA). Chromatographic separation was performed with a Zorbax SB-C18 analytical column (2.1 x 50mm, 1.8 μm, Agilent) at 45°C using 0.1% formic acid in water (A), 0.1% formic acid in acetonitrile (B), and a gradient from 30% to 75% B at 0.1-5 min, followed by a 0.5 min wash step at 100% B and equilibration with 100% A for 7.3 min at a flow rate of 0.2 mL/min. Samples were analyzed in SRM mode with Q1 and Q3 set to unit resolution, 100 ms dwell time and a 1.47 s cycle time. Ion spray voltage (IS) was set to 5500 V, temperature (TEM) to 500°C, ion source gas 1 and 2 (GS1, GS2) to 35, curtain gas (CUR) to 35, and declustering potential (DP) to 70 eV. Data was acquired with Analyst 1.7 (Sciex). The most intense fragment ions for cefoperazone and ceftiofur were determined, the final method included six fragment ions for cefoperazone and eight ions for ceftiofur. Collision energies were optimized at 15 eV for cefoperazone and 25 eV for ceftiofur. 44 fecal and 15 plasma samples of mice were analyzed in three technical replicates. |
| informatics | |
| instruments | AB SCIEX QTrap 5500 |
| species | Mouse |
| massModifications | none |
