Phenformin

Glipizide (GP, 98%), glimepiride (GM, 99%), glyburide (GB, 99%), tolbutamide (TB, 99%), gliquidone (GQ, 98%), gliclazide (GL, 99%), carbutamide (CB, 98%), tolazamide (TLZ, 96%), acetohexamide (AH, 98%), chlorpropamide (CPM, 99%), glibornuride (GBN, 98%), repaglinide (RGLN, 99%), rosiglitazone (RGLT, 98%), phenformin (PF, 95%), metformin (MFM, 97%), zirconyl chloride octahydrate (ZrOCl₂·8(H₂O), 98%), meso-tetra (4-carboxyphenyl) porphine (H₂TCPP, 97%), N, N-Dimethylformamide (DMF), and dopamine hydrochloride (DA, 98%) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd (Shanghai, China). The anti-SUs monoclonal antibody (anti-SUs mAb, 5 mg/mL) and coating antigen (SUs-OVA, 2 mg/mL) were prepared in our lab. 2-(N-morpholino) ethanesulfonic acid (MES, 99%) and bovine serum albumin (BSA, 98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nitrocellulose membranes (UniSart CN140) were received from Sartorius Stedim Biotech GmbH (Goettingen, Germany). Sample pads (SB08), polyvinylchloride backing plates (SMA31-40), and absorbent pads (CH37K) were supplied by Shanghai Liangxin Co., Ltd. (Shanghai, China).
The FIC-Q1 test strip analyzer was purchased from Suzhou Helmen Precision Instruments Co., Ltd. (Suzhou, China). The XYZ-3060 Dispensing Platform was supplied by BioDot, Inc. (Irvine, CA, USA). The CTS-300 automatic slitting machine and the ZQ-2000 strip cutter were purchased from Shanghai kinbio Tech. Co., Ltd. (Shanghai, China). The high-speed refrigerated centrifuge (GL-23M) was made by Hunan Xiangyi Technology Co., Ltd. (Changsha, China).

CN-Cbl (Sigma Aldrich, Cat# V900445) was dissolved in ddH₂O as a stock solution at 15 mM. Metformin and phenformin (Sigma Aldrich, Cat# D150959 and Cat# PHR157) were prepared in ddH₂O as stock solutions at 1 M and 0.2 M, respectively. Varied doses of biguanides were applied in this study according to previous studies^{18 (link),38 (link)} and the level of tolerance of tested organisms to the drugs. In brief, 50 mM Metformin was used in the on-plate treatment for ∆nhr-114 worms, and 200 mM was used in all bacterial treatments. The same volumes of ddH₂O were used as vehicle (Veh). phenformin at 4 mM was used in all bacterial treatments for feeding Pacdh-1::GFP worms, LC-MS/MS measurement, growth measurement and qPCR. phenformin was used at an optimized concentration, 2 mM, in the Keio Collection screen for feeding Δnhr-114 worms to ensure sufficient bacterial growth. The highest dose of phenformin (5 mM) was used in on-plate treatments or growth inhibition assays to screen for the strongest phenformin-resistant strains. All reagents were filtered with 0.22 μm membranes prior to use and stored at −20 °C.

Keio E. coli deletion collection clones were grown overnight in 500 μL of soy medium containing 0.05 nM B12, 2 mM phenformin, and 50 μg/mL kanamycin in 96-well deep-well plates. The overnight bacterial culture was concentrated to 25 μL (30 μL for those wells on the margin) and seeded onto B12-deficient 96-well plates. Approximately 30 synchronized L1 of Δnhr-114 animals were placed onto each well of the plates, and worms were allowed to develop for 96–120 h at 20 °C before observation. The fecundity phenotype in each well was scored from 0 (sterile) to 5 (fertile), as noted in Supplementary Data 1.