Neomycin sulfate

Healthy adult female C57BL/6 mice weighing 18-22g were used in this study. Mice were housed in wire-bottomed, wire-lid cages, allowed access to chow and water ad libitum, and acclimatized in a temperature-controlled room (25±2°C) with 12-hour light and dark cycles for 1 week before experiments. To induce intestinal microbiota dysbiosis, healthy mice were treated with broad-spectrum antibiotics as described previously [15 (link)]. Briefly, broad-spectrum antibiotics cocktail, consisting ampicillin (1g/L, Sigma), neomycin sulfate (1g/L, Amresco), metronidazole (1g/L, Sigma) and vancomycin (0.5g/L, Amresco), was administered to mice ad libitum via drinking water for 1, 3, 5, 7 or 14 days respectively. At each time point, mice were anesthetized for intestinal paracellular permeability assay, and then sacrificed for sample collections. Samples were used for the following experiments.

Ampicillin (1 g/L, Amresco), neomycin sulfate (1 g/L, Amresco), vancomycin hydrochloride (0.5 g/L, Vancocin), and metronidazole (1 g/L, Alfa Aesar) were added into the drinking water (ABX) of the mice for 4 weeks [25 (link)]. Microbial depletion was confirmed by examining the presence of living microorganisms in aerobic or anaerobic culture. Water containing antibiotics was changed twice a week, and treatment was stopped 2 days prior to gavage of Prevotella. For preparation of the bacterial inoculum, Prevotella was grown on fluid thioglycolate medium (Oxoid) at 37°C under anaerobic conditions for 3 days before use. After centrifugation, the bacteria were suspended in the fluid medium. Mice were gavaged with 200 μL of inoculum (dose 1 × 10⁸) and received the doses every other day for 4 weeks as previously described [15 (link), 26 (link)]. One hour prior to the bacterial gavage, mice were injected intraperitoneally with 3 mg of cimetidine HCl (Sigma-Aldrich) in 100 μL PBS to inhibit stomach acid secretion to improve the colonization [26 (link)].

Gut microbiota is experimentally disrupted by administering broad-spectrum antibiotics (ampicillin, neomycin, metronidazole and vancomycin) in the drinking water [12 (link), 13 (link), 15 (link), 17 (link)]. Since this route of administration would result in severe dehydration and subsequently affect the host immunity [33 (link), 34 (link)], we used a modified protocol. The mice were given 0.2 ml of the following broad-spectrum antibiotics– 10 mg/ml ampicillin (Amresco), 10 mg/ml neomycin sulfate (Amresco) and 5 mg/ml metronidazole (Hualu Holding Co., Ltd.)–twice daily for 3 weeks by oral gavage. Vancomycin was excluded since it might have had an impact on the pneumococcal infection. To ensure that the other antibiotics had no effect on pneumococcal infection, and to test any potential antibacterial effect of murine blood and tissues, S. pneumoniae was co-cultured with the sera, and the lung or liver homogenates of the gut microbiota-disrupted mice, prior to initiating the infection (S1 Table).