Exposure to Antibiotics Affects Saponin Immersion-Induced Immune Stimulation and Shift in Microbial Composition in Zebrafish Larvae

Abstract

In the last decades, pollution of the environment by large scale use of antibiotics in agriculture and human medicine have led to increased antimicrobial resistance in both the environment and the host animal microbiome. Disturbances in the host microbiome can result in impaired immunity and reduced resilience of aquaculture species. Here, we investigated whether environmentally measured levels of the commonly used antibiotics ciprofloxacin and oxytetracycline influences the host microbiome and susceptibility toward saponin-induced immune stimulation in larval zebrafish. Firstly, neutrophil and macrophage reporter zebrafish larvae were exposed to different concentrations of soy saponin by immersion. A dose-dependent increase in neutrophil presence in the intestinal area was observed together with increased expression of immune genes il1b, tnfa, il22 and mmp9. To investigate the effect of antibiotics, larval zebrafish were immersed in ciprofloxacin or oxytetracycline in the presence or absence of a low dose of saponin. In vivo imaging revealed that antibiotic treatment did not reduce the number of neutrophils that were recruited to the intestinal area upon saponin exposure, although it did tend to lower pro-inflammatory cytokine levels. Microbial sequencing of whole larvae revealed that exposure to a low dose of saponin already shifted the microbial composition. The combination of oxytetracycline and saponin significantly increased α-diversity compared to the controls. In conclusion, the current study provides evidence that the combination of low levels of antibiotics with low levels of anti-nutritional factors (saponin) can induce inflammatory phenotypes and can modify the microbiota, which might lead to altered disease susceptibility.

Intestinal Microbiota and Immune Modulation in Zebrafish by Fucoidan From Okinawa Mozuku (Cladosiphon okamuranus)

“…After quantifying RNA by a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), cDNA was generated from 1 μg of RNA using Superscript™ III First Strand Synthesis Systems (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The diluted cDNA corresponding to 125 ng of RNA was used as a template for each reaction of quantitative polymerase chain reaction (qPCR) using the ABsolute TM qPCR SYBR® Green Mix (Thermo Fisher Scientific, Waltham, MA, USA) as previously described ( 27 ). The sequences of the primer used in this study can be found in Table S1 .…”

“…Tg (mpeg1:mCherry/mpx:eGFPi 114 ) (28,29) and wild-type zebrafish were maintained in Zebtec family tanks (Tecniplast, Buguggiate, Italy) under continuous flow-through at 28 • C (14/10-h light/dark cycle) and fed daily with Tetramin Flakes (Tetra, Melle, Germany). For the experiments using zebrafish larvae, embryos were obtained from the adult transgenic zebrafish by natural spawning and raised with embryo medium (E3) water as described previously (27). OM fucoidan powder (>95% pure fucoidan) extracted from C. okamuranus as described previously (30) was provided by South Product Co., Ltd., Okinawa, Japan.…”

“…Tg (mpeg1:mCherry/mpx:eGFPi 114 ) zebrafish larvae were anesthetized with MS-222 (tricaine methane sulfonate) solution and embedded in 1% low melting point agarose (Thermo Fisher Scientific, Waltham, MA, USA), as previously described (27). Larvae were imaged as whole mounts with a Leica M205 FA Fluorescence Stereo Microscope.…”

“…Zebrafish offer an ideal in vivo model to investigate how fucoidan affects host immunity and microbiota under normal (unchallenged) conditions because of their compatibility with live visualization ( 26 ). Using a double-transgenic zebrafish model combined with next-generation sequencing of 16S rRNA genes, we have recently shown that microbiota modulation by antibiotics can significantly affect host inflammatory immune responses in zebrafish larvae immersed in saponin ( 27 ). In this study, we exploited this approach to investigate how OM fucoidan can affect immune response and microbiota composition of zebrafish larvae.…”

Ciprofloxacin promotes polarization of CD86+CD206‑ macrophages to suppress liver cancer

“…Although increased expression levels of CD86, TNF-α and IL-1β were observed, the decreased expression of CD206 in macrophages after treatment with ciprofloxacin and MCIP also exerted some tumor-inhibitory effects. However, other studies reported that ciprofloxacin could promote the expression of TNF-α and did not affect the expression of IL-1β in animal models (46). The changes in these cytokines were not consistent with the classical model of LPS-induced M1-like macrophage polarization (47).…”