PJZ_57_6_2535-2541

In-Vitro and In-Vivo Antibacterial Effects of Saxifraga umbellulata var. Pectinata on Escherichia coli Isolated from Yaks

Kuanhui Liu1, Shihui Xie1, Ting Li1, Reem M. Aljowaie4, Mohamed S. Elshikh4 and Kun Li2,3*

1Wuhu Institute of Technology, Wuhu 241000, China

2Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China

3MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China

4Department of Botany and Microbiology, College of Science, King Saud University, P.O. 2455, Riyadh 11451, Saudi Arabia

ABSTRACT

Yaks are economically important animals in plateau regions. Bacterial diarrhea especially caused by drug-resistant pathogens like Escherichia coli has been a persistent issue among them. In this study, we explored the antibacterial effects of Saxifraga umbellulata var. Pectinata (SUP) on E. coli isolated from yaks. Results showed that the MIC and MBC of SUP against E. coli was 8 mg/mL and 16 mg/mL, respectively. The growth inhibition curve demonstrated that SUP significantly inhibited the growth of E. coli in a dose-dependent manner. Animal studies showed that SUP increased the survival rate of animals by alleviating intestine damage. In infected animals, villus height (P<0.0001) and the ratio of villus height/crypt depth (P<0.001) were lower, while crypt depth was significantly higher (P<0.0001). However, animals treated with SUP showed significant increase in villus height (P<0.0001), the ratio of villus height/crypt depth (P<0.0001) and decrease in crypt depth (P<0.001). Organ bacterial loads demonstrated that animals fed with SUP had markedly lower bacteria loads in heart (P<0.5), liver (P<0.01), spleen (P<0.5), lung (P<0.001), duodenum (P<0.05), jejunum (P<0.01), ileum (P<0.01), cecum (P<0.01), colon (P<0.01) and rectum (P<0.05). Crystal violet staining revealed that SUP inhibited the biological film formation of E. coli (P<0.0001). Membrane permeability analysis of E. coli indicated that SUP markedly increased bacterial leakage (P<0.0001). This study demonstrates that Saxifraga umbellulata var. Pectinata can inhibit E. coli both in vitro and in vivo by affecting the bacterial biofilm and membrane permeability, which may provide insights for the development of novel anti-E. coli drugs or preventive measures against diarrhea in plateau yaks.

Article Information

Received 15 April 2024

Revised 10 June 2024

Accepted 24 June 2024

Available online 26 September 2024

(early access)

Published 19 September 2025

Authors’ Contribution

KHL and KL research idea and methodology. KHL, SHX and TL reagents, materials, and analysis tools. KL writing original draft. RMA, MSE and KL writing review and editing. KL visualization and supervision. All authors read and approved the final manuscript.

Key words

Anti-microbial, Diarrhea, Escherichia coli, MIC, MBC, Saxifraga umbellulata var. Pectinata, Yak

DOI: https://dx.doi.org/10.17582/journal.pjz/20240415062301

* Corresponding author: [email protected]

0030-9923/2025/0006-2535 $ 9.00/00

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Yaks are economically important and essential bovine ruminant on the Qinghai-Xizang plateau (Li et al., 2023; Chen et al., 2022). These animals provide milk, meat, fur or related products and as well as serving as means of transport for native people (Lu et al., 2023; Wang et al., 2023). However, diarrhea poses a frequent problem in these animals, leading to severe losses and affecting ruminant health (Li et al., 2022). Diarrhea in cattle, especially calf diarrhea is a world-wide issue in the cattle industry (Choi et al., 2021; Rasheed et al., 2022; Anwar et al., 2022). Previous studies have confirmed that pathogens such as viruses (bovine viral diarrhea virus, torovirus), bacteria (Escherichia coli, Salmonella spp.), and parasites (Cryptosporidium parvum, Giardia duodenalis) are factors that lead to diarrhea in animals (Chang et al., 2021; Shi et al., 2020; Gelalcha et al., 2022; Arsenault et al., 2022; Ali et al., 2024; Taghipour et al., 2022). Among them, E. coli is a commonly detected bacterial pathogen that bring severe challenges to public and livestock health (Frankel and Ron, 2018; Li et al., 2023). Moreover, an increasing number of multi-resistant bacteria have been isolated from food animals, further complicating the issue due to limited available antibacterial agents (Roth et al., 2019; Ullah et al., 2023).

Traditional Chinese medicines are valuable and effective means for curing diseases and enhancing health (Chi et al., 2021). Among them, many herbs have antibacterial effects, such as Andrographis paniculata, Sanguisorba officinalis L. and garlic (Dai et al., 2019; Zhou et al., 2021; Tesfaye, 2021). The long history of Xizang medicine has integrated medical systems developed from traditional Chinese medicine and other medicines like Arabian medicine, which have greatly contributed to the health of plateau herdsmen (Huang et al., 2023).

The Xizang medicine of Saxifraga umbellulata var. Pectinata (SUP) is a representative plateau perennial herb, which belongs to Saxifragaceae family. In regions with altitude over 3 km, this herb is a recognized traditional Xizang medicine named Songdi used for treating liver and gallbladder diseases as well as digestive diseases (Huang et al., 2023). Previous reports have indicated that SUP extraction has antibacterial activity, hepatoprotective effect (Huang et al., 2023). However, not much information is available about the antibacterial effect of SUP on E. coli isolated from yaks. Therefore, we conducted this study to investigate the in vitro and in vivo antibacterial effect of SUP on E. coli isolated from yaks.

Materials and Methods

Bacterial isolate

Multi-drug resistant E. coli was previously isolated from diarrhea yaks and stored in the clinical veterinary laboratory of Nanjing Agricultural University. This bacterium was resistant to penicillin, ampicillin, erythromycin, tetracycline, streptomycin and gentamicin.

Preparation of the SUP extracts solution

SUP (200 g) was obtained from Tibetan medicine factory (Lhasa, China). Ethyl acetate extraction of SUP was performed according to previously described protocols (Hou et al., 2022; Liu et al., 2022). Initially the herbs were crushed and soaked with 2 L of ethanol (75%) overnight. The following day, herbs were heated to 85 ℃ and subjected to reflux extraction for 2h, followed by filtration. The SUP herbs were extracted and filtered three times and all the products were mixed together. At last, the mixed SUP products were dried and reconstituted in sterile water at 1g/mL for further use.

In vitro antibacterial effect of SUP on E. coli

The anti-E. coli activity of SUP was examined via commonly utilized methods of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) detection (Han and Guo, 2012; Parvekar et al., 2020). In a 96-well plate, 100 μL of SUP at various concentrations (0, 512 mg/mL, 256 mg/mL, 128 mg/mL, 64 mg/mL, 32 mg/mL, 16 mg/mL, 8 mg/mL, 4 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL and 0.25 mg/mL) was added to an equal volume of bacteria solution (106 CFU/mL). Subsequently, 50 μL of LB medium (Hangzhou Binhe Microorganism Reagent Co., Ltd, China) was added to each well and the plate was incubated at 37 ℃ for 18 h. The MIC endpoint was determined when no visible growth of E. coli was observed in the well. To determine the MBC, 100 μL of medium from wells before and after MIC well, and MIC well plated onto LB agar plates and incubated at 37 ℃ overnight. The MBC endpoint was reached when the plates had fewer than 0.25 x 105 CFU (0.1%) colonies.

Next, we evaluated the growth inhibition curve of E. coli in response to different doses of SUP. In sterile tubes containing 4.90 mL LB medium, 50 μL of bacteria solution (106 CFU/mL) was added along with 50 μL of SUP at concentration of 0, MIC, 2MIC and 4MIC mg/mL. Each concentration had 18 independent repeat tubes. The tubes were then incubated at 37 ℃ in shaker and at time point of 0, 2h, 4h, 6h, 8h, 10h and 12h, three tubes of each concentration were sampled to check OD600 value.

The effect of SUP on the biological film and membrane permeability of E. coli

The effect of SUP on the biofilm was assessed using crystal violet staining (Bai et al., 2022). Ina 96 well plate, 100 μL of E. coli (OD600=0.05) was mixed with 100 μL of SUP (4MIC) and incubated at 37 ℃ for 6 h and 12 h. Then the OD620 value was measured, and the plate was washed by PBS three times and fixed with 200 μL methanol for 15 min. Finally, the plate was washed with PBS for three times and stained with 0.1% crystal violet for 30 min. Finally, the plate was washed with PBS and 200 ul of 30% glacial acetic acid added for measuring absorbance at 540 nm. Three independent repeats were performed for all wells, and equal volume of sterile water was added to control wells. The ratio value of OD540/OD620 was calculated to determine the effect of SUP on the biofilm.

To evaluate the effect of SUP on the membrane permeability of E. coli, 100 uL bacteria solution (106 CFU/mL) was mixed with SUP (4MIC) in a 96-well plate, and then incubated at 37 ℃. The OD260 and OD280 values of supernatant were examined at 0, 2, 4 and 8 h. Three independent repeats were set for all wells, and an equal volume of sterile water was added in control wells.

Animal experiments

A total of 39 male Kunming mice (five weeks, average weight of 23.5±1.3 g) were obtained from Qinglongshan animal breeding (Nanjing, China). The mice were reared and housed in the animal facility having free access to feed and water. All of the mice were given three days for acclimatization and then grouped into control (C), infection (I) and treatment (T) groups. Mice in group I and T were intra-peritoneally infected with a bacteria solution (107 CFU/mL), while group T received treatment with SUP (200 mg/Kg) for three days. Group C and I were treated with an equal volume of sterile water. On the second day, three mice from each group were euthanized to collect organ and intestines samples. Daily weights and mortality of mice were recorded. Those collected tissues were ground with sterile PBS, and then used for bacteria culture on LB agar plate. Colony forming units (CFUs) were counted to analyze tissue bacterial loads. Additionally, the jejunum was employed for pathologic analysis.

Pathologic analysis

The jejunum from Kunming animals was fixed in paraformaldehyde (4%) and then subjected to H & E staining in Pinuofei Biological Technology (Wuhan, China). An Olympus CX23 microscope (Olympus Co., Japan) was used for pathologic analysis. Statistical analysis of villus height and crypt depth of mice in C, I and T were performed.

Statistical analysis

Non-parametric tests were conducted using IBM SPSS (27.0) software. Data are presented as means ± SD and statistical significance was determined at P < 0.05.

Results

In vitro antibacterial effect of SUP on E. coli

The MIC and MBC of SUP anti-against E. coli were 8 mg/mL and 16 mg/mL, respectively. The growth inhibition curve clearly demonstrated that SUP significantly inhibited the growth of E. coli, indicating a dose-dependent bactericidal effect (Fig. 1).

Crystal violet staining showed that SUP significantly inhibited the formation of E. coli biofilm at 6 h (P<0.0001) and 12 h (P<0.0001) (Fig. 2A). Membrane permeability analysis of E. coli revealed that SUP markedly increased bacterial leakage at 4 h (P<0.0001) and 8 h (P<0.0001) (Fig. 2B).

In vivo antibacterial effect of SUP on E. coli

Animal study revealed that E. coli infection led to mice mortality within 2-24h, while treatment with SUP saved the animals lives (Fig. 3A). Weight analysis showed that E. coli caused weight loss in mice, while animals

treated with SUP showed slightly higher body weights (Fig. 3B). Pathologic analysis indicated that E. coli obviously disrupted the integrity of intestinal villi in mice, whereas SUP alleviated intestinal damage in animals (Fig. 3B). Villus height (P<0.0001) and the ratio of villus height/crypt depth (P<0.001) in group I were markedly lower than in group C, while crypt depth in group I was significantly higher (P<0.0001). However, animals treated with SUP presented noticeably increased villus height (P<0.0001), ratio of villus height/crypt depth (P<0.0001), and decreased crypt depth (P<0.001) (Fig. 3B).

Organ bacterial loads showed that E. coli loads in the heart (P<0.5), liver (P<0.001), spleen (P<0.001), lung (P<0.001), kidney (P<0.001), duodenum (P<0.001), jejunum (P<0.001), ileum (P<0.001), cecum (P<0.01), colon (P<0.01) and rectum (P<0.001) in group I were significantly increased. Interestingly, animals fed with SUP exhibited markedly lower bacteria loads in heart (P<0.5), liver (P<0.01), spleen (P<0.5), lung (P<0.001), duodenum (P<0.05), jejunum (P<0.01), ileum (P<0.01), cecum (P<0.01), colon (P<0.01) and rectum (P<0.05) (Fig. 4).

Discussion

Cattle are important food-producing ruminants, providing nutritious products for citizens. Therefore, cattle disease not only harm animal health, but potentially threaten the protein food supply. Especially on the cold plateaus, yaks are crucial food resources (Li and Liu, 2022). E. coli is a common opportunistic pathogen causing diarrhea, resulting in significant economic losses to farming industry due to medical costs and animal deaths (Zhang et al., 2022). With antibiotic abuse in veterinary and medical practices, drug-resistant E. coli has been detected in the environment, animals and people (Hu and Cheng, 2016). There is an urgent need to screen novel antibacterial drugs with fewer side effects.

Medicinal herbs have been popularly used for thousands of years due to their antimicrobial properties, offering promising alternatives to conventional antibacterial drugs (Alanazi et al., 2023). Previous research has found herbs such as Chrysanthemum, Lagotis brachystachya and Oak bark for their anti-E. coli effects (Kim et al., 2013; Hou et al., 2022; Šukele et al., 2022). Consistent with these findings, our study confirmed that SUP could inhibit the growth of multi-drug resistant E. coli from yaks both in vitro and in vivo (Figs. 1, 3). In vitro studies showed that SUP could inhibit E. coli at 8 mg/mL, with MBC was 16 mg/mL. The growth inhibition curve indicated that SUP (32 mg/mL) could nearly completely inhibited bacteria growth (Fig. 1). Similar to a previous a study reported gut injuries caused by E. coli (Ismael et al., 2023), the strain of E. coli isolated from yaks proved lethal to mice causing severe intestine damage. However, our in vivo results showed that SUP decreased mice mortality by mitigating intestine damages and reducing bacteria loads (Figs. 3, 4).

Furthermore, we investigated the anti-E. coli mechanism of SUP by examining the biofilm and membrane permeability of E. coli. Biofilms consist of numerous bacterial cells aggregated together with extracellular matrix, which can shield bacteria from antibacterial agents and confer resistance to drugs (Lu et al., 2021), as well as protect from the host immune system (Roy et al., 2018). Following treatment for 6 h and 12 h, SUP significantly inhibited the biofilm formation (Fig. 2A), suggesting that SUP may hinder E. coli survival by impeding the biofilm formation. Our results are consistent with previous studies that have shown various herbs can inhibit the bacterial biofilm formation (Hou et al., 2022; Lu et al., 2019). Membrane integrity and permeability are crucial for bacteria growth (Yang et al., 2021), as a compromised membrane can result in the leakage of cell contents (Xu et al., 2017). In this study, proteins and nucleic acids were detected in E. coli treated with SUP (Fig. 2B), indicating that SUP could increase the permeability of E. coli membrane.

CONCLUSION

In this study, we demonstrated that Saxifraga umbellulata var. Pectinata could inhibit E. coli in vitro and in vivo by affecting the biological film and membrane permeability of bacteria. These findings provide insights that could potentially lead to develop novel anti-E. coli drugs or prevent measures for diarrhea in plateau yaks.

Declarations

Acknowledgement

The authors extend their appreciation to the Researchers Supporting Project number (RSP2024R418), King Saud University, Riyadh, Saudi Arabia.

Funding

The current study was funded by the Natural Science Research Project of Education Department of Anhui Province “Study on the preparation and application in animal production of immune adjuvant nanoparticles of CP-PLGA” (Grant No. KJ2021A1334).

Ethical statement

All the experiment procedures were conducted in accordance with the guidelines and approval of the Ethics Committee of Nanjing Agricultural University (NJAU.No20240226021).

Statement of conflict of interest

The authors have declared no conflict of interest.

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