Zhixiu ZHU, Xinhua JIANG, Shunzhou DENG*, Wenbo ZHANG, Bei WANG, Huihong LI

1. College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China;

2. Nanchang Golden Pastoral Animal Health Services Co., Ltd., Nanchang 330013, China

Staphylococcus is widely distributed in water, feedstuff,drinking water, ground, and object surface. It parasitizes skin, mucosa, intestinal tract, respiratory tract and mammary gland. Pathogenic Staphylococcus aureus might induce two kinds of diseases. One is purulent disease, such as wound infection of animals, abscess, cellulitis, arthritis,mastitis and sepsis. Staphylococcus aureus is the major pathogeny of these diseases.The other is toxic diseases. The polluted feedstuffs might induce enteritis, toxic vomiting of animals and human, and toxin shock syndrome of human[1].

For a long period of time, antibiotics have been abused to prevent diseases caused by bacterial infection,so that there are more and more pathogenic strains carrying tetracycline and erythromycin resistance genes or producing penicillinase[2].And strain resistance becomes more and more strong. Strong resistant bacteria methicillin-resistant Staphylococcus aureus (MRSA) also appears[3]. The resistant superbug NDM-1 reported recently might be cureless[4]. Therefore, Food and Agriculture Organization (FAO),World Trade Organization(WTO) and developed countries has become more and more strict on the use of antibiotics; it is regulated that the antibiotics for human can not be used for animals. Under this situation,it is extremely important to find environmental biological drugs without poison or drug resistance.

Probiotics for animals have no side effects on human and animals,cause no environmental pollution, enhance bacterial resistance and destroy microbe ecology. Thus, probiotics for animals have wide application prospect and cause more and more attention.

In this research, Bacillus subtilis was isolated from the intestinal tract of grass carp. In vitro inhibitory effects of the metabolite of Bacillus subtilis on Staphylococcus aureus were researched. This research provided a strong theory basis for the control of diseases induced by Staphylococcus aureus.

Materials

Animals

Wild grass carp in Poyang Lake was 2-3 kg in weight.

Reagents

Staphylococcus aureus was stored in College of animal science and technology, Jiangxi Agricultural University; 10×PCR buffer and MgCl2were both purchased from Beijing Time Technologies Co., Ltd.; dNTP and DNA Marker were both purchased from Beijing Time Technologies Co.,Ltd.; Taq DNA polymerase was purchased from Promega; DNA gel extraction kit was purchased from Shanghai Sangon Biotech Engineering Co., Ltd.; agarose was purchased from New England Biolabs; 10 mg/mL ethidium bromide was purchased from Bio-Rad Laboratories; basic nutrient broth and nutrient agar were both selfmade;trace fermentation tube and test paper with drug susceptibility were purchased from Hanghzou Tianhe Microbial Co.,Ltd.

Instruments

AIR TECH bechtop was purchased from Antai Company of Sujing Group; COCI optical microscope was purchased from Chongqing Optical Instrument Factory; Motic digital microscope was purchased from Motic China Group Co., Ltd.; electro-heating standing-temperature cultivator was purchased from Shanghai Yuejin Scientific Instruments Factory; vertical steam sterilizer was purchased from Shanghai Sheng’an Medical Instrument Factory; PCR instrument was purchased from Bio-Rad; electrophoresis apparatus, Horizontal Gel System and glue mould were all purchased from Bio-Rad; WP750 microwave oven was purchased from Guangdong Galanz Company; Versa Doc 1000 Gel Imaging System was purchased from Bio-Rad; Oxford cup was purchased from Qindong Taiying Test Products Company ( inside diameter: 6 mm, outside diameter: 8 mm, length: 10 mm); high -speed centrifuge was purchased from Shanghai Anting Scientific Instrument Factory; pipettes (0.5-10 l, 5-200 l)were produced in Poland.

Methods

Isolation and identification of Bacillus subtilis

Isolated culture of Bacillus subtilis Fresh and healthy grass carps’ intestines were washed by 0.85% sterilized saline water for 4-5 times, and placed in 80 ℃water bath for 20 min.After cooling, sterile operation was carried out by inoculating loop. Intestinal mucosa was inoculated to general nutrition agar medium,and put in biochemical incubator at 37 ℃.After cultivation for 24 h, growth characteristics and colony characteristics of bacteria were observed. Typical colonies were selected. After Gram staining and microscopic examination,micro-morphology of thalli was observed. Single colony after Gram staining was selected and inoculated to general nutrition agar plate. The isolated bacterial strain was stored in a refrigerator at-4 ℃.

Biochemical identification of Bacillus subtilis The isolated bacteria were inoculated to biochemical fermentation tubes by conventional bacterial biochemical test method[5], and cultured in biochemical incubator at 37℃for 24-48 h.The biochemical characteristics were observed and recorded.

Drug sensitive test of Bacillus subtilis The isolated bacteria were inoculated to broth culture medium, and cultured in biochemical incubator at 37℃for 24 h. Drug sensitive test was carried out by the method of He Xiaoqing[6].Size of inhibition zone was detected.

Detection and sequence analysis of 16S rRNA of Bacillus subtilis Two pairs of primers were designed: A: 5’-TGA GTT TGA TCC TGG CTC AG-3’B:5’-CTA CCA GGG TAT CTA ATC CT-3’C:5’-AGAGTT TGA TCC TGG CTC AG-3’D:5’-AAG GAG GTG ATC CAG CCG CA-3’. PCR detection of isolated bacteria was carried out.After 1%agarose gel electrophoresis,sample was sent to Shanghai Sangon Company for sequencing. Homologous analysis of the obtained sequence and sequence in GenBank was carried out by BLAST. The similarity between tested strain and known strain was compared in corresponding domain sequence. According to the search results of homologous sequence, sequence of corresponding bacteria was downloaded to match the tested bacteria by Clustal X1.8 software. Molecular phylogenetic analysis of MEGA4.0 software was carried out in order to form phylogenetic tree.

In vitro inhibitory effects of Bacillus subtilis on Staphylococcus aureus Detection of inhibition zone Staphylococcus aureus after broth culture for 24 h was evenly coated to agar medium, and then sterile Oxford cup was slowly put. The broth culture of isolated bacteria (2.8×108/ml) was centrifuged. After aseptic filtration, the sterile metabolites were dipped to Oxford cup,cultured in biochemical incubator at 37 ℃for 24 h. The size of inhibition zone was detected.

Detection of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The sterile metabolites of isolated bacteria were added to test tubes with sterile broth medium in a degressive mode of 2-1, 2-2and 2-5. Then, broth culture of 1 ×108/ml Staphylococcus aureus was added. The tubers were put in biochemical incubator at 37 ℃for 48 h. Bacteriostatic phenomenon was observed and recorded.The reactants in tubes before maximum bacteriostatic concentration were inoculated to general agar medium, and cultured at 37 ℃for 24 h.Colony growth status were observed and recorded.

Results

Isolation and identification of Bacillus subtilis

Isolated cultivation results of Bacillus subtilis Colony characteristics of isolated bacteria:strain in general agar medium was flat, milk white and not transparent. As the cultivation time prolonged,colonies become dry(Fig.1).Microscopic characters of isolated bacteria: the observed bacteria were rod-shaped and in chain-like conformation. They were Gram-positive and produced spore(Fig.2).

Biochemical identification results of Bacillus subtilis Table 1 reported that the isolated bacteria were positive to nitrate,MR,V-P,starch and other reactions. This was different from CB-1 strain published by Li Xu-ji and isolated strain K published by Wen Jian-xin. Thus, due to the biological diversity, Bacillus subtilis varied greatly in their functions[10].

Drug sensitive test of Bacillus subtilis Table 2 reported that the isolated bacteria were sensitive to amikacin,cefalexin, ciprofloxacin and cefradine,was not sensitive to amoxicillin, ampicillin, penicillin G, piperacillin and cefepime.

Table 1 Physiological and biochemical characters

Table 2 Results of drug sensitive test mm

Detection and sequence analysis of 16S rRNA of Bacillus subtilis 700 and 1500 bp bands were detected by PCR (Fig.3), which was consistent with the predicted results. Fig.4 illustrated the sequencing data. Partial 16S rRNA sequence isolated from strain ZZX-001 was compared with NCBI. Results showed that the isolated strain ZZX-001 and reference strain had more than 99.4% homology. The isolated strain ZZX-001 had the highest homology with AB065370,AB440266 (Japan isolated strain),AY971364 (Beijing isolated strain),HM770882 (America isolated strain),JX123316 (Hubei isolated strain) and KF668463,which was 100%.The isolated strain ZZX-001 had the lowest homology with HM034816 (Xinjiang isolated strain DM-1) and JF499655(Sichuan isolated strain), which was 99.4% (Fig.5). Results showed thatthis isolated strain was Bacillus subtilis. Phylogenetic tree and sequence comparative analysis showed that this phylogenetic tree had two branches.One branch was ZZX-001; and the other branch was the rest reference strains(Fig.6).

In vitro inhibitory effects of Bacillus subtilis on Staphylococcus aureus Detection results of inhibition zone Transparent inhibition zone appeared around the Oxford cup (Fig.7). After detection,the size was 24 mm.

Detection of minimum inhibitory concentration and minimum bactericidal concentration Among the ten test tubes, the first five tubes had relatively clear supernatant; while the last four tubes showed an increasing trend of turbidity(Fig.8).The minimum inhibitory concentration(MIC)was 2.8×108×2-5/ml. Observation of the inoculated plates showed that I and plates had no colony; while the Staphylococcus aureus in the latter plates was increasing.The minimum bactericidal concentration (MBC) was 2.8×108×2-2/ml.

Discussions and Conclusions

According to the morphological characteristics of isolated bacteria, the physiological and biochemical characteristics, drug sensitive test results,and 16S rRNA detection and sequence analysis, Bacillus subtilis was identified, which was a species of Bacillus. It had long history of preparing fermented foods. Due to the nonpathogenic character and no production of toxin and heat sensitization protein, Bacillus subtilis was approved to be food safety strain by Food and Drug Administration of the United States and the Ministry of Agriculture of China[6]. Bacillus subtilis had extremely strong stress-resist ability and disease resistance[7], which could restrict the growth of a variety of animal and plant pathogens. Using Bacillus subtilis as a biocontrol bacterium in food, plant and animal has become a research hotspot.

The isolated bacteria had relatively strong inhibitory effects onStaphylococcus aureus; and the diameter of inhibition zone reached 24 mm,which could be used for the control of Staphylococcus aureus. When the isolated bacteria were used to cure diseases induced by Staphylococcus aureus,the insensitive drugs could also be adopted, such as amoxicillin,ampicillin, penicillin G, piperacillin and cefepime. However, sensitive drugs should be avoided such as amikacin,cefalexin,ciprofloxacin and cefradine.

Reports showed that Bacillus subtilis could produce various antibacterial substances, such as lipopeptide antibiotics[11], antimicrobial peptide[12],antibacterial polypeptide[13]and lantibiotics[14-15]. All these antibacterial substances had inhibitory effects on various bacteria, virus and pathogenic bacteria[16], which were ideal substitute goods for antibiotics. In this research,the bactriostasis effects of isolated bacteria on Staphylococcus aureus were only restricted within in vitro.Further research was still needed on the metabolite and antimicrobial mechanism of isolated bacteria.

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