Antimicrobial Activity of 11 Insects Extracts Against Multi Drug Resistant (MDR) Strains of Bacteria and Fungus

Objective: 11 kinds of insects, which are traditional Chinese medicine (TCM), were detected by antimicrobial experiments to find new natural antibacterial extractives. Method: The body or shell of insects were extracted by two stage extraction solvent. Firstly, the body were impregnated two times with dichloromethane solvent, and then, soaked with 70% methanol and extracted two times. Disk diffusion test and Broth Dilution Assay were used to test MIC (MIC-minimum inhibitory concentration) value of these extractions. Multi Drug Resistant (MDR) Strains of Bacteria and Fungus were used as detected strains in the test. Result: Of all eleven species of insects, extractives of the shell of Cryptotympanapustulata Fabricius and the body of Mole cricket have significant antibacterial activity on Staphylococcus aureus and Mycobacterium tuberculosis; the different organic solvents components of C. Fabricius and M.cricket also have inhibit effects on S.aureus and M.tuberculosis; The MIC of the ethyl acetate fraction of M.cricket on M.tuberculosis was even reached to 0.01 g/mL. Moreover, the effect of water extractives from C. Fabricius on M.tuberculosis was 0.01 g/mL eighter. Discussion: The discovery of new antimicrobial medicine is always an urgent and successive need in the field of Medicine. Insects is a new field for the discovery of antibiotics.


Introduction
As we all know, the emergence of drug-resistant bacterial strains has caused the infection treatment more difficult and complicated. The discovery of new antimicrobial medicine is always an urgent and successive need in the field of Medicine [1]. Traditional antibiotics have been unable to meet the current needs of treatment; the research and development of new antibiotics cannot keep up with the rate of infection caused by pathogenic microbes. Therefore, more and more scientists are paying attention to the research and development of new antibacterial drugs [2,3]. Insects are an important part of the Chinese traditional medicine (TCM), such as Cryptotympanapustulata Fabricius, the shells of the C. Fabricius is a kind of common TCM, which has the efficacy of dispersing wind and relieving heat, clearing the lung, clearing out the rash, relieving the antichloristic and clearing the liver, relieving the antitussive, relieving asthma, breathing the spasmodic and retreating the eyes. It is commonly used in the treatment of wind and fever, cough and sore throat. Pain, epilepsy in children, convulsions, convulsions, rubella, tetanus and other diseases [4,5]. Most insects are also used as part of Chinese medicine prescription, and have special function of agaist infection diseases based on Compendium of Materia Medica (Table 1). However, few scientists have studied the anti-infection effect of insects deeply.
In recent years, the antibacterial and anti-inflammatory effects of insects were notified by some  [6][7][8]. For example, in 2012, Sun Long studied the activity of antimicrobial peptides from two species of Coleoptera insects, and found that their antibacterial peptides had certain antibacterial activity [9]. In 2009, Li Xiu-hua studied the antibacterial activity of the metabolites of nematode bacteria, and found a metabolite of bacteriostatic to gray mould [10].
In the study, 11 kinds of insects were extracted by the two stage solvent extraction method. And the antibacterial activity of the insect extracts were detected by Broth Dilution Assay. Five kinds of Multi Drug Resistant (MDR) Strains of Bacteria and Fungus were used as experimental strains. The effective components were successively separated by different extraction methods, and the antibacterial activity of the extracts from various extraction methods was studied in vitro to provide basic datas for understanding the chemical composition and the special function of agaist infection diseases of Chinese medicine prescription.

Matereial
The dry shell or bodies of 11  Mueller-Hinton broth and agar were bought from Shanghai Ruichubio Reagent Co., Ltd.

Preparation of crude extractives.
The crude extractives of insects were extracted by two stage solvent extraction method [11][12][13]. Insects were lyophilisation, crushed and dipped with dichloromethane for 2 times, each time for 5 days, and the leach solution was recovered to recover the insect body. The fat soluble components of insects were obtained after solvent extraction. The above recovered insects were dried to constant weight, then soaked in 2 times with 70% methanol for 3 days. After solvent extraction, insect extract II was obtained. 11 extracts were labelled and placed in 4 o C. for preservation. 3 different insects (one of them was added nothing as a blank control). Then, the zone diameter of inhibition were tested after 24 hours culture at the corresponding temperature to observe the growth rate. However, M tuberculosis is growing slower than other strains, and its observation was generally two days).

Broth Dilution Assay.
Broth Dilution Assay were used to test the susceptibility of bacteria. To antibiotics. Here, it were used to test MIC of the organic solvents extracts of insects [14,15]. 6 concentration gradients were tested, each gradients were inoculated with S aureus and M tuberculosis respectively. Cultured at 37 o C. and shake for 24 h. And repeated twice. All those experiments were carried out in laboratories with biosecurity level II. DMSO were included in every experiment as control.

Disk diffusion test of insects extractives
Among the 11 crude extracts, only extractive of C fabricius and M crickets extracts have inhibit effects to S aureus and M tuberculosis ( Table 2). Mole cricket -----Note: * indicates that the phenomenon of bacteriostatic is not obvious.

Disk diffusion test of organic solvents extracts
All 7 organic solvents components of C fabricius and M cricket were ineffective to Enterococcus faecalis ATCC29212; the components of No 1,3,4,6 show inhibit effects on S aureus and M tuberculosis (Table 3).

Broth Dilution Assay analyse the effective parts of insects
Four parts of M cricket and C fabricius have obvious inhibit effect on S aureus and M tuberculosis. The MIC of No.3, the ethyl acetate parts of M cricket, is 0.1 g. mL -1 and 0.01 g.m L -1 on S aureus and M tuberculosis separately; however, the MIC of No.4, ethyl acetate parts of C fabricius, is 0.01 g. mL -1 on S aureus. Moreover, No 6, the effect of water extract parts from C fabricius, is also 0.01 g.mL -1 on M tuberculosis. All those four components have strong inhibit effect on S aureus and M tuberculosis (Table  4).

Conclusion
Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis, is a broad-spectrum antibiotics tolerance bacteria strains [16,17]. Staphylococcus aureus ATCC6538 is a strain of Methicillin-resistant Staphylococcus aureus (MRSA), which can cause different kind of infections in human [18][19][20]. It is a life-threatening pathogen to patients, because it has resistance to almost all the commonly used antibiotics. S aureus and M tuberculosis are both the Multi Drug Resistant (MDR) Strains.
In our study, the Bacteriostatic of butanol parts of M crickets have inhibit effect on S aureus and M tuberculosis. The ethyl acetate parts of M crickets also have strong inhibit effect on that two kinds strains; However, the other insect's extracts had no inhibit effect on the those bacteria strains and M albican ATCC10231. It can be found that the chemical components of n-butanol part of C fabricius have strong antibacterial activity to S aureus and T bacilli; The MIC of ethyl acetate extracts of C fabricius was 1 * 10 -2 g mL -1 , which shows that ethyl acetate extracts of C fabricius contains some materials have effects on S aureus. These results indicate that insect extracts possess antibiotic activity and are potential natural antibiotics and have certain development prospects.
C falbricias is a kind of TCM, and usually used as part of TCM prescription to cure tuberculosis [21]; such as Inner Xiao Sheng Xuan Decoction; it is also commonly used in the treatment of wind and fever, cough and sore throat. Pain, epilepsy in children, convulsions, convulsions, rubella, tetanus and other diseases [4,5]. In our study, we found that the shell of C falbricias have inhibit effect on S aureus and M tuberculosis. Which can partly explain the function of C falbricias in TCM.
In this paper, the research on the antibacterial activity of insect extracts is limited to the insects that have been purchased. However, the insect resources of our country are extremely rich and the number is very large, which provides a new research resource for the further exploration of the antibacterial active substances in the body of medicinal insects, pests and pathogens. On the basis of this experiment, according to the established experimental method, the research object of the insect source will be extended to provide new ideas for us to find new natural antibiotics or antibiotic precursor compounds.