Antibacterial Activity of Vitex negundo L. against a Multidrug Resistant Pathogenic Bacterium

Aims: The aim of this present study is to investigate antibacterial activity of extracts and/or essential oils of Vitex negundo L. leaves to an unknown pathogenic bacterium have been resistant to various groups of antibiotics Study Design: The study was design followed by previously studied and manuals of antimicrobial susceptibility test. Methodology: For antimicrobial susceptibility test disks diffusion method were used according to the guideline of European Committee on Antimicrobial Susceptibility Testing followed by McFarland standards. Various groups of antibiotics were used for conforming resistant of this unknown pathogen. The zone of inhibition and MIC was compared and measures with National Committee for Clinical Laboratory Standards (NCCLS) guidelines and reference data of previously studied. Results: According to NCCLS and data to our study revealed that UB201201 (unknown) was a Original Research Article Mahfuz-Al-Mamun et al.; EJMP, 8(2): 97-106, 2015; Article no.EJMP.2015.094 98 multidrug resistant bacterium. Molecular identification discloses the bacterium were Bacillus cereus group. All extracts of Vitex negundo L. leaves (ethanol, chloroform, ethyl acetate and hexane) and essential oils were responsible for antibacterial susceptibility with inhibition zone of 29±0.7, 9±0.5, 8±0.3, 12±0.7, and 25±0.2 mm respectively. Conclusion: This study signifies ethanolic extract of Vitex negundo L. leaves as moderate antimicrobials and can be used for pharmaceutical and medicinal purpose. Therefore, isolation and identification of bioactive compounds from this plant will be an interest for human being.


INTRODUCTION
In recent years, the emergence of pathogenic microorganisms acquired resistance to a wide range of formerly efficacious antibiotics has become a major cause of concern both in hospital settings and in the community [1,2]. The antibioticresistant bacteria are germs that are not killed by commonly used antibiotics and when bacteria are exposed to the same antibiotics over and over, the bacteria can change and are no longer affected by the drug. Moreover, the risk of transferring antimicrobial drug resistance to nonresistant bacteria and the propagation of multidrugresistant (MDR) bacteria from agricultural to clinical and/or communityassociated settings are being debated by research, regulatory, and health authorities [3,4]. Despite emphasis being put in research of synthetic drugs, a certain interest in medicinal plants has been reborn, in part due to the fact that a lot of synthetic drugs are potentially toxic and are not free of side effects on the host [5]. Because of the side effects and the resistance that pathogenic microorganisms build against antibiotics, much attention has been paid to extracts and biologically active compounds isolated from plant species used in herbal medicine. Vitex negundo L. (Verbenaceae), is an important medicinal plant found throughout Bangladesh (known as Nishinda) using as an important source of natural drugs from ancient time (Ayurvedic and Unani Systems of Medicine) and a number of pharmacological and medicinal activities and properties have been attributed toit by several studies [644].The aim of the present study is to investigate antibacterial activity of extracts and/or essential oils of Vitex negundo L. leaves to an unknown pathogenic bacterium have been resistant to various groups of antibiotics.

Isolation of Bacteria and Antimicrobial Bioassay
Bacteria were isolated from an eighteen years diarrheal patient in Rajshahi Medical College Hospital, Rajshahi and subjected to pure culture by using nutrient agar media (Merck Ltd. Germany). The antibacterial activity of the test samples was tested by disc diffusion method according to Uropean Committee on Antimicrobial Susceptibility Testing (UCAST, Version 3, 2013) [45]. Bacterial strains grown on nutrient agar at 37°C for 24 hours were adjusted to the turbidity of the 0.5 McFarland standards (10 6 colony forming units/mL). Then, the standard inoculums were spread by sterile cotton on the surfaces of nutrient agar prepared for growth of the bacteria. The disks (6mm in diameter) were impregnated with 10 µL at various concentrations of the extracts and placed on the inoculated media. The petri dishes incubated overnight at 37°C for bacterial growth and inhibition zone were examined carefully. Commercial Ampicillin, Streptomycin, Trimethoprime, Ciprofloxacin, Azithromycin and Tetracycline discs were used as standard antibiotics. The antibacterial activity was determined by measuring the diameter of zone of inhibition by millimeter scale.

Collection and Extraction of Plant Material
The mature fresh plant leaves of Vitex negundo L. were collected from the region of kushtia, Bangladesh and washed properly with sterile distilled water and dried at room temperature. The dried powdered was prepared using blender machine. Shaded dry and crashed leaves (150 gm) were subjected to 1000 ml borosilicate glass vessel alone with methanol, chloroform, ethyl acetate and hexane (500 ml of each) as solvents. After 48h the extracts was filtrated and solvents were then evaporated using rotary evaporator until a concentrated extracts was obtained. Crude extracts were obtained kept at 4ºC until further assay. Water distillation method was used for extraction of essential oil.  [47] and 50 ng template DNA. The thermal cycler (Bio Rad I Cycler 1708740) was programmed for the initial denaturation step (94°C) of 5 min, followed by 44 cycles of 1 min denaturation along with 1 min primer annealing (37°C) and 2 min primer extension (72°C), followed by the 7 min primer extension (72°C) step. The amplified DNA was visualized by gel electrophoresis. After amplification of 16S r DNA from isolated bacteria were sequenced by automated DNA sequencer.

16S rDNA sequence analysis and molecular characterization of bacteria
The 16S rDNA sequence of UB201201 was used for taxonomical identification and phylogeny analysis. In taxonomical terms, molecular characterizations of the concerned isolates by 16S rDNA sequencing focus on following facts [48] (Table 3) were obtained through the analysis of concerned sequences in the GenBank database, EMBL Nucleotide Sequence Database and DDBJby using the algorithm BLASTN [49]. Phylogeny.fr [50] was used to construct Maximumlikelihood Phylogenetic Trees (PhyML) with our concerned sequence ( Fig. 1).

Bacterial Susceptibility Tests
The bacterium was resistant to antibiotics testified by the pathology lab Rajshahi Medical College Hospital and further test were done in Islamic University supported lab. Commercial Ampicillin, Streptomycin, Trimethoprime, Ciprofloxacin, Azithromycin and Tetracycline discs were used as standard antibiotics to test bacterial susceptibility (Table 1). Bacterial susceptibility test was also done by essential oil and different solvent extracts of Vitex negundo L. leaves ( Table 2). The antibacterial activity was determined by measuring the diameter of zone of inhibition by millimeter scale.

Molecular Characterization of the Bacterium
The accession number, nomenclature and taxonomical identifications of UB 201201 were obtained through 16S rRNA gene analysis from International Nucleotide Sequence Databases (INSD) ( Table 3) and Phylogeny.fr was used to construct Maximumlikelihood Phylogenetic Tree (Fig. 1). The molecular test identified it as Bacillus cereus strain mmm86 (AB709908.1) in Bacillus cereus group.

DISCUSSION AND CONCLUSION
The overuse of antibiotics has become a major problem for the emergence and dissemination of multidrug resistant strains of several groups of microorganisms in Bangladesh. Both gram positive and gramnegative bacteria pathogens have shown a remarkable ability to develop resistance to antimicrobial agents which have forced clinicians to seek alternative treatments for patients with serious grampositive and gram negative infections. Illnesses due to multidrug resistant gramnegative microorganisms pose an important clinical problem, resulting in significant morbidity and mortality worldwide [51,52]. In this study, the Table 1 shows that the bacterial strains UB201201 are multidrug resistant bacteria.

Fig. 1. Phylogenetic tree representing pathogenic bacteria of Bacillus cereus group
The phylogenetic tree is shown in cladogram (ignoring branch lengths  The 16S rDNA partial sequences of the unknown bacterial strain UB201201 generated in the present study were deposited in the International Nucleotide Sequence Databases (INSD) and its accession numbers, nomenclature and taxonomical identification were obtained through the analysis of concerned sequences in the GenBank Database (Table 3). It is mentionable that thecriteria for identification to the species level were defined as 16S rDNA sequence similarity of 99% with that of the prototype strain  [55]. MIC value were also compared with the MIC range of reference value have been previously reported and confirm resistant to these antibiotics [5658].
Bacillus cereus has emerged as one of the most virulent bacteria and the etiological agent of two distinct food poisoning syndromes [59], as the diarrhoealtype and the emetictype. Bacillus cereus infections occur mainly, though not exclusively, in persons predisposed by neoplastic disease, immunosuppression, alcoholism and other drug abuse, or some other underlying condition, and fatalities occasionally result. Reported conditions include bacteremia, septicemia, fulminant sepsis with hemolysis, meningitis, brain hemorrhage, ventricular shunt infections, infections associated with central venous catheters, endocarditis, pseudomembranous tracheobronchitis, endophthalmitis, pneumonia, periodontitis, empyema, pleurisy, lung abscess, brain abscess, osteomyelitis, salpingitis, urinary tract infection, dermatolymphangioadenitis associated with filarial lymphedema, and primary cutaneous infections [60,61]. It is a wellrecognized agent of mastitis and abortion in cattle, and can cause these conditions in other livestock [62]. Nevertheless, there is limited information available with respect to its antimicrobial agent susceptibility. Published data suggest that Bacillus cereus isolates are susceptible in vitro to gentamicin, vancomycin, clindamycin, chloramphenicol, and erythromycin [63,64]. Resistant to penicillin, ampicillin, trimethoprim andcephalosporin have also been reported [65]. The most possible reason behind multidrug resistances of Bacillus cereus that it produces a broad spectrumβlactamase which hydrolyzes β lactam rings of antibiotics and another important fact is that they are sporeforming bacteria allowing them to survive in different types of adverse environments.  Table 2). Among them ethanol extract reflect more bioefficacy and antibacterial potency (inhibitory zone 29±0.7 mm) against the identified bacterium depicting that it contains such bioactive compounds and/or phytochemical constituents that can retard the bacterial growth or kill Bacillus cereusstrainmmm86. So, we can say that the extractability of bioactive compounds is greatly influenced by polar compounds. Thus, the ethanol extracts of Vitex negundo L. contains such bioactive compound(s) that have the ability to inhibit the growth or to kill the concerned multi drug resistant bacterium (Bacillus cereus strain mmm86) and can be used for pharmaceutical and medicinal purpose. How extracts of Vitex negundo L. be responsible for antibacterial activity against multidrug resistant bacterium is unclear but it may be due to the presence of some compounds like β-Caryophyllene, Ag (Silver) nanoparticles, betulinicacidsetc. have been previously reported in several study [75 79]. Therefore, this study may be taken into consideration for future therapeutic and drug development purpose for which further research is needed.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.