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Highlights in BioScience             

ISSN: 2682-4043 

DOI:10.36462/H.BioSci.20215                                                                                                          
 

 

Highlights in BioScience                                                                                                                                                                        August 2020| Volume 3 

http://bioscience.highlightsin.org/ 

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Research Article 

 

 

Open Access 

 
 

 

 

 

1Department of Science Laboratory 

Technology, Biological Sciences Unit, 

Microbiology Option, the Oke Ogun 

Polytechnic, Saki, Nigeria. 
2Department of Pure and Applied Biology, 

P.M.B 4000, Ogbomosho, Ladoke Akintola 

University of Technology, Nigeria. 
3Department of Zoology, Parasitology Unit, 

University of Ibadan, Ibadan, Nigeria. 
4Genetics and Molecular Biology Unit, 

Department of Botany, University of Ibadan, 

Nigeria. 
5Department of Zoology, Cellular Parasitology 

Unit, University of Ibadan, Ibadan, Nigeria 

 

Contacts of Authors  

 

* To whom correspondence should be 

addressed: Adeoti O. Micheal 

 

Citation: Micheal AO, Olayemi AA, Adenike 

AK, Olabiyi AA, Olufemi OS, Olajumoke AE, 

Ademola AD, Joy OO. (2020). Antibiotic 

Effects of selected leaf extracts and molecular 

profile of Klebsiella pneumoniae and 

Enterobacter bugandensis strain (AdM2). 

Highlights in BioScience Volume 3. Article ID 

20205. dio:10.36462/H.BioSci.20215 
Received:  May 20, 2020 

 

Accepted:  July 18, 2020 

 

Published: August 05, 2020 
 

Copyright: © 2020 Micheal et al. This is an 

open access article distributed under the terms 

of the Creative Commons Attribution License, 

which permits unrestricted use, distribution, 

and reproduction in any medium, provided the 

original author and source are credited. 

 

Data Availability Statement: All relevant data 

are within the paper and supplementary 

materials 

 

Funding: The authors have no support or 

funding to report. 

 

Competing interests: The authors declare that 

they have no competing interests. 

Antibiotic Effects of selected leaf extracts and molecular 

profile of Klebsiella pneumoniae and Enterobacter 

bugandensis strain (AdM2) 

 
Adeoti O. Micheal

1, 2,5
*, Aboladale A. Olayemi

1
, Adeoye K. Adenike

1, 3
, 

Abiola A. Olabiyi
1
, Olufemi S. Olufemi

1,4
, Adedokun E. Olajumoke

1
, 

Adesina D .Ademola
1
 and Olaoye O. Joy

1
 

 

Abstract 

       Only healthy surface tissues of healthy animals that are constantly in 

contact with the environment can easily be colonized by various 

microbial species, including pathogenic ones. However, these 

microorganisms are usually the cause of opportunistic infections in 

animals. Two plants; Vernonia amygdalina (taxonomic ID number 

112605) and Ocimum gratissimum   (taxonomic ID number 112606) were 

identified at the Nigerian Federal Research Institute in Ibadan and 

subsequently concentrated with methanol, water and n-hexane. Twelve 

pre-slaughter healthy cattle in the same ratio of male to female were 

selected for this study. Samples from the mouth, skin, nose, vaginal and 

gastrointestinal tract were collected. Susceptibility patterns for 

antimicrobial agents were performed using disk-diffusion agar method. 

All isolates were subjected to bacteriological and molecular 

identification (PCR and sequencing techniques). Preliminary 

identification was carried out based on standard bacteriological, 

microscopic identification, biochemical criteria, while PCR and DNA 

sequencing techniques were used for molecular identification and 

analysis. Basic local alignment sequence tools (BLAST) were used to 

compare retrieved bacterial sequences to a set of previously published 

strains in the database. The morphological and biochemical profiles of 

six out of eight normal flora from different sites were Gram-negative. All 

bacterial cultures were oxidase-positive, including 7 catalase-positive 

cultures. The majority of isolated bacteria were moderately sensitive to 

all the extracts tested but resistant to Amoxylin. Noticeable amplification 

of 16S rRNA genes from Klebsiella pneumoniae strain EMB and 

Enterobacter bugandensis strain AdM2 was obtained. The amplification 

of the resistant genes of the two isolates was approximately 1,500 base 

pairs; however, some of the suspected virulence and antibiotic genes 

were within this base pair range. 

 

Keywords: Vernonia amygdalina, Ocimum gratissimum, Anti-bacterial, 16S 

rRNA, Cattle 

Introduction  

Antibacterial are kinds of antimicrobial agent used mainly against bacteria, 

for the treatment of bacterial infections [1]. The invention of antibiotics has 

assisted in the control of pathogenic bacteria until when most pathogens become 

resistant to the treatment. Increased alarming rate of Antibiotic resistance 

worldwide is causing difficulty and posing danger to humanity as various diseases 

and ailments are now difficult to treat and control [2].

https://creativecommons.org/licenses/by/4.0/


 

 

Micheal et al., 2020                                                                                                     Antibiotic Effects of leaf extracts of K. pneumoniae and E. bugandensis 

 

 

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In addition, majority of available synthetic 

antibacterial drugs are becoming less effective due to their 

side effects such as tendonitis, seizure, and Steven-Johnson 

syndrome [3]. The emergence of antibiotic resistance and 

the unbearable side effects (toxicity) of some of the 

commercially available antibiotics makes it imperative to 

search for newer, more effective and cheaper drugs that 

could serve as alternative therapy for the treatment of 

various infections and diseases [4]. Plants are reservoir of 

therapeutic substances which have a vital role in the 

sustenance of human health in the time past. World Health 

Organization (WHO) opines that plant parts or their potent 

make up are used in the productions of array of drugs in 

traditional therapies [5]. These plants have highly 

contributed to the development of human health and welfare. 

Simultaneously, there is an increase in data and huge 

patronage to herbal products round the world [6].  

Medicinal plants such as Ocimum gratissimum   have 

been affirmed to provide different culinary and medicinal 

properties which effect can be bacteriostatic and 

bacteriocidal on some bacteria. These effects have been 

attributed to the peptides, alkaloids, essential oils, phenols 

and flavonoids which are main constituents in these plants 

[7]. It is a therapeutic plant which has been used 

traditionally for the treatment of various disorder [8].  

Cattle are reared for various reasons for consumption 

and economic purposes .they are used to pull farm 

implements in farmland and rural areas. Cattles are 

herbivores which mean they do not eat only plants, grass and 

cereals. Microbiota is the total of microorganisms that 

resides on or within any of a number of human tissues and 

bio-fluids. In new born, it is a powerful stimulus for the 

development of the immune system. Unlike humans which 

are also colonized by many microorganisms, some microbes 

are normal flora in human they are commensal, they co-exist 

without being detrimental to humans; others to humans host 

are mutualistic [9]. This study was to provide an insight into 

molecular identification of two pathogenic organisms and 

document their antimicrobial susceptibility profiles to 

locally available plants. Gene sequencing technology will be 

used for strain identification, and classification. In addition, 

we aim to use some nucleotide sequence analysis tools to 

perform a basic phylogenetic and diversity analysis in order 

to evaluate the genetic variability of the flora studied. 

Materials and Methods 

Plant collection and identification  

Plants were selected based on ethno botanical 

information obtained from traditional medicine practitioners 

in Saki. All plants were collected around Challenge II area 

in Saki. Parts of the plants (leaves) were used for extraction. 

The identification was carried out in Forest Research 

Institute of Nigeria (FRIN), Ibadan with voucher specimen 

of 112605 and 112606 for Vernonia amygdalina and 

Ocimum gratissimum   respectively.  

Extraction and isolation of active agents 

The materials of each was washed with distilled water 

and dried at room temperature by exposure to atmosphere 

for weeks. The dried materials were pulverized into fine 

powder using electric blender and packed into a plastic 

container with screw cap for further use. Four types of 

extracts (aqueous, methanol, methanol plus water, n-hexane) 

were done. 100 g of each bitter leaf powder were weighed 

into three different sterile bottles and was soaked with 500 

ml of methanol; n-hexane and 250 ml of methanol plus 250 

ml of distilled water. 50 g of each scent leaf powder were 

also weighed into three different sterile bottles and was 

soaked with 250 ml of methanol, n-hexane and 175 ml of 

methanol plus 175 ml of distilled water. The mixture was 

stirred carefully with sterile stirrer and was covered with foil 

paper. The bottles were kept at room temperature for 3 days.  

The aqueous solution was prepared on the night of 

extraction by weighing 100g of bitter leaf powder in another 

bottle and was soaked with 500 ml of water and 50 g of 

scent leaf powder was also weighed in another sterile bottle 

and soaked with 250 ml of distilled water and covered with 

foil paper. The mixtures were extracted by sieving through 

muslin cloth, cotton wool and later filtered using Whatman 

filter paper into clean sterile bottles. The aqueous prepared 

was boiled for 15 minutes and it was allowed to cool and 

was extracted like it was done to the previous ones. The 

extracts filtrates were concentrated using rotary evaporator 

at 45°C at the Department of Pharmaceutical Chemistry 

Laboratory in University of Ibadan, Oyo State, Nigeria. 

Determination of the antibacterial activity of the extract 

Normal flora samples were collected from twelve cattle 

(6 males and 6 females) from different sites (nose, mouth, 

anus, vagina and skin) from the abattoir situated in Foofo, 

Saki, Ibadan, Oyo State, Nigeria. Swab sticks were used for 

the collection of specimen from different site from each 

cattle and were well labeled for recognition. Nutrient agar 

was prepared according to manufacturer’s instructions. The 

specimens were directly streak on the prepared agar in 

different plates after solidifying and then incubated for 24 

hours at room temperature. After 24 hours, the growth from 

the media incubated was transferred on MacConkey Agar 

and Eosin Methylene Blue for sub-culturing till pure culture 

is formed. Isolates were also tested on MRS and SSA agar 

but there was no growth. 

Minimum inhibitory concentration 

Each test organisms from serial dilution were streaked 

onto various plates and carried out in a septic condition and 

the plates were labeled accordingly. Disc diffusion method 

were introduced by using disc(sterilized perforated disc 6 



 

 

Micheal et al., 2020                                                                                                     Antibiotic Effects of leaf extracts of K. pneumoniae and E. bugandensis 

 

 

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mm in size seeded with different concentrations of the 

extract of 2 g, 4g and 6 g which is diluted into 10 ml of each 

methanol, n-Hexane, methanol + aqueous and aqueous and 

Amoxylin which is the control used was also diluted in 100 

ml of distilled water. Then the seeded disc was placed on the 

surface of the inoculated agar medium. The inoculated 

plates were incubated at 37
0
C the organism’s susceptibility 

test with plant extracts were recorded after 24 hours by 

measuring the mean diameter of the clear zone of inhibition 

using Vernier caliper in millimeter (mm). 

Biochemical tests for bacterial identification 

Isolates were subjected to catalase and oxidase tests, 

which was identified using Bergey's determinative 

bacteriology manual [10]. In order to perform a catalase test, 

the production of gas bubbles has been used as an indication 

of a positive result. Isolate was placed on a glass slide with 

an inoculating loop 2 % of Hydrogen Peroxide was drop on 

the isolates and bubble isolates were recorded positive and 

vice versa. The oxidase test was used to detect the enzyme 

cytochrome oxidase activity. A purple coloration indicates 

positive results while no color change indicates oxidase 

negative results. Inoculums of each isolate were smeared on 

a clean filter paper and oxidase reagent (tetramethyl-

phenylenediaminedihydrochloride) was drop on each isolate. 

Then colour changes were observed. 

DNA extraction and 16S PCR 

Bacterial cells broth was prepared and lysed through a 

ZR Bashing TM Lysis Tubes.  750µl lysis was pipetted in 

form of solution to the tubes. DNA extraction from bacterial 

samples was carried out using the ZYMO bacterial DNA 

extraction kit according to the protocol used by the 

manufacturer. The DNA quantity and quality was measured 

using gel electrophoresis techniques. Conventional PCR was 

applied to all the samples to amplify the 16S rRNA gene 

using previously reported protocol (Figure 1) [11].  

 
Figure 1: Agarose gel electrophoresis showing positive 

amplification of 1,500 bp fragment of 16S rRNA gene of 

Klebsiella pneumoniae performed with strain-specific 

primer. Lane M: 200-1037 bp DNA ladder. Strain lane 

G1:1500bp from cattle while G2: AdM2 (1000-1700) 

isolated from cattle. G3 and G4 (Isolates from other sources: 

rain and well water respectively). 

The PCR results were sequenced by using Sanger 

sequencing technique and its products were subjected to 

analysis by Basic Local Alignment Search Tools [12] 

(BLAST) and similar sequences were retrieved from the 

National Centre for Biotechnology Information (NCBI) 

database [13]. The phylogenetic analysis was performed 

using MEGA 5.2 version [14].  

Ethical considerations 

Experiments on animals have been conducted, 

according the research protocol reviewed by animal ethics 

committees of Microbiology Option, the Oke Ogun 

Polytechnic, Saki, Nigeria. 

Results and Discussion  

The resultant nucleotides sequences of the 16S rRNA 

was matched to Klebsiella pneumonia and Enterobacter 

bugandensis (AdM2) with accession number MK719814 

and MN213349 (Figure 2).  

Figure 2: The BLAST results of 16S bacterial sequences 

obtained in the current study. 

 

The antibacterial activity of both V amygdalina (bitter 

leaf) and Ocimum gratissimum   (scent leaf) were found to 

be dosage and extraction solvent dependent. Methanolic 

extract of both plants at their highest tested doses possessed 

more antibacterial activities compared to the other extracts 

from different solvents when tested against the bacteria 

strain of Klebsiella pneumoniae and E. bugandensis 

(AdM2).  This might attributed to the fact that methanol 



 

 

Micheal et al., 2020                                                                                                     Antibiotic Effects of leaf extracts of K. pneumoniae and E. bugandensis 

 

 

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extracted more of the bioactive constituents of the plant 

compared to other solvents. The methanolic extract of V 

amygdalina appeared more effective in the concentrations of 

6g/ml when tested on K. pneumonia and showed moderate 

zone of inhibition on Enterobacter bugandensis compared 

to the other tested doses, though the trend revealed dose 

dependent activity, i.e the higher the concentration the 

higher the effectiveness of the plant extract. The aqueous 

extract showed little to no inhibitory activity on the tested 

organisms while ciprofloxacin used as control inhibited the 

growth than the extracts (Table 1). Zones of inhibition 

produced by methanolic extract of Ocimum gratissimum   

ranged from 7. 00 ± 0.00 at 2g/ml to 14.75±0.05 at 6g/ml 

against on K. pneumonia, followed by n-hexane extract 

ranged from 4.30±0.15 at 2g/ml to the highest dose of 6g/ml 

with value of 5.75±0.20, methanol + aqueous extract also 

showed moderate inhibitory activity while aqueous extract 

was found not to inhibit the growth of the test organisms at 

lower concentrations. However, at higher concentration, 

zones of inhibition were observed for the Enterobacter 

bugandensis ranges from 1.30±0.02 at 4g/ml and 2.85±0.03 

at 6g/ml (Table 2).

 

Table 1: Antibacterial activities of bitter leaf extract on K. pneumonia and E. bugandensis (AdM2). 

Leaf  extract Concentration 

(g/ml) 
Mean diameter of zone of inhibition of bacteria in mm (±SEM) 

  
Methanol Methanol + Aqueous n- Hexane 

Aqueous 

only 

Control 

(ciprofloxacin) 

K. pneumonia 

2.0g/ml 9.75 ± 0.03 4.75±0.02 3.30±0.15 1.00±0.00 22.00± 0.00 

4.0g/ml 11.35±0.02 5.00±0.00 3.10±0.22 1.40±0.08 24.00±1.00 

6.0g/ml 16.75±0.05 5.25±0.02 3.75±0.03 1.50±0.05 27.30±1.00 

E. bugandensis 

2.0mg/ml 4.50±0.05 2.50±0.08 2.00±0.00 0.00±0.00 10.00±0.00 

4.0g/ml 5.00±0.00 3.60±1.06 2.90±0.14 1.00±0.00 11.00±0.00 

6.0g/ml 6.30±0.10 4.30±0.14 3.30±0.00 1.00±0.02 15.00±1.00 

 

 

Table 2: Antibacterial activities of scent leaf extract on K. pneumonia and E. bugandensis (AdM2). 

Leaf  extract 
Concentration 

(g/ml) 
Mean diameter of zone of inhibition of bacteria in mm (±SEM) 

  
Methanol 

Methanol + 

Aqueous 
n- Hexane Aqueous only 

Control 

(ciprofloxacin) 

  2.0g/ml 9.75 ± 0.03 4.75±0.02 3.30±0.15 1.00±0.00 22.00± 0.00 

K. pneumonia 4.0g/ml 11.35±0.02 5.00±0.00 3.10±0.22 1.40±0.08 24.00±1.00 

 6.0g/ml 16.75±0.05 5.25±0.02 3.75±0.03 1.50±0.05 27.30±1.00 

E. bugandensis 

2.0mg/ml 4.50±0.05 2.50±0.08 2.00±0.00 0.00±0.00 10.00±0.00 

4.0g/ml 5.00±0.00 3.60±1.06 2.90±0.14 1.00±0.00 11.00±0.00 

6.0g/ml 6.30±0.10 4.30±0.14 3.30±0.00 1.00±0.02 15.00±1.00 

Veronia amygdalina produces a variety of flavonoids 

and bitter lactones which contribute to the bioactivities of 

this plant [15]. V. amygdalina serves well as a low cost are 

readily available source of important nutrients to human 

[16]. The result of the current study demonstrated that the 

six out of eight normal flora from different sites isolated 

from cattle were bacterial culture while others were positive 

according to the standard bacteriological identification and 

microscopic identification. The sensitivity tests show that 

there is high resistance of test organism against antibacterial 

activity of plant extracts. This means that scent leaf and 

bitter leaf extracts are more effective as antibacterial agent 

against test organism. Sequencing analysis was done to 

subject a Deoxyribonucleic Acid, Polymerase Chain 

Reaction to test organism so as to understand its features, 

structure, function and evolution [17,18].Some of the 

suspected antibiotic resistance genes and virulence genes 

were (hylA, tetA, tetE and blaTEM) respectively with base 

pairs of 1-1.5kb [19–21]. Phylogenetic analysis of retrieved 

bacterial isolates indicates a high level of identification of 

16S rRNA sequences for bacterial genus and species 

(Figure 3). Such results could confirm the usefulness of 

using such genomic regions in the identification and 

characterization of bacterial isolates.



 

 

Micheal et al., 2020                                                                                                     Antibiotic Effects of leaf extracts of K. pneumoniae and E. bugandensis 

 

 

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Figure 3:  Phylogenetic trees of Enterobacter bugandensis (A) and Klebsiella pneumoniae (B) isolates from normal cattle 

flora compared to similar 16SrRNA gene sequences.  

 

Conclusion  
Most of the sensitive extracts were unable to inhibit the 

growth of the organisms and are presumably drug-resistant. 

Scent leaf and bitter leaf extract appeared more effective 

than their previously reported actions. Notwithstanding, V. 

amygdalina possesses various bioactivities with low or 

absence of side effects having great health promoting 

effects. It is however more advantageous to incorporate V. 

amygdalina into health supplement for both human and 

animal benefits.  

Acknowledgement 
The Authors wish to acknowledge the abattoirs 

attendants for the supports during the course of this 

research. Also, Fehintola Oluwaseun deserves to be 

mentioned; for processing and retrieval of sequences from 

NCBI database. 

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