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\begin{document}


\def\RunningAuthor{Krishna Chaithanya K et al.}
\firstPage{3024}
\articleType{Original Article}
\receivedDate{02 Mar 2020}
\acceptedDate{01 May 2020}
\revisedDate{02 Apr 2020}
\journalVolume{2020, 11}
\journalIssue{3}
\journalDoi{ijrps.v11i3.2399}
\copyrightYear{2020}
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\title{\textit{In vitro} antibacterial potential of acetone leaf extract of \textit{Otostegia integrifolia Benth} against human selected bacterial pathogens}
\author{Kidu~Hintsa\textsuperscript{1},  
            Tekleweyni~Tadege\textsuperscript{1},  
            Tesfay~Weletnsae\textsuperscript{1},  
            Gopalakrishnan~V~K\textsuperscript{1},  
            Kamalakararao~K\textsuperscript{2}, 
            Krishna~Chaithanya~K\textsuperscript{*}\textsuperscript{1}~\\[5pt]{\textsuperscript{1}Department of Chemistry, College of Natural and Computational Sciences, Aksum University, Axum, Tigray region, Ethiopia}~\\{\textsuperscript{2}Department of Biochemistry, Adikavi Nannaya University, Rajamahendravaram,  Andhra Pradesh, India}}

\begin{abstract}
\textit{Otostegia integrifolia Benth (O. integrifolia)} is the endogenous medicinal plant of Ethiopia mostly used for the treatment of Stomach ache, tonsillitis, hypertension, malaria, ascariasis, and lung diseases. The current study was focused on phytochemical analysis and evaluation of the antibacterial activity of \textit{O. integrifolia Benth} leave extracts against selected human bacterial pathogen by the agar well diffusion and microtube broth dilution method. Phytochemical investigation was carried out for the identification of secondary metabolites responsible for antibacterial activity. \textit{In vitro }antibacterial potential of \textit{O.integrifolia Benth }leaf organic extracts against human pathogenic gram-negative (\textit{E. coli, K. pneumonia, V. cholera}) and positive bacteria (\textit{B.subtilis, E. faecalis, S.aureus)} were assessed by agar well diffusion, and bacterial inhibitory concentration of effective plant extracts was determined by 96 well plate broth dilution assay. Among all the tested organic leaf extracts, the acetone leaf extract of \textit{O.integrifolia Benth} exhibited a promising broad spectrum of in vitro antibacterial activity against all tested multiple drug-resistant bacteria pathogens with significant MIC values of \textit{K.pneumoniae }(2.144\ensuremath{\mu }g/ml), \textit{V. cholera} (2.025\ensuremath{\mu }g), \textit{B.substilis} (2.604 \ensuremath{\mu }g), \textit{and S.aureus} (3.028 \ensuremath{\mu }g), respectively. The significant antibacterial activity of acetone leaf extracts of \textit{O. integrifolia Benth} was due to the existence of flavonoids and phenolic compounds. The current studies demonstrated that the broad-spectrum antibacterial activity of acetone leaf extracts of \textit{O.integrifolia Benth }might be helpful for the isolation of novel potent antibacterial agents against infectious bacterial pathogens without any side effects.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Medicinal plants,\newline Antibacterial agents,\newline Human pathogenic bacteria,\newline Agar well diffusion,\newline Broth dilution method
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Krishna~Chaithanya~K~\\ Phone:\ +251 944121156~\\  Email:\ krishnachaitanyawc@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
        \textsc{DOI:}\ \href{https://doi.org/10.26452/\@journalDoi}{\textcolor{blue}{\underline{\smash{https://doi.org/10.26452/\@journalDoi}}}}\par%
        \vspace*{-11pt}\hrulefill\\{\fontsize{9.12}{10.12}\selectfont Production and Hosted by}\par{IJRPS |~\href{www.ijrps.com}{www.ijrps.com}}\par%
        \vspace*{-2pt}{\fontsize{9.12}{10.12}\selectfont\textcopyright\ \@copyrightYear\ $|$ All rights reserved.}\par%
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\section{Introduction}
Multidrug-resistant Enterobacteriaceae family such as \textit{Escherichia coli, Klebsiella pneumonia, Enterococcus faecalisis, and Vibrio cholera }produces extended-spectrum \ensuremath{\beta }-lactamases(ESBLs) enzymes (EC-3.5.2.6). They have shown higher hydrolyzing activity against \ensuremath{\beta }-lactam ring containing cefotaxime and oxyimino-\ensuremath{\beta }-lactam substrates such as ceftazidime, ceftriaxone, and cefepime, causing multidrug-resistant nosocomial and community-acquired infections \unskip~\citep{759119:18050797,759119:18050549}. Recent reports suggested that the ESBL-producing Entero bacteriaceae family was causing inflammatory and urinary tract infections (UTI) and bloodstream associated infections \unskip~\citep{759119:18051504}. \textit{E.coli}  is a gram-negative bacteria which, when inhabitated in the human intestine tract during immune suppression of host, causes lower urinary tract infection and septicemia \unskip~\citep{759119:18050707}. \textit{K. pneumonia }is a gram-negative bacteria belonging to the Enterobacteriaceae family is inhabitated as intestinal flora of humans. It is also found in water and soil food, and it causes hospital-acquired urinary tract and wound infections \unskip~\citep{759119:18050790}. \textit{V. cholerae} is a gram-negative bacteria belonging to the Vibrionacea family. It inhabitates in brackish water producing both enterotoxin and cholera toxin (CT), causing severe diarrhoea \unskip~\citep{759119:18050555}.  \textit{Enterococcus faecalis} is a gram-positive bacteria belonging to the Enterococcaceae family. It exists as commensal as well as an opportunistic pathogen causing clinical infections such as urinary tract infection and meningitis \unskip~\citep{759119:18050796}. \textit{Bacillus subtilis} is a gram-positive bacteria belonging to the Bacillaceae family. It inhabitates in soil and also in the digestive tract of humans and generally harmless as it might occasionally cause human eye infections \unskip~\citep{759119:18050704}. 

The multiple drug resistance in human pathogenic bacterial species has been developed due to excessive intake of commercially available synthetic antibiotics for the treatment of bacterial infections. Since two decades acquiring and spreading of multiple drug resistance among different bacterial pathogenic species depends on the interaction between bacterial cellular components and chemical structure of synthetic and semi-synthetic antibiotics, the genetical ability of bacterial species to acquire and transmit resistance among the bacterial species and between host and bacterial species. Inappropriate usage of antibiotics, host pathogenic virulent characteristics, environmental factors, and antibiotics are allied with adverse side effects of the host, including immunosuppression and allergy \unskip~\citep{759119:18052070} .

In rural Ethiopia, 80\% of human population and 90\% of livestock majorly depends on medicinal plants for the treatment of communicable and non-communicable diseases \unskip~\citep{759119:18050549,759119:18050795,759119:18050800}. The traditional usage of medicinal plants in Ethiopia is an integral part of the culture and lifestyle of Ethiopian people \unskip~\citep{759119:18050554}.


\begin{table*}[!htbp]
\caption{\boldmath {Phytochemical Screening of Petroleum ether, chloroform, acetone and ethanol extracts of \textit{O. Integrifolia Benth} leaves.} }
\label{tw-131cab218c8c}
\centering 
\begin{threeparttable}

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\begin{tabulary}{\linewidth}{LLLLLL}
\tbltoprule \rowcolor{kwdboxcolor}{\multirow{2}{*}{S.No}} & {\multirow{2}{*}{Plant Constituent \mbox{}\protect\newline }} & \multicolumn{4}{p{\dimexpr(\mcWidth{3}+\mcWidth{4}+\mcWidth{5}+\mcWidth{6})}}{\cAlignHack Extract}\\
\rowcolor{kwdboxcolor} &  & Petroleum ether extract & Chloroform extract & Acetone extract & Ethanol extract\\
\tblmidrule 
1 &
  Alkaloids &
  + &
  + &
  + &
  -\\
2 &
  Coumarins &
  + &
  + &
  + &
  +\\
3 &
  Flavonoids \mbox{}\protect\newline  &
  + &
  ++ &
  +++ &
  ++ \mbox{}\protect\newline \\
4 &
  Phenolics compound &
  + \mbox{}\protect\newline  &
  ++ &
  +++ &
  ++\\
5 &
  Terpenoids &
  + &
  + &
  ++ &
  ++\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{NB:  (+) score indicate slight positive reactionfor Secondary metabolites.}
    
\item{(++) score indicate definitive positive reaction for Secondary metabolites.}
    
\item{(+++) indicate significant reactions were obtained for Secondary metabolites.}
    
\item{(-)Absent}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Zone of inhibition (mm) of organic leaf extracts \textit{O.integrifolia Benth} against Gram negative and positive bacteria} }
\label{tw-9decdaa6c696}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
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\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.1424\linewidth-2\tabcolsep}p{\dimexpr.19\linewidth-2\tabcolsep}p{\dimexpr.1095\linewidth-2\tabcolsep}p{\dimexpr.10249999999999996\linewidth-2\tabcolsep}p{\dimexpr.1161\linewidth-2\tabcolsep}p{\dimexpr.11639999999999999\linewidth-2\tabcolsep}p{\dimexpr.1004\linewidth-2\tabcolsep}p{\dimexpr.1227\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Plant  \mbox{}\protect\newline Extract/ \mbox{}\protect\newline  & Concent & \multicolumn{6}{p{\dimexpr(.6675999999999999\linewidth-2\tabcolsep)}}{Zone of inhibition (mm)  of organic  leaf extract  \textit{O.integrifolia Benth} against Gram negative and positive bacteria}\\
\rowcolor{kwdboxcolor}Standard & ration(\ensuremath{\mu }g/ ml) &  &  &  &  &  & \\
\rowcolor{kwdboxcolor} &  & \textit{B.subtilis} \mbox{}\protect\newline ATCC 3915 & \textit{E. feacalis} \mbox{}\protect\newline ATCC \mbox{}\protect\newline 29212 & \textit{S.aureus} \mbox{}\protect\newline ATCC \mbox{}\protect\newline 29213. & \textit{E.coli} \mbox{}\protect\newline ATCC 25922 & \textit{K.Pneumonia} ATCC \mbox{}\protect\newline 700603 & \textit{V. cholera} \mbox{}\protect\newline ATCC \mbox{}\protect\newline 39315\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.1424\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{O.I.L.PE}} &
  100mg &
  NA &
  NA &
  10 mm &
  NA &
  NA &
  NA\\
 &
  250mg &
  NA &
  NA &
  11 mm &
  NA &
  12mm &
  13 mm\\
 &
  500mg &
  NA &
  NA &
  13 mm &
  NA &
  14mm &
  15 mm\\
\multicolumn{1}{p{\dimexpr(.1424\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{O.I.L.CH}} &
  100mg &
  7mm &
  NA &
  8 mm &
  NA &
  NA &
  10mm\\
 &
  250mg &
  9 mm &
  8 mm &
  10 mm &
  NA &
  NA &
  11mm\\
 &
  500mg &
  13 mm &
  10mm &
  15 mm &
  NA &
  NA &
  13mm\\
\multicolumn{1}{p{\dimexpr(.1424\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{O.I.L.AC}} &
  100mg &
  10 mm &
  NA &
  11 mm &
  NA &
  NA &
  10 mm\\
 &
  250mg &
  15 mm &
  NA &
  15 mm &
  NA &
  10 mm &
  15 mm\\
 &
  500mg &
  18 mm &
  NA &
  16 mm &
  NA &
  12 mm &
  18 mm\\
\multicolumn{1}{p{\dimexpr(.1424\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{O.I.L. ET}} &
  100mg &
  NA &
  NA &
  11 mm &
  NA &
  10 mm &
  10 mm\\
 &
  250mg &
  NA &
  NA &
  12 mm &
  NA &
  12 mm &
  12 mm\\
 &
  500mg &
  NA &
  NA &
  13 mm &
  NA &
  15 mm &
  13 mm\\
Ciproflaxin &
  20mg &
  38 mm &
  38 mm &
  36 mm &
  26 mm &
  31 mm &
  35mm\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{a: Indicates the  Zone size includes 6-mm well ; NA- No zone of inhibition.}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Minimum Inhibitory Concentration (MIC) of \textit{O. integrifolia Benth} leaf extracts on tested bacterial strains } }
\label{tw-c26c6d2dfbfb}
\def\arraystretch{1.1}
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\tbltoprule \rowcolor{kwdboxcolor}{\multirow{2}{*}{Plant Extract \mbox{}\protect\newline }} & \multicolumn{4}{p{\dimexpr(\mcWidth{2}+\mcWidth{3}+\mcWidth{4}+\mcWidth{5})}}{MIC (\ensuremath{\mu}g/ml) of acetone leaf extracts of the \textit{O. Integrifolia Benth }against tested multidrug resistant bacteria. \mbox{}\protect\newline }\\
\rowcolor{kwdboxcolor} & \textit{B.substilis} \mbox{}\protect\newline ATCC 3915 & \textit{S.aureus} \mbox{}\protect\newline ATCC \mbox{}\protect\newline 29213 & \textit{K.pneumoniae} \mbox{}\protect\newline ATCC \mbox{}\protect\newline 700603 & \textit{V. cholera} \mbox{}\protect\newline ATCC \mbox{}\protect\newline 39315\\
\tblmidrule 
Acetone extract &
  2.604 &
  3.028 &
  2.144 &
  2.025\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
Plants can synthesize the bioactive secondary metabolites such as alkaloids, flavonoids, tannins and phenolic compounds, saponins and glycosides \unskip~\citep{759119:18050794}. Different researchers in research laboratories and pharmaceutical companies screen various medicinal plants against multiple drug-resistant bacterial and fungal species. \unskip~\citep{759119:18050552}. Consequently, these conditions have been obligatory for microbiologists to search for novel antimicrobial agents from various medicinal plants \unskip~\citep{759119:18050547,759119:18050548}

O.integrifolia Benth belongs to the family Lamiaceae endemic plant of Ethiopia and popularly known as an Abyssinian rose in Ethiopia. The dried leaves of O. integrifolia Benth plant are being used as fire fumigate for the eradication of insects in rooms and cooking vessels  \unskip~\citep{759119:18050792}.  Traditionally, this plant is being used in all regions of Ethiopia for curing tonsillitis, malaria, and ascariasis \unskip~\citep{759119:18050551}. The roots and leaves are being used for treating respiratory diseases \unskip~\citep{759119:18050706} . The objective of the current study was to evaluate the in vitro antibacterial activity of O. integrifolia Benth leaf extracts against tested multi-drug resistant human bacterial strains.
    
\section{Materials and Methods}
\textbf{Plant material collection and authentication}

The leaves leaves of \textit{O. integrifolia Benth} was collected in the month of October 2017, Axum city, Central Zone of Tigray region. The plant material was identified and authenticated by the Department of Biology, Addis Ababa University, and voucher specimen number of KH 001 was deposited in the National Herbarium for future reference.


\bgroup
\fixFloatSize{images/12861e3d-9de5-4b48-9019-29ea1d0f32df-upicture1.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/12861e3d-9de5-4b48-9019-29ea1d0f32df-upicture1.png}{\includegraphics{images/12861e3d-9de5-4b48-9019-29ea1d0f32df-upicture1.png}}{}
\makeatother 
\caption{\boldmath {The Zone of inhibition of petroleum ether leaf extracts \textit{O. integrifolia Benth} of against tested multiple drug-resistantbacteria by the agar well diffusion method.} 1-3 Gram positive bacteria ; 4-6 Gram positive bacteria}
\label{f-430167b157b8}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/5c9a8b9d-e825-4046-a0e1-f67faeb5c2b6-upicture2.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/5c9a8b9d-e825-4046-a0e1-f67faeb5c2b6-upicture2.png}{\includegraphics{images/5c9a8b9d-e825-4046-a0e1-f67faeb5c2b6-upicture2.png}}{}
\makeatother 
\caption{\boldmath {The zone of inhibition of chloroform leaf extracts \textit{O. integrifolia Benth }of against tested multiple drug-resistant bacteria by the agar well diffusion method.} 1-3Gram positive bacteria ; 4-6 Gram positive bacteria}
\label{f-4688a4aad624}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/e83dee0f-95ad-4a5d-93ba-d0864de6ffe8-upicture3.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/e83dee0f-95ad-4a5d-93ba-d0864de6ffe8-upicture3.png}{\includegraphics{images/e83dee0f-95ad-4a5d-93ba-d0864de6ffe8-upicture3.png}}{}
\makeatother 
\caption{\boldmath {The zone of inhibition of acetone leaf extracts \textit{O. integrifolia Benth} of against tested multiple drug-resistant bacteria by the agar well diffusion method.} 1-3Gram positive bacteria ; 4-6 Gram positive bacteria}
\label{f-cd9055f15386}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/fc5bc777-1038-42a0-ac0c-d4480c3f1d05-upicture4.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/fc5bc777-1038-42a0-ac0c-d4480c3f1d05-upicture4.png}{\includegraphics{images/fc5bc777-1038-42a0-ac0c-d4480c3f1d05-upicture4.png}}{}
\makeatother 
\caption{\boldmath {Zone of inhibition of Ethanol leaf extracts\textit{ O. integrifolia Benth} of against tested multiple drug-resistant bacteria by the agar well diffusion method.} 1-3Gram positive bacteria ; 4-6 Gram positive bacteria}
\label{f-fc46a117b02c}
\end{figure*}
\egroup

\bgroup
\fixFloatSize{images/f05b6a72-9e51-412f-a590-18f179bdcc6d-upicture5.png}
\begin{figure*}[!htbp]
\centering \makeatletter\IfFileExists{images/f05b6a72-9e51-412f-a590-18f179bdcc6d-upicture5.png}{\includegraphics{images/f05b6a72-9e51-412f-a590-18f179bdcc6d-upicture5.png}}{}
\makeatother 
\caption{\boldmath {Percentage of inhibition of acetone leaf extract \textit{O. integrifolia Benth} against tested Enterobacteriace family compared with Ciproflaxin standard}}
\label{f-9b810d2025aa}
\end{figure*}
\egroup
\textbf{Preparation of extracts}

The fresh leaf material was washed thoroughly with distilled water, and then was shade dried at room temperature for two weeks. The dried leaves were coarsely powdered with an electrical grinder, and the powdered leaf material (100 g) was extracted by using various solvents such as petroleum ether, chloroform, acetone, and ethanol in a soxhlet apparatus \unskip~\citep{759119:18050556}.

\textbf{Phytochemical Analysis}

The preliminary standard phytochemical screening was carried out for identifying phytoconstituents such as alkaloids, coumarins, flavonoids, phenolic compound, and terpenoids \unskip~\citep{759119:18050705,759119:18050557}.

\textbf{Test microorganisms and microbial culture}

All tested bacterial strains such as gram-negative bacteria [\textit{E.coli} (ATCC 25922), \textit{K.pneumonia }(ATCC 700603), \textit{and V.cholera} (ATCC 39315)] and gram-positive bacteria [\textit{B.subtilis} (ATCC 3915) \textit{E.faecalis }(ATCC 29212), \textit{S.aureus} (ATCC 29213)] were procured from Clinical Bacteriology Laboratory, Ethiopian Health and Nutrition Research Institute (EHNRI), Addis Ababa, Ethiopia. The collected bacterial strains were subcultured for in vitro  antibacterial activity.

\textbf{Preparation and standardization of inoculums}

A loopful of bacterial strains from the stock culture was transferred into Mueller-Hinton broth (MHB) test tubes and allowed to incubate at 37 \ensuremath{^\circ}C for 24 hrs. 1 ml of prepared bacterial culture was inoculated in 5mL of Mueller-Hinton broth (MHB) and incubated till it reached the turbidity equal to that of the standard 0.5 McFarland solution. The 0.5 McFarland turbidity standard was considered to be equivalent to a cell density of 1 x 10\ensuremath{^{8  }}CFU /mL \unskip~\citep{759119:18050798}.

\textit{\textbf{In vitro}} \textbf{ antibacterial Assay}

\textbf{Agar well diffusion assay}

The antibacterial activity of the prepared organic plant extracts was determined by agar well diffusion method \unskip~\citep{759119:18050553}. The selected bacterial pathogens from nutrient broth were spread on the Mueller-Hinton agar (MHA) plates. Five wells of 6mm in diameter were made with a sterile cork borer, and about 100 -150 \ensuremath{\mu}L of 10\% of DMSO dissolved organic tested plant extracts (100 \ensuremath{\mu}g to 500 \ensuremath{\mu}g/mL) were filled in test wells and then allowed to be incubated at 37 \ensuremath{^\circ}C for 24hrs. The stranded Ciproflaxin (20 \ensuremath{\mu}g) was used as a positive control for both gram-positive bacteria and gram-negative bacteria and DMSO was used as a negative control. The antibacterial activity was determined by measuring the zone of inhibition in millimeters. The experiments were conducted in triplicates and the results were represented as mean \ensuremath{\pm} standard deviation.

\textbf{MIC (Minimum Inhibitory Concentration)}

The Minimum Inhibitory Concentration (MIC) of effective plant extracts, exhibiting significant antibacterial activity against tested gram-positive \textit{B.substilis and S.aureus} and gram-negative \textit{K.pneumoniae  and V.cholera }bacterial pathogens, was determined by Mueller{\textendash}Hinton broth microdilution method. Various concentrations of (50, 25, 12.5, 6.25, 3.15 and 1.562\ensuremath{\mu}g/ml) acetone leaf extract of O.integrifolia in 10\% of DMSO solution were placed into the 96 well plates. Then, 100 \ensuremath{\mu}l bacterial cell suspensions containing 10\ensuremath{^{8 }} CFU ml-1 were inoculated in each well/plate \unskip~\citep{759119:18050799}  and allowed to be incubated at 37 \ensuremath{^\circ}C for 24 hrs. The culture intensity of each well was read at 600 nm and compared with the untreated control. 

\textbf{Statistical Analysis}

The results were expressed as Mean \ensuremath{\pm} SD and the statistical differences between the Mean \ensuremath{\pm} SD of the control group and the experimental group were assessed by GraphPad Prism 6.0.
    
\section{Results and Discussion}
Plants consist of a wide range of biologically active secondary metabolites responsible for different biological activities. Hence the presences of biologically active ingredients in the plant extracts are used for the treatment of both infectious and non-infectious diseases. These biologically active ingredients include alkaloids, coumarins, flavonoids, phenolic compounds, and terpenoids. 

\textbf{Phytochemical screening }

The qualitative phytochemical analysis of various organic leaf extract of \textit{O.integrifolia Benth},  showing the presence or absence of alkaloids, coumarins, flavonoids, tannins/phenols, and terpenoids, was investigated. As shown in the Table~\ref{tw-131cab218c8c}, petroleum ether leaf extract of \textit{O.integrifolia Benth} showed the presence of an inadequate amount of secondary metabolites such as alkaloids, coumarins, flavonoids, tannins/phenols, and terpenoids. 

The chloroform and ethanolic leaf extract of \textit{O.integrifolia Benth}  contained a significant number of secondary metabolites such as alkaloids, coumarins, flavonoids, tannins/phenols, and terpenoids. The acetone leaf extract of O.integrifolia Benth showed the presence of a maximum number of alkaloids, coumarins, flavonoids, tannins/phenols, and terpenoids. The highest secondary metabolites were found in acetone extracts of \textit{O. }integrifolia Benth leaf which could be attributed to the promising broad spectrum of antibacterial activities. The secondary metabolites present in different organic extracts of \textit{O.integrifolia Benth }leaves have therapeutic importance for curing infectious diseases. Our results also correlated with the reports of \unskip~\citep{759119:18050550}. He reported that 80\% of methanolic leaf extract of\textit{ O. integrifolia Benth} consists of phenolic compounds and flavonoids. 

\textbf{Antibacterial activity of leaf extract of \textit{O. integrifolia Benth}}

Nowadays, medicinal plants are widely used as an alternative source for novel antibacterial agents against multiple resistant bacteria \unskip~\citep{759119:18050791}. Many secondary metabolites of plants have shown significant antibacterial activity by targeting various drug-resistant pathways. The development of multidrug resistance in many pathogens against conventional antibiotics is a serious threat as it increases morbidity and mortality of both developing and developed countries \unskip~\citep{759119:18050793}. 

Hence the current investigation has been aimed at the screening of secondary metabolites and evaluating the \textit{in vitro }antibacterial activity of the leaf extract of \textit{O.integrifolia Benth } against disease-causing, multidrug-resistant bacterial pathogens such as\textit{ E.coli }(responsible for gastroenteritis, urinary tract infections, and neonatal meningitis), \textit{K.pneumonia }(causative organism of pneumonia),\textit{ V.cholerae  }(characterized by severe diarrhoea), \textit{B subtilis} (causes human eye infections), \textit{S.aureus }(a wound infecting pathogen causes septicemia), \textit{and E.faecalisis} (causes urinary tract infection, bacteremia, bacterial endocarditis, and meningitis).

\textbf{Antibacterial activity of organic leaves extract of \textit{O.integrifolia Benth}}

The \textit{in vitro} antibacterial activity of the leaf extract of \textit{O.integrifolia Benth} at different concentrations (100, 250, and 500 \ensuremath{\mu}g/ml) and standard antibiotic Ciproflaxin (20 \ensuremath{\mu}g/ml) were shown in Table~\ref{tw-9decdaa6c696}. The \textit{O.integrifolia}  leaf extract shows the zone of inhibition (\textit{in vitro }antibacterial activity)  ranging from 7 mm to 18 mm diameter (which included 6 mm of agar wells). 

Significant results were shown by acetone leaf extract of \textit{O.integrifolia} against tested gram-positive and gram-negative bacteria.  

The antibacterial activity of petroleum ether leaf extract of \textit{O.integrifolia Benth} (as shown in Table~\ref{tw-9decdaa6c696} and Figure~\ref{f-430167b157b8}), exhibited a moderate zone of inhibition against the tested, gram-negative bacteria [\textit{V.cholera} in the range between (12mm-15 mm) \textit{and K.pneumoniae} (12-14 mm) at a concentration of 250 \ensuremath{\mu }g/ml-500 \ensuremath{\mu }g/ml)]  and gram-positive bacteria [(S.aureus (1-13 mm) at a concentration range of 100 \ensuremath{\mu }g/ml-500 \ensuremath{\mu }g/ml]. The \textit{in vitro}  antibacterial activity of petroleum ether leaf extract of \textit{O.integrifolia Benth} showed effective narrow spectrum against the tested, gram-negative \textit{K.pneumoniae and V. cholera} bacteria. 

The antibacterial activity of chloroform leaf extracts of \textit{O.integrifolia Benth }(as shown in Table~\ref{tw-9decdaa6c696} and Figure~\ref{f-4688a4aad624}), exhibited significant zone of inhibition against the gram-positive bacteria  [\textit{S.aureus} (8-15 mm), \textit{B.subtilis }(7-13 mm), \textit{E.faecalis} (8-10 mm) at a concentration of 100 \ensuremath{\mu }g/ml-500 \ensuremath{\mu }g/ml] and moderate zone of inhibition against  the gram-negative bacteria [\textit{V.cholera} (10-13 mm) at a concentration of 100 \ensuremath{\mu }g/ml-500 \ensuremath{\mu }g/ml]. 

The antibacterial activity of acetone leaf extract of \textit{O.integrifolia Benth}  (as shown in Table~\ref{tw-9decdaa6c696} and Figure~\ref{f-cd9055f15386}) exhibited prominent significant zone of inhibition against the tested gram-positive bacteria [\textit{B.subtilis} (10- 18 mm) \textit{S. aureus} (11-16 mm) at a concentration of 100 \ensuremath{\mu }g/ml-500 \ensuremath{\mu }g/ml] and moderate zone of inhibition against gram-negative bacteria] \textit{K.pneumoniae} (10-12 mm) \textit{and V.cholera} (10-15 mm) at a concentration of 100 \ensuremath{\mu }g/ml-500 \ensuremath{\mu }g/ml], but failed to inhibit the growth of gram-negative bacteria such \textit{E.coli} and gram-positive bacteria such as \textit{E.faecalis}. The acetone leaf extracts of \textit{O.integrifolia Benth} showed an effective broad spectrum of \textit{in vitro} antibacterial activity against both gram-positive and gram-negative bacteria.  

The antibacterial activity of ethanolic leaf extracts of \textit{O.integrifolia Benth }(as shown in Table~\ref{tw-9decdaa6c696} and Figure~\ref{f-fc46a117b02c}) had shown significant zone of inhibition against gram-positive bacteria [\textit{B.subtilis} (10-15 mm), \textit{S.aureus }(11-18 mm) at a concentration of 100-500 \ensuremath{\mu }g/ml] and moderate inhibition against gram-negative bacteria [\textit{K.pneumoniae }(10-12 mm) at 250-500 \ensuremath{\mu }g/ml \textit{and V.cholera} (10-13 mm) at 100 \ensuremath{\mu }g/ml-500 \ensuremath{\mu }g/ml. 

The ethanolic leaf extracts of \textit{O.integrifolia  Benth} showed an effective broad spectrum of \textit{in vitro } antibacterial activity against both gram-positive and gram-negative bacteria and did not show any activity against gram-negative bacteria such as \textit{E.coli }and  gram-positive bacteria such as \textit{E.faecalis}.

\textbf{MIC of the effective plant extracts}

The Minimal inhibition concentration was carried out for the evaluation of the inhibitory potential of the plant extract against the most susceptible tested bacteria. Only the tested bacteria which were highly susceptible to the acetone leaf extracts of \textit{O.integrifolia Benth} was taken for determining the MIC. As shown in Figure~\ref{f-9b810d2025aa} and Table~\ref{tw-c26c6d2dfbfb}, the acetone leaf extracts of \textit{O.integrifolia Benth} shows maximum zones of inhibition of 75\%, 70\%, 60\%, and 55\% against \textit{V.cholera, K.pneumoniae, S.aureus, and B.substilis} with MIC values of 2.025 \ensuremath{\mu}g/ml, 2.144 \ensuremath{\mu}g/ml, 3.028 \ensuremath{\mu}g/ml, and 2.604 \ensuremath{\mu}g/ml.
    
\section{Conclusions}
The results of the current study revealed that the acetone leaf extract of \textit{O.integrifolia Benth}  showed a broad spectrum of \textit{in vitro} antibacterial activity against tested gram-positive bacteria such as \textit{B.subtilis, S.aureus,}  and gram-negative bacteria such as \textit{K. pneumonia and V.cholera}. The phytochemical analysis of acetone leaf extract of \textit{O.integrifolia Benth} had shown the presence of alkaloids, coumarins, flavonoids, tannins and phenolics compounds, and terpenoids, which are responsible for potential \textit{in vitro} antibacterial activity. This study suggests that the acetone leaf extract of \textit{O.integrifolia Benth} has shown promising \textit{in vitro} antibacterial activity and phytochemical constituent which are useful for further studies to untangle novel treatment strategies for diseases associated with a bacterial infection. 
\section*{Acknowledgements}We, the authors, thank the Department of Chemistry, Aksum University, Ethiopia, for providing laboratory facilities.

\textbf{Financial Support}

This project work was funded by the Ministry of Education, Ethiopia, in the form of Student Project work.

\textbf{Conflict of Interest}

The authors confirm that this article content has no conflict of interest.
    

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