• Users Online: 143
  • Print this page
  • Email this page


 
 
Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 18  |  Issue : 4  |  Page : 340-342

Preparation of modified selective medium for isolation of Enterococcus faecalis in pure culture from heavy sources (rapid diagnosis)


Department of Microbiology, College of Medicine, University of Babylon, Hilla, Iraq

Date of Submission07-Jul-2021
Date of Acceptance01-Sep-2021
Date of Web Publication18-Dec-2021

Correspondence Address:
Jawad Kadhim Tarrad AL-Khafaji
Department of Microbiology, College of Medicine, University of Babylon, Babylon Province, Hilla.
Iraq
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/MJBL.MJBL_47_21

Get Permissions

  Abstract 

Subject: This research focuses on the preparation of a new modified selective medium for the primary isolation of Enterococcus faecalis in pure culture from heavy and clinical sources. Materials and Methods: The modified selective medium was prepared and named as Enterococcus Jawad (EJ)–Medium. The new medium was prepared by adding 0.4 g of sodium azide, 2 mL of 0.1% crystal violet (CV), and 60 g of sodium chloride in a liter of Brain-Heart Infusion Agar (BHIA), and the pH of the medium was adjusted to 8.6. The primary isolation of E. faecalis from heavy bacterial sources and clinical infections was performed by culturing all collected samples on EJ-medium and comparing them with other approved selective media, such as Azide-BHIA, CV-blood agar, and Enterococcus selective agar (ESA), to estimate the ability of these media in isolating E. faecalis (during primary isolation) in pure culture. Results: The results showed that the EJ-medium had a high rate of isolation, 100%, of E. faecalis in pure culture from heavy sources (human feces and sewage water) and from clinical sources (urine, bedsore, and blood) when compared with other culture media that were already used for isolation of this organism. The Azide-BHIA and CV-blood agar showed no high purity in an initial isolation of the organism. Conclusions: From these results, we concluded that the EJ-medium is highly efficient in the primary isolation of E. faecalis in pure culture by one step from different environmental and clinical sources; thus, it does not need to be diagnosed by classical methods that are expensive and consume a long time. Since Azide-BHIA, CV-blood agar, and ESA were not exactly selective media for the isolation of E. faecalis in pure cultures from heavy sources, their use was limited in clinical cases.

Keywords: Enterococcus faecalis, modified selective media, purified isolation


How to cite this article:
AL-Khafaji JK. Preparation of modified selective medium for isolation of Enterococcus faecalis in pure culture from heavy sources (rapid diagnosis). Med J Babylon 2021;18:340-2

How to cite this URL:
AL-Khafaji JK. Preparation of modified selective medium for isolation of Enterococcus faecalis in pure culture from heavy sources (rapid diagnosis). Med J Babylon [serial online] 2021 [cited 2022 Jan 18];18:340-2. Available from: https://www.medjbabylon.org/text.asp?2021/18/4/340/332751




  Introduction Top


The enterococci are primarily commensal residents in the intestine of humans and animals. The species Enterococcus faecalis is a common inhabitant of the human intestinal microflora and the genitourinary tract of men and women.[1] The species is recognized as an important causative agent of health care–associated infections, including urinary tract infections (UTIs), postsurgical wound infections, bacteremia, endocarditis, meningitis, and neonatal sepsis.[2]

E. faecalis is implicated in about 10% of cases and up to 16% of nosocomial UTI. The species is responsible for a significant proportion of cases of bacterial endocarditis, accounting for 5–20% of reported series. Bacteremias due to this organism are more common than infective endocarditis. Bedsores and wound infections often occur in elderly patients with serious underlying medical conditions or in immunocompromised individuals who have undergone antimicrobial therapy.[3]

Because of their ability to resist most antibiotics and antiseptics, the strains of E. feacalis are found on patients’ beds, medical devices, hospital floors, and water polluted with these bacteria, creating a health problem. In the past decade, E. faecalis has become one of the main pathogens of hospital-acquired infections, especially among patients who have been a long time in the hospital. These bacteria cause many human diseases, such as UTIs, wound infections, bedsores, endocarditis, and other infections.[4] Therefore, they require rapid and accurate diagnostic methods, the most important of which is the primary isolation of E. faecalis from different sources.

The aim of the study was to prepare a new modified selective medium for pure isolation and rapid identification of E. faecalis in a short time.


  Materials and Methods Top


Preparation procedure of the new medium

Crystal violet (0.1 g; CV; Oxoid/United Kingdom) was dissolved in 100 mL of distilled water; then, it was sterilized by filtration using a 0.22 nm membrane filter. The solution was stored in a dark bottle at 4°C.

Sodium azide (0.4; Oxoid/United Kingdom) and sodium chloride (60 g) were added to 1000 mL of Brain-Heart Infusion Agar (BHIA; Oxoid/United Kingdom); then, they were sterilized by autoclave for 15 min.

After autoclaving, 5 mL of fresh human blood and 2 mL of 0.1% CV solution were added to the melted medium at 45°C with gentile mixing.[3],[5],[6],[7]

The pH of the medium was adjusted to 8.6 with sodium hydroxide (0.1 M, Oxoid/United Kingdom) solution.[8],[9]

The new medium was named EJ-medium.

Collection of specimens

The study was conducted at Teaching General Hilla Hospital, Babylon governorate. Two hundred fifty samples were collected from different sources. Of these, 125 stool samples were collected from the patients who were admitted to the hospital. The samples were taken from the patients by standard procedures as described by Collee et al.,[5] and 125 samples were collected from sewage water of the hospital and AL-Uhodyia canal at Hilla city by standard methods described by Tang and Stratton.[10]

Different specimens were also collected from the same patients: 30 samples of urine from UTI cases, 25 samples of patients with bedsores, and 10 blood samples from patients with endocarditis. These samples were collected by the procedures mentioned by Tille[4] and Collee et al.[5]

Comparative study

The primary isolation of E. faecalis from heavy sources was performed by culturing each collected sample on EJ-medium separately, and other culture media such as CV-blood agar, Azide-BHIA, and ESA medium were inoculated with the samples.

The estimation of the ability of EJ-medium in the isolation of E. faecalis in pure culture was conducted in the following manner: (i) The bacterial growth appeared on the EJ-medium compared with on other selective culture media. (ii) The mixed cultures that developed on other culture media were again inoculated on EJ-medium.

Standard biochemical tests mentioned in approved microbiological references[4],[7],[11] were performed to confirm the bacterial isolates as E. faecalis.


  Results and Discussion Top


Previously, the primary isolation of E. faecalis was carried out by using some selective media, such as ESA medium, or using blood agar medium and BHIA that are supplemented with some bacterial inhibitors either with CV only[6] or with sodium azide only.[7]

These media are not exactly selective media for the isolation of E. faecalis in pure cultures. This fact explains that the media contain either only Na-Azide that leads to the inhibition of Gram-negative bacteria only or only CV that results in the inhibition of Gram-positive bacteria only; therefore, some species of other bacteria can be grown on these media through primary isolation.

The new medium, EJ-medium, is composed of enrichment base medium (BHIA-base) that is supplemented with both the inhibitors, Na-azide and CV, so that both Gram-negative and Gram-positive bacteria are inhibited, except E. faecalis, which is already resistant to CV. The new medium also contains a high concentration of salt, and the alkaline condition is not a favorable environment to grow most types of microorganisms, so most contaminants can be removed in the first step.

The new modification, including BHIA-base medium, was chosen rather than other media because it enriched the medium. Both the inhibitors, the sodium azide and the CV, were added to the BHIA-base in the same medium, instead of adding each inhibitor separately in ordinary medium. Fresh human blood that had not been previously added was added to the new medium. Another new modification involved increasing the salt concentration of the medium by making the final concentration of sodium chloride 6.5% and creating an alkaline environment with pH 8.6.

Our results show that the EJ-medium is highly significant in clearance form in 100% pure isolation of E. faecalis from heavy sources (stool and sewage water) and from clinical sources (urine, bedsore, and blood) when compared with classical culture media that are already used for the isolation of this organism. The Azide-BHIA and CV-blood agar show mixed cultures that indicate the growth of bacterial contaminants and low purity in the isolation of E. faecalis from different sources; see [Table 1] given next.
Table 1: : Purity of bacterial growth on EJ-medium compared with other culture media for isolation of E. faecalis from different sources

Click here to view


EJ-medium showed high efficiency in the isolation of E. faecalis in pure culture from sources of high contamination and clinical infections. This means that the new medium contains an enriched medium that promotes the growth of E. faecalis, and it contains both inhibitors, so the Gram-positive and Gram-negative bacteria cannot grow on this medium. In addition, the pH of the medium and high sodium chloride concentration does not permit the growth of contaminants, such as bacteria and fungi, except E. faecalis, which is resistant to both inhibitors and can grow under these harsh saline and alkaline conditions. Therefore, the EJ-medium is considered as an excellent selective medium for the isolation of this bacterium from different sources either environmentally or clinically.

The new medium can be easily applied in different fields.

  1. This new medium can be used by researchers and postgraduate students who are involved in the easy isolation and diagnosis of these bacteria.


  2. It can be used by laboratory workers in our country’s hospitals to isolate and diagnose diseases caused by these bacteria.


  3. The new medium can be used in health and epidemiological studies in the community without much hassle.


In brief, the EJ-medium has many advantages, such as its materials are easily available; it is cheap, easy to use, easy to prepare, and highly efficient in the isolation of E. faecalis without any microbial contamination. Because this medium is highly efficient and gives 100% pure isolates, it does not need to be diagnosed by conventional methods (does not require chemical and physiological tests), which are costly and exhausting for academic researchers. It also reduces the material cost of the state, especially in the health and research sectors. Time is shortened, so researchers who use this medium do not need more time (is not time-consuming) for the diagnosis of this organism, but they diagnose it in the first step (in primary isolation); the bacteria can be obtained in a pure step and do not need other diagnostic tests to be performed. The new medium also reduces the effort exerted by the concerned parties in the preparation of the chemical solutions, the reagents, the culture media, and other materials to diagnose this bacterium.


  Conclusions Top


We can conclude the following points: (i) EJ-medium is excellent for the isolation of E. faecalis in pure culture from environmental sources and clinical cases. (ii) Azide-BHIA, CV-blood agar, and ESA are not suitable media in primary isolation of the organism from heavy sources but can be used in clinical cases with limited things.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Teixeira I, Facklam R. Enterococci. In: Manual Clinical Microbiology, 18th ed. Vol. I, 2003. USA: ASM. pp. 422-33.  Back to cited text no. 1
    
2.
Mundy IA, Sahm D, Ilmore M. Relationship between enterococcal virulence and antimicrobial resistance. Clin Microbiol Rev 2000;13:513-22.  Back to cited text no. 2
    
3.
AL-Khafaji JK. Bacteriological and genetic study of some isolates of Enterococcus faecalis isolated from clinical and environmental sources. Ph.D thesis, College of Science, Al-Mustansiriya University, Baghdad; 2005.  Back to cited text no. 3
    
4.
Tille PM. Baily and Scott Diagnostic Microbiology, 13 ed. USA: Elsevier; 2014.  Back to cited text no. 4
    
5.
Collee JG, Marmion BP, Fraser AG, Simmons, A. Mackie and McCartney Practical Medical Microbiology. 14 ed. UK: Churchill Livingstone; 1996.  Back to cited text no. 5
    
6.
AL-Barzangi SI. A genetic study on bacteriocin-producing Enterococcus faecalis. M.Sc thesis, College of Science, Baghdad University, Baghdad; 2001.  Back to cited text no. 6
    
7.
Manero A, Blanch A. Identification of Enterococcus species with biochemical key. Appl Environ Microbiol 1999;65:4425-30.  Back to cited text no. 7
    
8.
Murray BE. The life and times of enterococci. Clin Microbiol Rev 1990;3:46-65.  Back to cited text no. 8
    
9.
Huycke MM, Sahm DF, Gilmore GS. Multiple-resistant enterococci: The nature problem and agenda for the future. Emerg Infect Dis 1998;4:239-49.  Back to cited text no. 9
    
10.
Tang YW, Stratton CW. Advances Techniques in Diagnostic Microbiology. USA: Springer; 2006.  Back to cited text no. 10
    
11.
Macfaddin JF. Biochemical Tests for Identification of Medical Bacteria, 3rd ed. USA: Lippincott; 2000.  Back to cited text no. 11
    



 
 
    Tables

  [Table 1]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results and Disc...
Conclusions
References
Article Tables

 Article Access Statistics
    Viewed48    
    Printed0    
    Emailed0    
    PDF Downloaded13    
    Comments [Add]    

Recommend this journal