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Development and Evaluation of Modified Biochemical Medium for Rapid Identification of Enterococcus spp. Pornchat Boonlai 1 , Piyawut Chukaew 1 , Pussadee Tobunluepop 1 , Anantaporn Chunpong 2 , Anek Pootong 1 1 Department of Medical Technology, Faculty of Allied Health Science and

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Development and Evaluation of Modified Biochemical Medium for Rapid Identification of Enterococcus spp.

Pornchat Boonlai1, Piyawut Chukaew1, Pussadee Tobunluepop1, Anantaporn Chunpong2, Anek Pootong1

1Department of Medical Technology, Faculty of Allied Health Science and

2Microbiology Department, Thammassat University Hospital, Thammasat University

Because of the characteristics, including colony morphology and catalase reaction, of genus Enterococcus andgenus Streptococcus were similar. However, their antimicrobial susceptibility tests were different. As routine drug susceptibility was performed simultaneously with identification, this might lead to using the inappropriate antimicrobial drug in the test. Thus, the aims of the study were to modify and evaluate the biochemical medium for rapid identification of Enterocoocus spp. Bile esculin medium containing 2% and 4% bile salt were added with final concentration of 1% to 5% of NaCl. These modified bile esculin (MBE) were used to identify 3×108 CFU/ml of the standard stains of Streptococcus spp. and Enterococcus spp. by incubating at 37ºC for 4 hr. A total of 163 Streptococcaciae clinical isolated were tested with MBEs and conventional method. The MBE formulas that could distinguish Enterococcus spp. from Streptococcus spp. were MBE-A (3% NaCl + 2% bile salt-BE), MBE-B (4 % NaCl + 2% bile salt-BE) andMBE-C (3 % NaCl + 4% bile salt-BE). The sensitivity of MBE-A and MBE-B were 73.1% and 45.2% at 3 hr of incubation period, respectively. The specificity of both medium was 100%. The sensitivity and specificity of MBE-C were 66.7% and 100% at 4 hour, respectively. Although MBE had a low sensitivity but its specificity was 100%. Therefore, in case of a positive result, MBE could be useful as a guideline for appropriate drug susceptibility test.

2. Comparison of MBEs and conventional method for identification of clinical isolate of Enterococcus

Clinical isolates of Enterococcus (N=93) was positive by MBE as shown in following table. With MBE-A and B, 3 and 1 isolates of Streptococcus gr. D gave weak positive, respectively

Objectives

- To develop and evaluate the biochemical medium for rapid identification of Enterocoocus spp.

Methods

1. Modified Bile esculin medium (MBE)

Bile esculin mediums with NaCl at 1-5% final concentration were used to identified the standard stains of Streptococci and Enterococci with

- Inoculums: 3×108 and 1.5x108 CFU/ml - Temperature: 37ºC

- Incubation time: 1-4 hours

2. Identification of clinical isolates using MBEs and conventional method

A total of 163 Streptococcaciae clinical isolated from patients of Thammasat University hospital were tested with MBEs and conventional method and the result was compared.

The sensitivity of MBE-A and MBE-B were 73.1% and 45.2% at 3 hour of incubation, respectively. The specificity of both medium was 100%. The sensitivity and specificity of MBE-C were 66.7% and 100% at 4 hour, respectively.

Results

  • 1. Modified Bile esculin medium (MBE)
  • 1.1 Optimum recipes of MBE were
  • MBE-A: 2% Bile salt-BEwith 3% NaCl
  • MBE-B: 2% Bile salt-BEwith 4% NaCl
  • 3. MBE-C: 4% Bile salt-BEwith 3% NaCl
  • 1.2 Optimum condition
  • Inoculums: 3×108 CFU/ml - Temperature: 37ºC
  • - Incubation time: 3-4 hours

Conclusions

- MBE had 100% specificity

- MBE could be useful as a guideline for appropriate drug susceptibility test and should be developed for use in routine microbiology laboratory.

Acknowledgements

We would like to thank Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University for technical support. Anek Pootong is supported by grant from TRF.

References

  • Qadri SM, Flournoy DJ, Qadri SG. Sodium chloride-esculin hydrolysis test for rapid identification of enterococci. J Clin Microbiol 1987; 25(6): 1107-1108.
  • Bosley GS, Facklam RR, Grossman D. Rapid identification of Enterococci. J Clin Microbiol 1983; 18(5): 1275-1277.
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