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Journal of Clinical Microbiology, August 2005, p. 4296-4297, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.4296-4297.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

FAST-TRACK COMMUNICATIONS

Nonfermenters under the Influence: Ethanol Disinfection of a Saline Dispenser Caused Misidentification by the Vitek 2 System Using the New GN Cards

Received 25 April 2005/ Returned for modification 2 May 2005/ Accepted 3 May 2005

Since its introduction in 1997, the Vitek 2 system (bioMérieux, Marcy-l'Etoile, France) has established itself as a stable and accurate automated system for rapid bacterial identification. Bacterial suspensions and test cards containing a panel of substrates are both placed into the instrument, where the cards are automatically inoculated, sealed, incubated, and read to detect biochemical reactions. From the start, the ID-GNB card has been used for the identification of gram-negative bacilli. This card gives a final result in 3 hours but, as was shown by several studies, is better able to identify Enterobacteriaceae than nonfermenters, probably because of their slower metabolisms (2-4).

To improve overall identification results for gram-negative bacilli, the recently developed GN card is now being introduced worldwide. For this card, the Vitek 2 system uses colorimetric technology instead of fluorescence-based methods to detect biochemical reactions. After the Vitek instrument is adjusted by installation of new readers and a database update so that the card can be used, a greater diversity of species can be identified in both Enterobacteriaceae and nonfermenter groups (1).

When we started using the new GN card, we found that Pseudomonas aeruginosa and Acinetobacter baumannii were constantly misidentified as Burkholderia cepacia and Klebsiella oxytoca, respectively. This phenomenon did not occur while using ID-GNB cards, which were processed identically to GN cards.

A few random experiments focused our attention on the bottle-top dispenser used to transfer sterile saline (0.45% sodium chloride solution) to the test tubes used for suspension preparation. The dispenser can be autoclaved for sterilization or decontamination according to the manufacturer's guidelines. For practical reasons however, we applied a local protocol in which at the end of the day the dispenser was rinsed with an 80% ethanol solution and left to soak in ethanol overnight. The next morning it was rinsed with sterile distilled water and suspension solution before use.

Because we suspected that residual ethanol in or on parts of the dispenser interfered with bacterial identification, we took samples from a bottle of saline solution to culture them for contaminants and to test them for the presence of ethanol. This bottle had been in use for 2 days, meaning that on two consecutive mornings it had been connected to the freshly rinsed dispenser. No contaminants could be demonstrated in the saline. However, ethanol was detected in a concentration of 0.4 g/liter (0.4 promille), which is consistent with 250 µl of 80% ethanol in a 500-ml saline bottle (5 to 6 drops). We subsequently inoculated GN cards with four different strains of P. aeruginosa and three different strains of A. baumannii from suspensions containing concentrations of ethanol ranging from 0 to 0.4 g/liter.

Table 1 shows the effect of ethanol on the final results. Very low quantities of ethanol were enough to cause misidentification, although for one strain of P. aeruginosa the reactions appeared unaffected.


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  		TABLE 1. Results of testing bacterial suspensions containing ethanol with the Vitek 2 GN card

We concluded that residual ethanol present on the outside of the filling tube attached to the main body of the bottle-top dispenser interfered with the reactions or the viability of the bacilli in the GN cards. The problem was solved after we changed to using disposable materials for saline dispensing and abandoned the practice of ethanol disinfection.

ACKNOWLEDGMENTS

bioMérieux kindly provided the Vitek 2 GN cards.

REFERENCES

    1
  1. Funke, G., and P. Funke-Kissling. 2004. Evaluation of the new VITEK 2 card for identification of clinically relevant gram-negative rods. J. Clin. Microbiol. 42:4067-4071.[Abstract/Free Full Text]
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  2. Funke, G., D. Monnet, C. deBernardis, A. von Graevenitz, and J. Freney. 1998. Evaluation of the VITEK 2 system for rapid identification of medically relevant gram-negative rods. J. Clin. Microbiol. 36:1948-1952.[Abstract/Free Full Text]
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  3. Joyanes, P., C. M. del Carmen, L. Martinez-Martinez, and E. J. Perea. 2001. Evaluation of the VITEK 2 system for the identification and susceptibility testing of three species of nonfermenting gram-negative rods frequently isolated from clinical samples. J. Clin. Microbiol. 39:3247-3253.[Abstract/Free Full Text]
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  4. Ling, T. K., P. C. Tam, Z. K. Liu, and A. F. Cheng. 2001. Evaluation of VITEK 2 rapid identification and susceptibility testing system against gram-negative clinical isolates. J. Clin. Microbiol. 39:2964-2966.[Abstract/Free Full Text]
Marjan J. Bruins*
Maurice J. H. M. Wolfhagen
Laboratory of Clinical Microbiology  and Infectious Diseases,
Isala Klinieken Rhijnvis Feithlaan,
62 8021 AM Zwolle, The Netherlands

* Phone: 31 38 424 3111,Fax: 31 38 424 3146,E-mail: m.j.bruins{at}isala.nl Back

Journal of Clinical Microbiology, August 2005, p. 4296-4297, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.4296-4297.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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