Journal Identification = ABC Article Identification = 1446 Date: May 30, 2019 Time: 1:26 pm
doi:10.1684/abc.2019.1446
350 To cite this article: Farfour E, Henry A, Razillard A, Cardot E, Limousin L, Cahen P, Jolly E, Vasse M, Mathonnet D. Rapid identification ofEscherichia colicolonies from clinical sample inoculated on CHROMagar Orientation media (Becton Dickinson).Ann Biol Clin2019; 77(3): 350-2 doi:10.1684/abc.2019.1446
Letter
Ann Biol Clin 2019; 77 (3): 350-2
Rapid identification of Escherichia coli colonies from clinical sample inoculated on CHROMagar Orientation media (Becton Dickinson)
Identification rapide d’Escherichia coli à partir d’échantillons cliniques ensemencés sur milieux CHROMagar Orientation (Becton Dickinson)
Eric Farfour1 Amandine Henry1,2 Anthony Razillard1 Emilie Cardot1 Lucie Limousin1 Pierre Cahen1 Emilie Jolly1 Marc Vasse1 Damien Mathonnet1
1Service de biologie clinique, Hôpital Foch, Suresnes, France
2Service de biologie, Hôpital A. Mignot, Le Chesnay, France
Article received January 06, 2019, accepted April 29, 2019
Abstract. CHROMmagar Orientation media (Becton Dickinson) was devel- oped and validated for the culture of urinary samples. It allows a direct identification ofE. colicolonies without additional tests. As CHROMmagar Orientation media is superior to non-chromogenic media for the distinction of enterobacterial colonies, it is used for the inoculation of a large variety of samples in clinical laboratories. Direct identification of E. colicolonies cul- tured from these samples is not validated by the manufacturer. The difference in microbial ecology and the nature of the sample may impact CHROMagar Orientation performances for this use. We evaluated these media for the direct identification ofE. colicolonies from 410 samples (excluding urine). Its sen- sitivity of 99% allows a direct identification ofE. colicolonies cultured from a wide variety of samples. On-site testing using a large number of representa- tive samples, allows laboratories to assess agar media performance and adapt their uses. Suppliers who are aware of frequent and non-recommended use of their culture media should perform tests and if conclusive, adapt their technical instructions.
Key words: Escherichia coli, CHROMagar Orientation media, clinical sample
Résumé. Le milieu de culture gélosé chromogénique CHROMagar Orientation (Becton Dickinson) a été développé et validé pour la culture des prélève- ments urinaires. Il permet l’identification directe sans tests complémentaires des colonies de E. coli. En raison de sa supériorité par rapport au prélève- ment non-chromogénique pour différencier les colonies d’entérobactéries, il est utilisé pour l’ensemencement d’une grande variété de prélèvements par les laboratoires. Mais, l’identification directe des colonies deE. colin’est pas validée à partir de ces échantillons. En effet, l’écologie microbienne différente selon l’origine des prélèvements et la nature même des échantillons peuvent avoir une incidence sur les performances de la gélose CHROMagar Orienta- tion. Nous avons évalué ce milieu pour l’identification directe des colonies de E. colià partir de 410 prélèvements non urinaires. La sensibilité évaluée à 99 % permet cette utilisation. La réalisation d’essais sur site à partir d’un nombre élevé d’échantillons représentatifs permet au laboratoire de valider les performances des géloses. Les fournisseurs ayant connaissance de l’utilisation fréquente et non recommandée de leur milieu de culture devraient effectuer des tests et, s’ils sont concluants, adapter leurs instructions techniques.
Mots clés : Escherichia coli, milieu de culture CHROMagar Orientation, échantillon biologique
Correspondence :E. Farfour
<ericf6598@yahoo.fr>
Journal Identification = ABC Article Identification = 1446 Date: May 30, 2019 Time: 1:26 pm
Ann Biol Clin, vol. 77, n◦3, mai-juin 2019 351
Rapid identification ofEscherichia colicolonies
CHROMagar Orientation media (Becton Dickinson) was designed for the culture of urinary sample in the early 1990’s. The media composition supplies the growth of most frequently encountered micro-organisms recovered from urinary sample. It allows to assess both cultural and bio- chemical characteristics of bacterial colonies. Indeed, the media contains chromogenic substrates which release dif- ferently colored compounds upon degradation by specific bacterial enzymes. ThusE. colicolonies are rapidly recog- nized by their characteristic coloration in pink or dark-rose while Klebsiella spp, Enterobacter sppand Serratia spp colonies are colored in blue to dark blue andProteus spp, Providentia sppandMorganella spp in pale to beige sur- rounded by brown halos. Using this mediaE. colicolonies presenting characteristics coloration and morphology can be identified without additional tests.
Moreover, CHROMagar Orientation is superior to CLED agar or combination of blood and MacConkey agars, for the isolation, differentiation and counting of urinary tract pathogens [1-3]. Chromogenic media were consequently developed for other specific use, such as screening of multidrug resistant organisms (methicillin resistantStaphy- lococcus aureus, ESBL producingenterobacteriaceae) or rapid identification of micro-organisms (Staphylococcus aureus, Candida species) with superior results to non- chromogenic media. As a consequence of their superiority for differentiation of colonies, CHROMagar Orientation media are now frequently used in clinical laboratory in addition to non-chromogenic media or in replacement of Drigalski media for inoculation of a wide variety of clinical samples. However, bacterial epidemiology is largely dis- tinct in urinary tract, digestive or respiratory infections for instance. Furthermore, the nature of the sample can affect the quality of the agar and the coloration of the colonies. For these samples, CHROMagar Orientation agar can only be used as a help for colonies differentiation. And additional tests are required forE. coliidentification [4].
We assess the reliability of CHROMagar Orientation for direct identification, without additional testing, of E. coli recovered from a wide range of clinical specimens, exclud- ing urinary sample. Consecutive clinical samples usually inoculated on CHROMagar Orientation agar between April and July 2018 were included. CHROMagar Orientation agar followed the workflow process of the laboratory for others cultures media. They were incubated at 35◦C +/- 2◦C in aerobic atmosphere and protected from light. The media were read after 12 to 48 hours of incubation. All colonies presenting E. coli characteristics (pink or dark- rose round shaped millimetric colonies) were identified using MALDI-TOF mass spectrometry (Bruker Daltonic, Bremen, Germany) as recommended by the manufacturer.
Overall, 410 clinical samples, including 177 subcultures of positive blood culture, 83 gastric liquid, 15 respiratory
samples, 76 suppurations, 39 digestives samples and 20 others samples (biopsies. . .), yield growth of colonies pre- senting E. colicharacteristics. All but four isolates were identified asE. coliby MALDI-TOF mass spectrometry:
– two isolates recovered from positive blood culture were identified asPantoea agglomeransandHafnia alvei;
– two isolates recovered from digestives samples were identified as Hafnia alvei and Citrobacter freundii. The culture age of the latter was about 12 hours at time of identification.
Overall, CHROMagar Orientation media sensitivity was 99.0%.
CHROMagar Orientation represent a useful tool for rapid and direct identification of E. colicolonies cultured from a wide range of clinical sample without additional test- ing. The nature of the clinical specimens does not affect the quality and performance of the agar plates. For a wide variety of human samples CHROMagar Orientation sensitivity is similar to that reported for urinary sam- ple (96.9%) [2, 5]. Misidentification are rare and might be due to rarely encountered micro-organisms or young colonies. They could be avoided by confrontation with other laboratories results, i.e. antibiotics susceptibility test- ing. In case of discrepancies, additional tests or a second method of identification should be performed. Moreover, young colonies should not be identified without addi- tional testing as they may not have achieved all of their characteristics. Indeed, the manufacturer recommends an incubation of 20 to 24 hours at 37◦C [5]. This duration was not respected in our study as CHROMagar Orientation were integrated to the laboratory workflow and reading of CHROMagar Orientation was not dissociated from others media. However, our results suggest a high robustness of this media.
As part of the accreditation process, on-site tests using a sufficient number of representative samples allow to assess reagents performance. Also, suppliers who are aware of fre- quent and non-recommended use of their reagent should perform tests and update their technical instructions if conclusive.
Conflict of interest: None of the other authors has any conflict of interest to disclosure.
References
1. Hengstler KA, Hammann R, Fahr AM. Evaluation of BBL CHROMa- gar Orientation medium for detection and presumptive identification of urinary tract pathogens. J Clin Microbiol1997 ; 35 : 2773-7.
2. Merlino J, Siarakas S, Robertson GJ, Funnell GR, Gottlieb T, Bradbury R. Evaluation of CHROMagar Orientation for differentiation and presump- tive identification of gram-negative bacilli and Enterococcus species. J Clin Microbiol1996 ; 34 : 1788-93.
Journal Identification = ABC Article Identification = 1446 Date: May 30, 2019 Time: 1:26 pm
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3. Samra Z, Heifetz M, Talmor J, Bain E, Bahar J. Evaluation of use of a new chromogenic agar in detection of urinary tract pathogens. J Clin Microbiol1998 ; 36 : 990-4.
4. Ohkusu K. Cost-effective and rapid presumptive identification of Gram-negative bacilli in routine urine, pus, and stool cultures:
evaluation of the use of CHROMagar orientation medium in conjunc- tion with simple biochemical tests. J Clin Microbiol 2000 ; 38(12) : 4586-92.
5. Becton Dickinson. Instructions for use - ready-to-use plated media BD CHROMagar Orientation Medium PA-257481.03. September 2011.
