Comparison of bioMérieux API 20NE and Remel RapID NF Plus, identification systems of type strains of Ralstonia pickettii
Catherine C. Adley, Microbiology Laboratory, Department of Chemical and Environmental Sciences, University of Limerick, Limerick, Ireland (e-mail: firstname.lastname@example.org).
Aims: Comparison of two commercial miniaturized rapid systems for the identification of Ralstonia pickettii strains.
Methods and Results: Varying identification results were encountered using the bioMérieux API NE system and the Remel IDS RapID NF Plus commercial systems for R. pickettii. To compare these two systems, eight strains of R. pickettii were purchased from different commercial culture collections. Additionally, 32 industrial and eight clinical isolates, initially identified using the Vitek Junior (bioMérieux) were tested. Total number of isolates tested was 48. The API 20NE identified 29 isolates, as R. pickettii but was unsuccessful with 19 isolates. The Remel IDS RapID NF Plus identified 46 isolates as R. pickettii. One clinical and one industrial isolates was identified as non-R. pickettii with both systems.
Conclusions: The above results indicate that the use of API 20NE system for examining the identification of R. pickettii strains is inconsistent.
Significance and Impact of the Study: This study demonstrated that the RapID NF Plus is more accurate as an inexpensive identification system for the identification of R. pickettii, a potential emerging organism of medically and industrial importance.
Identification of Gram-negative nonfermenting rods by conventional methods is often difficult and time consuming, and commercial systems do not always provide reliable identification, especially for some genera or species (Wauters et al. 1995; Kiska et al. 1996; Van Pelt et al. 1999). Moreover, taxonomic studies have resulted in the description of an increasing number of new genera and species involved in nosocomial infections and requiring additional tests for identification. This was true, for instance, for the new genus Ralstonia (Vandamme et al. 1999; De Baere et al. 2001; Gilligan et al. 2003). The Rapid NF plus system utilizes conventional biochemical assay for the identification of medically important Gram-negative, nonfermentative bacteria and has been evaluated in several studies (Kitch et al. 1992; Kiska et al. 1996).
Ralstonia is a new genus that includes former members of Burkholderia species (Burkholderia pickettii and Burkholderia solanacearum). These organisms have been renamed as Ralstonia pickettii, Ralstonia solanacearum respectively. The genus was separated from Burkholderia, by phenotypic characteristics, cellular lipid and fatty acid analysis, rRNA-DNA hybridization, and phylogenetic analysis of 16S rDNA nucleotide sequences (Yabuuchi et al. 1995). Ralstonia spp. are aerobic Gram-negative, oxidase-positive, nonfermentative rod, found in water and soil (Gilligan et al. 2003). Anderson et al. 1990 identified R. pickettii in biofilm formation in plastic water piping. Its potential to form biofilm is potentially because of its ability to produce homoserine lactone molecules (Adley and Saieb 2005). It has been identified in ultrapure water in industrial systems (Kulakov et al. 2002) and in the Space Shuttle water system (Koenig and Pierson 1997).
Ralstonia pickettii has also been identified as an opportunistic pathogen in nosocomial infections, especially among immunosuppressed patients (Lacey and Want 1991; Wertheim and Markovitz 1992). Nosocomial infection outbreaks with R. pickettii have been reported mainly in association with contamination of hospital supplies (Gardner and Shulman 1984; McNeil et al. 1985; Roberts et al. 1990; Lacey and Want 1991; Maki et al. 1991; Raveh et al. 1993; Labarca et al. 1999) and with contaminated chlorhexidine skin cleansing solutions (Kahan et al. 1983; Maroye et al. 2000). The emergence of R. pickettii in high-purity water systems necessitates re-looking at this organism.
In this context, it is important to evaluate commercial system for their abilities to detect the presence of R. pickettii. Here we evaluated two commercial systems, the bioMérieux API 20NE (bioMérieux, Marcy L'Etoile, France) and Remel RapID NF Plus (Remel, Lenexa, KA, USA), for the ability to identify isolates of R. pickettii.
We initially undertook these studies because we needed a relative inexpensive accurate method to identity R. pickettii isolates, as an ambiguity was observed in the identification of isolates in the laboratory using methods other than the bioMérieux Vitek Junior.
Material and methods
Analysis was carried out on 48 strains of R. pickettii obtained from different sources. The type strains of R. pickettii, a clinical strain, were purchased from five different culture collections (see Table 1). One additional clinical strain, R. pickettii ATCC 49129 (Ralston et al. 1973; Yabuuchi et al. 1995), deposited as Pseudomonas cepacia (Burkholder) Palleroni and Holmes, American Type Culture Collection (Manassas, VA, USA) and a further soil strain from a rice field in Senegal (Garcia et al. 1977; Vandamme et al. 1999) of R. pickettii and deposited in two separate culture collection repositories, CCM 2846, Czech Collection of Microorganisms (Masaryk University, Brno, Czech Republic) and CCUG 18841 were analysed (Tables 1 and 2).
Table 1. Comparative identification of Ralstonia pickettii strains using bioMérieux API 20NE, Remel RapID NF Plus and the bioMérieux Vitek Junior systems
Table 2. Comparative identification of Ralstonia pickettii strains (eight clinical and 32 industrial) using bioMérieux API 20NE and Remel RapID NF Plus systems
| ULI 187||97·70||98·38|
| ULI 188||95·10||99·99|
| ULI 798||95·10||99·99|
| ULI 807||84·10||99·99|
| ULI 171||84·10||99·99|
| ULI 821||84·10||99·94|
| ULI 797||84·10||98·38|
| ULI 788||80·40||99·99|
| ULI 800||80·40||99·99|
| ULI 169||80·40||99·99|
| ULI 165||67·90||99·99|
| ULI 174||67·90||98·38|
| ULI 193||61·70||98·38|
| ULI 796||60·00||98·38|
| ULI 801||56·90||99·99|
| ULI 791||56·90||99·99|
| ULI 785||53·10||99·99|
| ULI 790||44·80||98·38|
| ULI 181||39·50||99·99|
| ULI 804||24·50||99·90|
| ULI 794||6·40||00·00|
| ULI 185||5·70||98·38|
| ULI 166||0·00||99·94|
| ULI 819||0·00||99·99|
| ULI 159||0·00||99·38|
| ULI 806||0·00||99·99|
| ULI 167||0·00||99·94|
| ULI 784||0·00||99·99|
| ULI 818||0·00||99·99|
| ULI 163||0·00||98·38|
| ULI 795||0·00||98·38|
| ULI 162||0·00||99·99|
| ULC 298||90·10||99·99|
| ULC 297||70·03||99·94|
| ULC 277||61·70||99·99|
| ULC 244||56·70||99·99|
| ULC 193||56·70||99·34|
| ULC 194||56·70||99·99|
| ULC 421||28·50||99·99|
| ULC 279||0·00||0·00|
In addition, eight clinical strains were obtained from the collection of the microbiology laboratory of the Limerick Regional Hospital, isolated from the cystic fibrosis bench. The remaining strains consisted of 32 isolates from a high-purity water system from an industrial setting.
Classical phenotypic tests were performed using the bioMérieux API 20NE system (http://www.biomerieux.com), consisting of eight enzymatic tests and 12 assimilation tests for the identification of nonfastidious Gram-negative rods. The Remel RapID NF Plus commercial system (http://www.remelinc.com) consists of 18 test scores based on microbial degradation of specific substrates. Both systems were used according to the protocol supplied by the manufacturers.
The analysis was carried out in triplicate for each test and using different batch lots. Among historic nonfermentative Gram-negative rods Tatum et al. (1974) devised two biovars, Va-1 and Va-2, according to their utilization of lactose and maltose, R. pickettii has been equated with biovar Va-2 (Pickett and Greenwood 1980). Lactose utilization was observed on MacConkey agar (Oxoid) at 35°C for 24 h. Lactose is broken down to give an acid product that turns the indicator deep red. Bacteria unable to ferment lactose utilize peptone, raising the pH of the medium, turning the indicator a buff colour (Tatum et al. 1974). Maltose utilization was recorded according to the API 20 NE system.
Results and discussion
The ID results obtained for the purchased strains are presented in Table 1, the clinical and industrial strains are presented in Table 2. All isolates were Gram-negative rods. Our results showed that these strains were biovar Va-2 (lactose and maltose negative).
In our studies, out of 40 strains (eight clinical and 32 industrial isolates) identified initially using the Vitek Junior, which is an expensive system and not all testing laboratories would have access to this system, the API 20NE misidentified 17 of 32 isolates as non-R. pickettii, cut-off points lower then 50%, whereas RapID NF Plus identify 38 isolates as R. pickettii. An important and unexpected result of this study was that the two instruments evaluated, differed in their rates of correct ID of R. pickettii, for which the Remel RapID NF system performed significantly better.
Eight purchased strains of R. pickettii were tested. The bioMérieux Vitek Junior identified all purchased strains as R. pickettii. The purchased strains were also tested using the API 20NE system, six strains of R. pickettii JCM 5969, NCTC 11149, DSM 6297, ATTC 49129, CIP 73.23 and CCUG 3318 were identified as R. pickettii, whereas CCM 2846 and CCUG 18841 were not (Table 1). The identification of these two identical purchased strains of R. pickettii, with the API 20NE system was inconsistent due to the Gelatine hydrolysis and assimilation of Mannitol biochemical tests that varied. Ralstonia pickettii strains were identified (‘good identification’, profile API 20NE 1041465 or ‘low discrimination’, profile API 20NE 0045457). Considering that all of these purchased isolates were catalogued as identical strains of R. pickettii, these results necessitated addressing this issue by the use of another method for confirming the identification of the strains.
In contrast, our results show that the Remel RapID NF system is more accurate in the identification of R. pickettii. It reconfirmed the identity of the eight purchased strains and the 38 of 40 industrial and clinical laboratory strains as R. pickettii. The Remel RapID NF system results were reproducible when compared with the bioMérieux API 20NE system prior to supplemental testing. Because the RapID NF Plus system is enzyme based, its ability to identify weakly oxidizing R. pickettii strains may be enhanced compared with those of the other commercial systems (Kiska et al. 1996). All strains were positive for glutmyl-β-naphthylamide, a key test for the identification of R. pickettii by this system. Using the RapID NF Plus system, the strains were identified as R. pickettii (‘adequate identification’, profile 400414). For the purposes of this study, RapID NF Plus identification of R. pickettii was taken as more accurate and repeatable.
In general, the study demonstrated insufficient accuracy of the API 20NE system. A study by Kiska et al. (1996) assessed the accuracy of the API Rapid (since replaced by the API 20NE) and the Vitek GNI systems, by studying 150 nonfermenting bacteria, including 58 isolates of Burkholderis cepacia. Their study included the RapID NF Plus and the Remel Uni-N/F Tek and N/F Screen. Again, the overall performances of these systems were relatively poor, accuracies ranged from 57 to 80%, with the Rapid NF Plus being best for identifying nonfermenters in general.
In view of our results, it is observed that the use of API 20NE system for identification of R. pickettii was inconsistent. Overall, RapID NF Plus identified the R. pickettii strains in this investigation with greater reliability.
The RapID NF plus system was truly rapid, easy to use and interpret. Its use of carbon substrate assimilation enables it to provide more accurate identification of medically important bacteria than do other commercially available system. As no international gold standard for the routine laboratory identification of R. pickettii exists to date, the definition of a suitable identification system remains a topic for further investigation.
The authors would like to acknowledge the assistance afforded by the personnel of the Microbiology Laboratory at the Mid-West Regional Hospital, Limerick, Ireland, and the local company that provided the industrial R. pickettii strains and Vitek Junior analysis.