In this prospective study, we have examined the suitability of partial 16S rRNA gene sequencing for the identification of aerobic nonfermenting gram-negative bacilli in the diagnostic laboratory. The study was designed to compare phenotypic with molecular identification for nonfermenting, gram-negative rod-shaped isolates of clinical relevance (isolates of P. aeruginosa were excluded from the study). Results of phenotypic identification (API 20 NE and VITEK 2 fluorescent system) were compared to the results of sequencing.
16S rRNA gene sequencing is more accurate for the identification of gram-negative nonfermenters than API 20 NE and VITEK 2: 92% of isolates were assigned at the species level by sequencing, compared to 54% and 53% by API and VITEK 2, respectively. In 12 of 45 (API 20 NE) and 11 of 57 (VITEK 2) isolates with identification to the species level by phenotypic procedures, sequence analysis yielded discrepant results. For API 20 NE, we concluded that sequencing was correct for 7/12 strains, and 5/12 strains remained unresolved. For VITEK 2, it was concluded that the molecular approach delivered the correct result for 9/11 strains, and 2 strains remained unresolved.
For the API 20 NE, a result seems reliable when a species assignment is reported as an excellent or very good identification; 18 of 20 and 15 of 18 cases in these categories were correctly identified when compared to the results of sequencing. In the categories “good species identification” and “acceptable species identification,” 10 of 15 cases and 2 of 5 cases were correct, respectively. Thus, if identification is of lower quality, the result is less reliable and the isolate should be subjected to sequence analysis if accurate identification is of concern. For the VITEK 2 fluorescent system, excellent species identifications are reliable; results of 33 of 37 cases in this category were consistent with molecular analysis. For the other categories, the numbers of isolates were too small to allow for firm conclusions.
One major problem of phenotypic test systems is that the available databases are limited. Of the 107 strains analyzed in the present study, 16 and 46 of the isolates corresponded to species not included in the API 20 NE and VITEK 2 fluorescent card databases, respectively. In other words, while the API 20 NE covers the majority of nonfermenters isolated, the VITEK 2 database needs to be expanded. More recently, efforts have been undertaken to enlarge the VITEK 2 database. The revised VITEK 2 database is associated with a new colorimetric detection card and covers 159 taxa (versus 101 for the ID-GNB card). Twenty of the 46 strains not identified in our study due to noninclusion of the corresponding species in the database are now included in the expanded VITEK 2 database. Evaluation with a large collection of 655 gram-negative strains (including 144 nonfermenters) gave encouraging results (12
). Further studies, testing the system under routine laboratory conditions, and comparing the results with genotypic methods are required before firm conclusions can be drawn on the quality of the enlarged VITEK 2 database.
16S rRNA gene sequencing has distinct benefits compared to phenotypic identification procedures: (i) it is not restricted to a specific group of bacteria as public databases such as GenBank cover the whole spectrum of phylogenetic diversity; (ii) novel, not yet described species can be assigned to a group of related bacteria; (iii) results are in general unambiguous and not dependent on strain variation or individual interpretation. We have recently demonstrated the potential of partial 16S rRNA gene sequencing for identification of gram-positive microorganisms and we have proposed algorithms for integration of molecular identification procedures into the diagnostic work flow (2
). In the present study, we have extended our efforts to include 16S rRNA gene sequence analysis for accurate identification of gram-negative nonfermenting bacilli.
16S rRNA gene sequencing has some limitations. It is in part compromised by a low phylogenetic power at the species or subspecies level (26
). In the present study, 35% of isolates could not be unambiguously assigned to a single species. This was particularly true for members of the Burkholderia cepacia
complex, the Acinetobacter baumannii-A. calcoaceticus
complex and close relatives, some members of the genus Pseudomonas
, the genus Achromobacter
, and the genus Ralstonia
. For the Burkholderia cepacia
complex, the recA
gene seems more appropriate for recognizing the different genomovars (19
). For Acinetobacter
, complete 16S rRNA gene sequencing has been proposed for identification (15
). More recently, the 16S-23S intergenic spacer region has been shown to differentiate closely related bacteria within the Acinetobacter baumannii-A. calcoaceticus
The quality of public databases, such as GenBank, is critical. Sequences are deposited independently of their quality, e.g., regardless of the correct assignment, the length of the sequence, or the number of ambiguous nucleotides. Of particular concern is that sequences in public databases may be assigned to a naming which possibly no longer is valid due to taxonomic changes or which never has been validly published before. As an example, several sequences are annotated under Acinetobacter calcoaceticus
in GenBank; however, this is an old denomination which phenotypically corresponds to members of the Acinetobacter baumannii-A. calcoaceticus
). Correct interpretation of sequence data also requires some familiarity with taxonomy and recent taxonomic changes. As an example, a sequence of an isolate with 100% homology to Burkholderia multivorans
but 12 mismatches to Burkholderia cepacia
should not be reported as B. multivorans
per se. Rather, the result reported should reflect that B. multivorans
regarded as a member of the B. cepacia
is the second most common species of the B. cepacia
complex in cystic fibrosis infections (20
Despite all these minor limitations, 16S rRNA gene sequencing does not lead to false identification, and with some knowledge about taxonomy, a sequence can unambiguously be assigned. In this study, partial 16S rRNA gene sequencing has been compared with two commercially available systems (API 20 NE, VITEK 2) for identification of nonfermenting gram-negative rods. The majority of strains could not be accurately identified by phenotypic profiling, as species assignment was found to be reliable only when an excellent (or very good) species identification according to the system's criteria was achieved; this was the case in 35% of the isolates. In practical terms, molecular identification is more laborious than phenotypic identification; results of sequencing are usually available within one to two working days. Given these considerations, we have developed an algorithm for the effective and proper identification of gram-negative nonfermenters in the diagnostic laboratory (Fig. ). Thus, when API 20 NE or VITEK 2 do not yield an excellent (or very good) species identification, nonfermenters should be subjected to 16S rRNA gene sequencing if adequate species assignment is of concern.
Algorithm for the identification of nonfermenting gram-negative bacilli.