Biochemical and Susceptibility Tests Useful for Identification of Nonfermenting Gram-Negative Rods

Abstract

Six hundred nineteen strains of nonfermenting gram-negative rods were tested for alkaline phosphatase, benzyl-arginine arylamidase, pyrrolidonyl arylamidase, ethylene glycol acidification, and susceptibility to desferrioxamine and colistin. The results were highly discriminant. Therefore, the proposed tests may be helpful for the identification of this group of organisms.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 of some genera or species (7,17,19). Moreover, recent taxonomic studies 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 (2, 3, 14), the former "Flavobacterium" group (12, 13), and others. In this study, we investigated a large number of strains for six biochemical and susceptibility characteristics that are rarely or not at all included in conventional schemes. Alkaline phosphatase (PHO), benzyl-arginine arylamidase (BAA), and pyrrolidonyl arylamidase (PYR), three enzymatic tests, are sometimes included in commercial systems, but they are not systematically reported in existing identification schemes (4, 6, 11). Susceptibility to desferrioxamine has been proposed for the identification of coagulase-negative staphylococci (9) but not for gram-negative bacilli. Acid production from ethylene glycol was described in corynebacteria (20) but, as far as we know, has not been evaluated in nonfermenters. We have also included susceptibility to colistin, although susceptibility to polymyxin B is often mentioned in conventional schemes (4, 6, 11), but low colistin content disks may yield slightly different results.Five hundred fifty-three clinical isolates of nonfermenting gram-negative rods and 66 reference strains, including type strains, were tested. They included 41 species or Centers for Disease Control and Prevention (CDC) groups (Table 1). Only fast growers on tryptic soy agar were investigated. Moraxella spp. and other fastidious nonfermenters were excluded, as were Acinetobacter spp. because the latter can be identified on the basis of a few simple routine tests. All tests and cultures were performed at 30°C. For enzymatic reactions, diagnostic tablets from ROSCO (Taastrup, Denmark) were used in accordance with the manufacturer's instructions with slight modifications. Tablets were put in 0.5 ml of a 3 to 4 McFarland bacterial suspension and incubated for 4 h. Reading of BAA and PYR was done after addition of 1 drop of cinnamaldehyde reagent. PHO turned yellow when positive; no color or a pale yellowish color was considered negative. Determination of acid production from ethylene glycol was performed as previously described (20). Susceptibility to desferrioxamine was determined on Mueller-Hinton agar using 6-mm-diameter paper disks loaded with 250 g of desferrioxamine (9). Testing for su...

Molecular and phenotypic features for identification of the opportunistic pathogens Ochrobactrum spp.

“…Pyrrolidonyl arylamidase (PyrA), L-proline arylamidase (ProA) and alkaline phosphatase (PHOS) had been previously described as very useful tests for the identification of non-fermenting Gram-negative rods (Laffineur et al, 2002). The authors found that 100 % of the Ochrobactrum spp.…”

“…More generally, this study could be considered as an improvement of the identification of Gram-negative non-fermenting rods by conventional methods. Indeed, their identification is often difficult, and the commercial systems are not always reliable, especially for some genera and species (Laffineur et al, 2002;VanPelt et al, 1999). Moreover, recent taxonomic studies resulted in the description of an increasing number of new taxa involved in nosocomial infections, and requiring additional tests for identification.…”

Chryseobacterium hominis sp. nov., to accommodate clinical isolates biochemically similar to CDC groups II-h and II-c

“…On the basis of this study, we propose that the 11 strains represent a novel species in the genus Chryseobacterium, for which the name Chryseobacterium hominis sp. nov. is proposed.Biochemical and morphological tests were performed as described previously (Laffineur et al, 2002;Schreckenberger et al, 2003); the morphological characteristics of the novel species are given in the species description. Inhibition zones were determined on tryptic soy agar around discs of tetracycline (30 mg), ciprofloxaxcin (5 mg), trimethoprim/cotrimoxazole (23.75 mg/1.25 mg), ampicillin (10 mg), temocillin (30 mg), cephalothin (30 mg), cefotaxime (30 mg), erythromycin (15 mg), gentamicin (10 mg) and colistin (10 mg).…”

Identification of Isolates within the Burkholderia cepacia Complex by a Multiplex recA and 16S rRNA Gene Real-Time PCR Assay

“…These isolates included Achromobacter xylosoxidans, Burkholderia gladioli, Pandoraea sputorum, Pseudomonas aeruginosa, Pseudomonas putida, Ralstonia mannitolytica, Ralstonia pickettii, Sphingomonas paucimobilis, and Stenotrophomonas maltophilia. These organisms can be difficult to identify (7) and may, at times, be clinically relevant (4). The GenBank accession numbers for the 16S rRNA gene sequences of all the isolates are EF427693 to EF427788.…”