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Acinetobacters do not possess unique biochemical characteristics to allow for unambiguous phenotypic identification. The genus is characterized by properties it does not possess, i.e., being oxidase negative, nonmotile, and nonfermentative. In 1986 Bouvet and Grimont (5) identified 12 genospecies by DNA-DNA hybridization. Three years later Bouvet and Jeanjean (6) reported new proteolytic strains that they designated genospecies 13 to 17. In the same year Tjernberg and Ursing (17) published information on unrelated strains that were also assigned to genospecies 13 to 15. An extensive set of substrate assimilation tests has become necessary for the unequivocal identification of the genospecies of acinetobacters (19). However, even commercially available multitest biochemical systems may not be able to discriminate between certain genospecies, such as genospecies 1, 2, 3, and 13 sensu Tjernberg and Ursing (13TU) (3), and Gerner-Smidt et al. (9) have suggested that these genospecies should be referred to as the Acinetobacter calcoaceticus-Acinetobacter baumannii complex.

The endemic presence of acinetobacters in our locality has been reported previously (16). During specimen processing we found an acinetobacter isolate that grew as mucoid colonies on Sabouraud dextrose agar, a glucose-containing fungal medium. This observation, coupled with the report of glucose-oxidizing acinetobacters being capable of growing in 5% dextrose at room temperature (13), prompted us to initiate a study of the growth of acinetobacters on solid media with glucose as an ancillary nutrient. We report, for the first time, on the ability of glucose-oxidizing acinetobacters to cause a unique brown discoloration of blood agar into which glucose is incorporated. In an attempt to use this positive phenotype as a simple means of identification, we identified by PCR the genospecies of isolates that exhibited the browning effect, as well as those that did not. Since certain strains of acinetobacters produce gelatinase (2), lipase (2), and phospholipase C (12), tests to determine production of these extracellular enzymes in the presence of glucose were also performed.

Clinical isolates and experimental conditions. Acinetobacters have been identified as oxidase negative, nonmotile, nonfermentative, gram-negative coccobacilli that grow well only under aerobic conditions (18). The ability to oxidize glucose was tested in Hugh and Leifson's medium, and the organisms were reported as glucose-oxidizing or non-glucose-oxidizing Acinetobacter spp.

Identification of genospecies by PCR. To assign acinetobacters to their respective genospecies, 42 of 199 clinical isolates were selected for PCR with primers G1 (5'-GAAGTCGTAACAAGG-3') and L1 (5'-CAAGGCATCCACCGT-3') (Gibco BRL, Life Technologies, Gaithersburg, Md.), which contain conserved sequences of the 16S to 23S rRNA spacer region, by modifications of protocols by Jensen et al. (11) and Nowak et al. (14). A fraction of a colony of each isolate was picked and suspended in a 50-µl reaction mixture containing GeneAmp PCR buffer (Perkin-Elmer) with 1.5 mM MgCl₂, 100 µM (each of the four) deoxynucleoside triphosphates (Pharmacia), 1% Triton X-100, and 1.25 ng of each primer per µl. The resulting mixture was heated at 94°C for 5 min. After the addition of 2.5 U of Taq polymerase (Perkin-Elmer), each tube was transferred to a Bio-Rad gene cycler for PCR under the following conditions: 94°C for 1 min, 55°C for 7 min, and 72°C for 2 min for 25 cycles followed by a terminal extension at 72°C for 7 min. Negative controls containing the reagents but no DNA, as well as the positive control A. baumannii ATCC 19606, were included with each batch of test samples. PCR products were separated by polyacrylamide gel electrophoresis (5% polyacrylamide) at 80 V for 2 h. The genospecies of the acinetobacter strains were identified based on the uniquely sized primary PCR fragments of each strain's DNA according to the method of Nowak et al. (14): A. baumannii (genospecies 2) possesses a band of 940 bp; Acinetobacter genospecies 3 possesses a band of 1,000 bp; and Acinetobacter lwoffii (genospecies 8 or 9) possesses a band of 1,300 bp. There were four acinetobacter isolates that showed a band of 1,100 bp, which did not fit into the classification scheme. Since these isolates also grew at 44°C, they were identified tentatively as Acinetobacter genospecies 13TU.

Results. A. baumannii ATCC 19606 and the majority of clinical isolates that oxidize glucose grew on horse blood agar containing 0.22 M glucose and produced a unique light-brown discoloration of the surrounding agar (Fig. 1). These results were reproducible with three different batches of media. The same browning effect could be demonstrated with a lower concentration of glucose (0.055 M) and was also evident on horse blood agar containing 0.22 M galactose, mannose, xylose, or lactose. The discoloration was present when sheep or human blood was used in place of horse blood but not on a similar agar base without the addition of blood. No discoloration was observed when acinetobacters grew on horse blood agar containing 0.22 M fructose, sucrose, mannitol, or glycerol or 0.34 M sodium chloride. Growth of acinetobacters on glucose-containing chocolate agar also did not cause any discoloration.

View larger version (105K): [in this window] [in a new window]   FIG. 1.   Browning effect of glucose-oxidizing acinetobacters on blood agar containing glucose. A. baumannii ATCC 19606 (1) and a clinical isolate of A. baumannii (4) caused a light-brown discoloration (indicated by arrows) on glucose-containing Columbia Agar Base with blood of horse (HoB+), sheep (ShB+), or human (HmB+) origin but not on plain agar with blood of horse (HoB), sheep (ShB), or human origin. The browning effect was not observed when other nonfermenters such as P. aeruginosa (2), Stenotrophomonas maltophilia (3), non-glucose-oxidizing A. lwoffii (5), Flavobacterium meningosepticum (6), and Moraxella osloensis (7) were cultured on glucose-containing blood agar. In addition, the discoloration was not seen on glucose-containing chocolate agar (Cho+) or Columbia Agar Base (CAB+) or on plain chocolate agar (Cho) and Columbia Agar Base.

View larger version (59K): [in this window] [in a new window]   FIG. 2.   Representative PCR profiles of the two most common Acinetobacter genospecies, A. baumannii (lanes 4 to 9) and A. lwoffii (lanes 1 to 3), with unique bands of 940 and 1,300 bp, respectively. Lane M, HaeIII-digested X174 DNA molecular size markers.

Discussion. A browning of the surrounding agar was observed when glucose-oxidizing acinetobacters were cultured on blood agar containing an aldose sugar (Fig. 1). This effect was not exhibited under high osmolarity or in the presence of ketose sugars. The browning effect is unlikely to be useful as a phenotypic trait for identifying the genospecies of acinetobacters, as it was not genospecies specific. Some strains of A. baumannii and A. lwoffii tested positive, while others of the same genospecies tested negative (Table 1). If the ability of an acinetobacter to cause browning is related to its ability to oxidize glucose, our work corroborates the report of Bouvet and Bouvet (4), who showed that each of these genospecies comprises glucose-oxidizing and non-glucose-oxidizing strains.