INTRODUCTION

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In recent years the evolution of human disease, in conjunction with the continuing improvement in detection and identification of bacterial pathogens, has yielded numerous previously unrecognized agents. Some of these agents include new Bartonella and Bordetella species; the new genera Afipia, Roseomonas, and Balneatrix; and other currently unclassified isolates (1-3, 8, 11-17). In 1983, the Special Bacteriology Reference Laboratory of the Centers for Disease Control and Prevention (CDC) received the first of 13 clinical isolates of unusual gram-negative, glucose-oxidizing bacilli that shared a unique phenotypic and cellular fatty acid (CFA) profile. These isolates were from male and female patients and from a variety of sources, including blood, bone, knee fluid, pulmonary, and duodenal and lymph node tissues.

Presented herein is a polyphasic study of this group, to which the provisional name CDC oxidizer group 3 (O-3) has been assigned. Included within this study are morphologic, biochemical, CFA, isoprenoid quinone, and in vitro antimicrobial susceptibility determinations.

	MATERIALS AND METHODS

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Bacterial strains. The O-3 isolates studied, along with their sources, geographic origins, and submission dates, are presented in Table 1. All strains were stored as suspensions in defibrinated rabbit blood in liquid nitrogen. Unless otherwise indicated, the strains were cultured on heart infusion agar supplemented with 5% rabbit blood (RBA) (BBL prepared media; BBL Microbiology Systems, Cockeysville, Md.) and incubated at 35°C in a candle jar.

Phenotypic tests. Biochemical testing was done by the methods of the CDC Special Bacteriology Reference Laboratory (17). The flagellum staining was performed by the Ryu stain method of Kodaka et al. (5), using a commercially available reagent (Carr-Scarborough Microbiologicals, Decatur, Ga.). With the exception of those for oxidase, catalase, growth temperature, and gelatin digestion, all biochemical tests were performed at 35°C in an aerobic incubator. The oxidase, catalase, and growth temperature tests were determined at 1 day and gelatin digestion was determined at 7 and 14 days of incubation. Five isolates were tested for growth on Trypticase soy agar with 5% sheep blood (BBL). Twelve isolates were tested for growth on Campy selective CVA media (BBL) at 35°C in an atmosphere of 5% hydrogen, 10% CO₂, and 85% nitrogen. The growth of these isolates was compared to their growth on RBA at 35°C in a candle jar atmosphere.

CFA analysis. Cells were saponified, and the liberated fatty acids were methylated and analyzed by capillary gas-liquid chromatography (GLC) (17). The amide-linked hydroxy acids that were not totally released by this saponification procedure were completely released by a subsequent acid hydrolysis of the methanolic aqueous layer after the methylation step (17). The identification of fatty acids and the determination of double bond positions in monounsaturated acids were accomplished by GLC and GLC-mass spectrometry (GLC-MS). The confirmation of hydroxy acids was accomplished by both acetylation and GLC-MS analysis, as described previously (17). Isoprenoid quinones were extracted from 100 mg of lyophilized cells and were analyzed by reverse-phase high-performance liquid chromatography and MS (6, 7).

In vitro antimicrobial susceptibility tests. Antimicrobial susceptibility profiles were determined by the broth microdilution method described by the National Committee for Clinical Laboratory Standards (NCCLS) (9), except that results were read after a 24-h incubation. The strains were streaked onto Trypticase soy agar with 5% sheep blood (BBL) and incubated for 18 to 24 h at 35°C in ambient air. Growth was taken from the plate with a sterile cotton-tipped swab and suspended in a tube of unsupplemented cation-adjusted Mueller-Hinton broth (Difco, Detroit, Mich.) to a density equivalent to a McFarland standard of 1.0. Eight-milliliter aliquots of the broth suspensions were added to 32 ml of sterile distilled water and inoculated into MIC plates with the MIC 2000 (Dynatech Laboratories, Chantilly, Va.). Microtiter MIC plates were prepared at the CDC according to the recommendations of the NCCLS (9) (final volume per well, 100 µl). The plates were incubated in ambient air for 24 h at 35°C, with a final inoculum concentration of approximately 5 × 10⁵ CFU/ml. The following organisms were used as controls: Escherichia coli ATCC 25922, E. coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, and Enterococcus faecalis ATCC 29212.

	RESULTS

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As indicated in Table 1, all O-3 strains were received from laboratories within the United States. The first was received in 1983, and most of the others were received after 1991. Isolates were obtained from a variety of sources, including blood, respiratory, bone, joint, and lymphatic tissues. Patients from whom these isolates were obtained ranged in age from 1 to 92 years, and no significant preference for either gender was noted. Clinical information was available on five patients, and no common underlying syndrome was identified.

The phenotypic characteristics of the O-3 strains in this study are presented in Table 2. Cells grown on heart infusion agar at 35°C for 18 to 24 h were thin, medium to slightly long curved rods with tapered ends (sickle-like) that sometimes formed rosettes (Fig. 1). All the strains grew slightly to moderately well on RBA that was incubated either aerobically or in a candle jar atmosphere for 18 to 24 h. Isolated colonies were circular, entire, translucent, and very punctate. Frequently, no hemolytic reaction was observed on RBA after overnight incubation at 35°C; however, five strains produced a faint green discoloration. Five strains were also tested for growth on Trypticase soy agar supplemented with 5% sheep blood, and all grew at rates and with morphologies similar to those obtained on RBA. All 13 strains were aerobic glucose oxidizers. All the strains produced acid from D-xylose, sucrose, and maltose; were oxidase positive; and hydrolyzed esculin. When examined at 2 days of incubation, all the strains were negative for growth on MacConkey agar; however, growth was detected at 3 to 7 days for five strains. All were negative for urease, indole, nitrate reduction, and gelatin hydrolysis. Four strains reduced 0.01% nitrite without gas formation. All the strains were motile by means of a single polar flagellum with a noticeably short wavelength (Fig. 2); however, motility was sometimes difficult to demonstrate.

View larger version (129K): [in this window] [in a new window]   FIG. 1.   CDC group O-3 strain G8822 with Gram stain, grown in heart infusion agar at 35°C for 24 h. Magnification, ×3,000.

View larger version (159K): [in this window] [in a new window]   FIG. 2.   CDC group O-3 strain G8822 with flagellum stain, grown in tryptone glucose yeast extract medium at 25°C for 24 h. Magnification, ×3,000.

Table 2 also contains phenotypic results for four similar glucose-oxidizing gram-negative species and CDC groups. The "Agrobacterium yellow group" was first described in 1985, and CDC groups O-1 and O-2 were first described in 1996 (12, 17). The strains designated Sphingomonas species represent isolates that phenotypically resemble Sphingomonas paucimobilis and Sphingomonas parapaucimobilis (17). O-3 is the only group in which a yellow growth pigment is not produced and the only group of predominately curved rods. Other tests useful in differentiating these taxa include those for oxidation of D-xylose, lactose, sucrose, and maltose; Christensen's urea hydrolysis; and 3-ketolactonate production.

To determine the growth ability of the O-3 group on Campylobacter selective medium, 12 isolates were tested for growth on Campy CVA. The results were then compared to their growth on RBA. All the isolates grew as well or, in most cases, better on the Campy CVA plates under microaerophilic conditions.

The CFA composition of the O-3 group is presented in Table 3. All 13 strains shared a unique profile characterized by relatively large amounts of 16:17c (8 to 22%), 16:0 (15 to 32%), and 18:17c (32 to 50%) with smaller amounts (1 to 10%) of 12:0, 3-OH-12:1, 14:0, 15:0, 18:0, Br-19:1, and 19:0cyc^11-12. In addition, 0 to 2% amounts of 3-OH-12:0, i-15:0, 2-OH-14:0, i-17:0, 17:18c, 17:16c, 17:0, 18:2, 18:19c, and 20:4 were observed in the CFA profile of the O-3 group. Upon acid hydrolysis of two representative strains, additional 1% amounts each of 3-OH-12:0 and 2-OH-14:0 were released, indicating that these acids are both ester and amide linked. No additional hydroxy acids were released upon acid hydrolysis. High-performance liquid chromatographic and MS data of the quinone extracts of three representative strains confirmed ubiquinone-10 (Q-10) as the major component, constituting approximately 90 to 100% of the total ubiquinone content. The quinone content of the O-3 group is most similar to that of Afipia species in that Q-10 is essentially the only quinone present, as studied by our methods (8). Therefore, the presence of Q-10, in combination with the described CFA profile, provides a rapid chemical identification of the O-3 isolates.

The results of antimicrobial susceptibility testing of all O-3 isolates against 19 antimicrobial agents are shown in Table 4. Based on NCCLS interpretative standards for the family Enterobacteriaceae (10), all the isolates tested were susceptible to the aminoglycosides (amikacin, gentamicin, and tobramycin), trimethoprim-sulfamethozaxole, and imipenem. Twelve of the 13 isolates were susceptible to chloramphenicol, with one isolate (G8743) yielding an intermediate MIC of 16 µg/ml. All the isolates, with the exception of G185, were resistant to most of the beta-lactams, including ampicillin, cefazolin, cefoxitin, cefotaxime, ceftriaxone, cefotetan, ceftazidime, and aztreonam, although clavulanic acid did appear to inhibit this activity. Resistance to tetracycline was variable. The ciprofloxacin and amoxicillin-clavulanate MICs clustered around the breakpoints, which makes the in vivo responses of these organisms to these antimicrobial agents difficult to predict.

	DISCUSSION

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CDC group O-3 represents 13 similar clinical isolates received by the Special Bacteriology Reference Laboratory between 1983 and 1994. The patient information submitted with these isolates indicates that this group may be isolated from males and females of all ages. Although these isolates often were obtained from sites associated with invasive disease (e.g., blood and lymph nodes), the limited amount of clinical information received makes it difficult to estimate their clinical significance. Additional studies are needed to better understand their pathogenic potential.

The duodenal tissue isolate is particularly interesting in that it was obtained from a patient with a diagnosis of duodenal ulcer and was submitted to CDC as a Campylobacter species. This suggests a potential for misidentification of the O-3 organism as a member of the genus Campylobacter in the clinical laboratory, especially since it grows well in Campylobacter selective medium.

O-3 strains are characterized by distinctive biochemical and CFA profiles, as well as cellular and flagellar morphology. However, the O-3 biochemical profile shares some similarities with other unclassified groups, including CDC groups O-1 and O-2, and the Agrobacterium yellow group (12, 17). Tests useful in differentiating among these groups include those for cellular morphology; oxidation of lactose, D-xylose, sucrose, and maltose; urea hydrolysis; 3-ketolactonate production; flagellar morphology; and presence of a yellow growth pigment. CFA analysis is also useful in differentiating the three CDC oxidizer groups and Sphingomonas. The utility of CFA analysis in identifying Agrobacterium yellow group strains will be better understood when more strains become available.

The antimicrobial susceptibility profiles of these isolates show a high level of in vitro resistance to many of the commonly used broad spectrum agents, including most of the beta-lactam drugs. The results for ciprofloxacin cluster around the breakpoint. In light of these findings, the clinical significance of O-3 isolates should be carefully assessed in determining appropriate antimicrobial therapy. At this time there is very little information available on the natural pathogenicity of these organisms.

The ultimate taxonomic classification of the O-3 group will require the use of molecular techniques, such as 16S rRNA sequencing and DNA-DNA hybridization analysis. At this time, we do not know whether the O-3 group represents one or more species. Some Special Bacteriology Reference Laboratory groups, such as DF-2 (now Capnocytophaga canimorsus) (2) and NO-2 (now Bordetella holmesii) (15), have been shown to be genetically homogeneous, whereas other groups, such as WO-1 and Pink Coccoid I through IV, have represented multiple species (4, 11, 14). We plan to initiate molecular taxonomic studies of these strains in the near future; however, the findings of this study should assist clinical microbiologists in the identification of this group, which will ultimately provide an increased understanding of its true clinical significance.