Use of 16S rRNA Sequencing for Identification of Actinobacillus ureae Isolated from a Cerebrospinal Fluid Sample

Abstract

Actinobacillus ureae, previously Pasteurella ureae, has on rare occasions been described as a cause of human infection. Owing to its rarity, it may not be easily identified in clinical microbiology laboratories by standard tests. This report describes a patient with acute bacterial meningitis due to A. ureae. The identity of the isolate was determined by means of DNA sequence analysis of a portion of the 16S rRNA gene.

Identification of pathogens is one of the main functions of a clinical diagnostic laboratory, and this is usually done on the basis of phenotypic testing. However, phenotypic tests do not always yield an unequivocal answer; either because one is working with an uncommon pathogen or else the biochemical profile of the organism is unusual. In these circumstances one can either give the organism a “best-fit” name or proceed to other methods of identification. We present a case that exemplifies this scenario.

Case report.

The patient was a 22-year-old male who presented to Groote Schuur Hospital in June 2000 with a 4-day history of headache, nausea, vomiting, rigors, and drowsiness. His past medical history included previous neurosurgery in 1994 and a depressed skull fracture in 1995 following a motor vehicle accident. Positive findings on examination included pyrexia, signs of meningeal irritation (neck stiffness and a positive Kernig's sign), and scars in the right frontal and parietal areas. No focal neurological signs were present, and no rhinorrhea or otorrhea was noted. The rest of the physical examination was normal.

Two blood samples for aerobic culture were taken, and a lumbar puncture was performed after a computed tomography (CT) scan of the head had excluded the presence of an intracranial mass lesion. The CT scan showed evidence of an old parietal depressed skull fracture and previous neurosurgery. No growth was detected in either of the blood cultures after 5 days of incubation. A full blood count showed that the patient had an elevated white blood cell count (15.9 × 10⁹ cells/ml). Examination of the cerebrospinal fluid (CSF) showed 1,365 neutrophils per mm³ and 234 lymphocytes per mm³. The protein concentration in the CSF was 1.0 g/liter, and the glucose concentration in the CSF was 1.1 mmol/liter (serum glucose concentration, 4.5 mmol/liter). No organisms were observed on Gram staining of the CSF prepared by cytocentrifugation. A diagnosis of acute bacterial meningitis was made, and the patient was started on intravenous ceftriaxone. He made a full recovery and was discharged well after 10 days of therapy.

The CSF was inoculated onto blood agar, onto boiled blood agar, and into serum broth and incubated at 35°C overnight in 5% CO₂. On the following day a small gram-negative bacillus was isolated from both agar plates and the serum broth. Although this isolate was initially thought to be an Haemophilus sp. on the basis of the morphology upon Gram staining, biochemical testing (Table 1) failed to confirm this.

TABLE 1.

Summary of some of the phenotypic characteristics of the CSF isolate compared to typical reactions observed with H. influenzae^a

Test	Result for:
	CSF isolate	H. influenzae
Oxidase	Positive	Positive
Catalase	Positive (weak)	Positive
Requirement for X factor	Positiveb	Positive
Requirement for V factor	Negativeb	Positive
ALA	Positiveb	Negative
Hydrolysis of urea	Positivec	Positive
Reduction of nitrate to nitrite	Positive	Positive
Acid from:
Glucose	Yes	Yes
Sucrose	Yes	No
Lactose	No	No
Maltose	Yes	Yes

^aData are from Holt et al. (8) and Koneman et al. (12).

^bThe test was performed twice.

^cPositive within 6 h.

Further identification of this organism was attempted with the BBL Crystal identification kit (Becton Dickinson, Sparks, Md.), resulting in the identification of Haemophilus influenzae. On the basis of some of the biochemical test results shown in Table 1, notably, the lack of a requirement for V factor as well as the positive δ-aminolevulinic acid (ALA) test result, this result was believed to be incorrect.

Since unequivocal identification of the isolate could not be established by routine phenotypic tests, a molecular biology-based approach with DNA sequence data was used. DNA was extracted from overnight broth cultures of the organism by previously described methods (3). A portion of the 16S rRNA gene was amplified with universal primers A193 and H210 (6). The PCR product was separated by electrophoresis on a 1.5% agarose gel dissolved in TAE (0.4 M Tris-acetate-0.01 M EDTA) buffer containing ethidium bromide. The DNA was visualized under UV light, and a 1.4-kb fragment was excised from the gel and purified (17). DNA sequence data were generated by automated laser fluorescence (ABI 3100; Applied Biosystems, Johannesburg, South Africa). On the basis of the sequencing data obtained with primer A193, further internal oligonucleotide primers were designed and both strands of a 336-bp portion of the amplicon were sequenced. A comparison of the DNA sequence with sequences in the National Center for Biotechnology Information database with BLAST software (1) showed 98.5% sequence identity with the published 16S rRNA sequences of Actinobacillus ureae. All four mismatches consisted of residues (nucleotides) whose identities were uncertain in the sequence in the database.

A. ureae is a small gram-negative bacillus which previously belonged to the genus Pasteurella (8). It is a commensal organism of the human upper respiratory tract, although it has been described as a cause of human infection. Altogether, 16 cases of infection with this organism have been reported in the English-language literature (10, 15, 19), and of these, 10 were cases of meningitis or meningoencephalitis. What is interesting is that in 8 of these 10 cases, the patients had a history of prior skull trauma or neurosurgery, as was the case with the patient described in the present case report. To the best of our knowledge, this is the third case of A. ureae infection reported in South Africa; the others were in a patient with septicemia reported from Tygerberg Hospital, Cape Town (14), and in a patient with meningitis reported from Johannesburg (9).

As expected, there was also significant sequence identity between the nucleotide sequence of the A. ureae strain isolated from the patient described here and other members of the genus Actinobacillus, such as A. suis (97%), A. equuli (96%), A. capsulatus (96%), A hominis (95%), and A pleuropneumoniae (92%). However, these organisms are all commensal organisms in various animals. They have rarely, if ever, been isolated from human clinical samples, and when they have been isolated, they have been isolated only in association with injuries caused by the respective animal host (2, 4, 12, 16). In addition, there are some distinct phenotypic differences between the other Actinobacillus species and A. ureae (Table 2). Given these differences, as well as the clinical features of the patient described here, it is unlikely that the isolate from the patient is one of the commensal organisms of the Actinobacillus genus in animals.

TABLE 2.

Phenotypic characteristics of various members of the genus Actinobacillus^a

Characteristic	Result or reaction for:
	CSF isolate	A. ureae	A. suis	A. equuli	A. capsulatus	A. hominis	A. pleuropneumoniae
Hemolysis (SBA)	None	None	Beta	None	None	None	Beta
Growth (MAC)	No	No	Yes	Yes	Yes	No	No
Catalase	Pos (weak)	Pos	Pos	Var	Pos	Neg	Var
Oxidase	Pos	Pos	Var	Var	Pos	Pos	Var
ONPG	Neg	Neg	Var	Var	Pos	Pos	Pos
Acid from:
Glucose	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Maltose	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Sucrose	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Lactose	No	No	Var	Yes	Yes	Yes	Var

^aData are from Ashhurst-Smith et al. (2), Holt et al. (8), Koneman et al. (12), and Peel et al. (16). Abbreviations: SBA, sheep blood agar; MAC, MacConkey agar; ONPG, ortho-nitrophenol-β-d-galactopyranoside; Var, variable reaction; Pos, positive; Neg, negative.

Descriptions of A. ureae suggest that it does not require X factor for growth (8, 12). The isolate described here is therefore unusual; as with the isolate described by Grewal et al. (7), it initially appeared to require X factor. The isolate from our patient was shown to be able to synthesize protoporphyrin intermediates from ALA, indicating that it is not dependent on exogenous X factor. The ALA-porphyrin test is considered to be a better indicator of a requirement for X factor since tests with discs impregnated with either X or V factor have been found to give variable results, depending on the nature of the medium used (13, 18). There is no report that the ALA-porphyrin test was conducted with the isolate described by Grewal et al (7).

Molecular biology-based techniques have proved useful for the detection and identification of organisms that are difficult or impossible to culture in vitro (11). In addition, analysis of DNA sequences has been used to name organisms that were unidentifiable by phenotypic testing (5). Although such molecular biology-based identification techniques are unlikely to supersede traditional phenotypic testing in a clinical laboratory, the approach described in this report can be considered an alternative when dealing with a phenotypically unidentifiable organism that is clinically significant. The molecular biology-based techniques of PCR and analysis of DNA sequence data are simple and within the scope of many clinical laboratories.

Nucleotide sequence accession number.

The nucleotide sequence data have been deposited in the GenBank nucleotide sequence database under accession number AF397302.