"Actinobaculum massiliae," a New Species Causing Chronic Urinary Tract Infection

We report on a new Actinobaculum species, "Actinobaculum massiliae,"isolated from the urine of an elderly woman with recurrent cystitis.Its phenotypic pattern was similar to those of both of the otherActinobaculum species described to date. On 16S rRNA sequencing,the Marseille isolate shared 95% homology with Actinobaculumsuis, 92 to 93% homology with Actinobaculum schaalii, 91 to92% homology with Arcanobacterium spp., and 87 to 90% homologywith Actinomyces species. A bootstrap value of 99% supportsthe node separating the Actinobaculum sp. from its closest neighbor(A. suis). In conclusion, on the basis of phenotypic, genotypic,and phylogenetic assessments, we show that the Marseille isolateis a previously unrecognized organism within the Actinobaculumgenus, and we propose placement of the organism in the taxon"Actinobaculum massiliae."

INTRODUCTION

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Introduction

Actinobaculum suis, initially described as Eubacterium suis(7), was reclassified as Actinomyces suis in 1992 (4). Its taxonomywas further modified in 1997 by Lawson et al. (3), who proposeda new genus, Actinobaculum, that includes two species: Actinobaculumschaalii and A. suis. A. suis is pathogenic for sows, leadingto cystitis (6) and abortion (9). The type strain of A. schaaliiwas isolated from the blood of a 64-year-old man with chronicpyelonephritis (3), suggesting that it may be an agent of urinarytract infection. Here we report on a new Actinobaculum speciesthat we named "Actinobaculum massiliae." The organism was isolatedfrom the urine of an elderly woman with recurrent cystitis.

CASE REPORT

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Case report

An 81-year-old woman presented with brachiofacial ischemic strokein the summer of 1999, and a bladder catheter was placed. Thecatheter was associated with cystitis and was removed 1 monthlater. A gram-positive bacillus was the only bacterium isolatedfrom her urine at that time. This bacterium, recovered by aprivate laboratory and thought to be a vaginal contaminant,was not identified, nor was it tested for its antibiotic susceptibility.Empirical therapy with amoxicillin-clavulanate was given for2 weeks, with partial and temporary resolution of the symptoms.Repeated episodes of pollakiuria with urge urinary incontinencewere reported starting in March 2000. In May 2001, a urine culturewas done when symptoms of cystitis, i.e., dysuria and pollakiuria,reappeared. At that time, a gram-positive bacillus was againisolated from the patient's urine, but this time it was identifiedas an Actinobaculum sp. by 16S rRNA amplification and sequencing.It was found to be susceptible to all antibiotics used for thetreatment of lower urinary tract infection. Despite successivetherapy with amoxicillin and trimethoprim-sulfamethoxazole,in October 2001 the same organism was recovered from the patient'surine at a high concentration (10⁶ bacteria/ml), and it wasthen resistant to trimethoprim-sulfamethoxazole. Rifampin therapy(900 mg/day) initiated in October 2001 failed, and it was laterassociated with in vitro resistance to rifampin (December 2001)and persistence of the pollakiuria. Cure was finally achievedwith doxycycline.

MATERIALS AND METHODS

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Materials and Methods

The strain isolated on sheep blood agar from the patient's urinein May 2001 was used for phenotypic, genotypic, and phylogeneticanalyses. Growth requirements and colony morphology were studiedwith cultures set up on Columbia sheep blood agar (BioMérieux,Marcy l'Etoile, France) and chocolate agar (BioMérieux)and incubated at 37°C in a 5% CO₂ atmosphere. In addition,we tested for the presence of growth under anaerobic conditionson sheep blood agar at 37°C (GENbag anaer; BioMérieux),and anaerobic conditions were confirmed by using the AnaerIndicatorstrip (Merck, Darmstadt, Germany). A mobility test was performedby the hanging-drop method. Oxidase and catalase activitieswere evaluated by routine procedures. Additional biochemicaltests were performed by inoculation of the API 20Strep (BioMérieux)and API Coryne (BioMérieux) systems according to theinstructions of the manufacturer, with the exception that theincubation time was increased to 72 h. The cell wall fatty acidcomposition was analyzed by gas chromatography (5).

DNA was extracted with the FastDNA kit (Bio 101, Carlsbad, Calif.)and a FastPrep120 grinder (Bio 101) according to the instructionsof the manufacturer. PCR amplification of the 16S RNA gene wasperformed with primers fD1 and rP2 (8) and Taq DNA polymerase(Gibco BRL, Life Technologies), also as specified by the manufacturer.The success of the amplification was determined by electrophoresisof the PCR products in a 1% agarose gel and staining with ethidiumbromide. The PCR products were purified by use of the QIAquickPCR purification kit (Qiagen, Courtaboeuf, France). Sequencingwas performed by using the dRhodamine Terminator Cycle SequencingReady Reaction kit with one of nine different primers and AmpliTaqDNA (Perkin-Elmer Biosystems, Warrington, England) with a 3100ABI Prism automated sequencer (Applied Biosystems, Courtaboeuf,France). Sequences derived from each primer were aligned, compared,and combined into a single 16S rRNA sequence by using Autoassemblersoftware (version 2.1; Applied Biosystems). The validity ofthe sequence obtained was assessed by comparison with two additionalsequences similarly obtained, but from two other PCRs of thesame template DNA. The sequence was compared with all eubacterial16S rRNA sequences available in the GenBank database by usingthe BLASTN 2.2.2 program available on the National Center forBiotechnology Information website (www.ncbi.nlm.nih.gov) (1).The 16S rRNA sequence of the Marseille isolate was aligned withthose of other members of the family Actinomycetaceae, and thedegree of 16S rRNA homology was determined by using the CLUSTWALW program supported by the DDBJ website (www.ddbj.nig.ac.jp).Sequences were edited by removal of the longer 5' and 3' endsso that their lengths matched that of the shortest sequence.The edited sequences were then analyzed by the distance matrixprogram (Kimura's correction) supported by the DDBJ website.With these sequences, additional trees were constructed by usingthe distance matrix, parsimony, and maximum likelihood programsof the Phylip package (2). The isolates recovered in Octoberand December were identified by 16S rRNA amplification and sequencingof a segment of about 500 bp with the following primers: 5'-CAGCAGCCGCGGTAATAC-3'and 5'-CACGAGCTGACGACA-3'.

Nucleotide sequence accession number. The 16S rRNA gene sequence of the Marseille "A. massiliae" isolatehas been deposited in the GenBank database under accession numberAF487679.

RESULTS

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Results

Primary isolation was obtained by inoculation of sheep bloodagar (BioMérieux) with 10 µl of midstream urineand incubation at 37°C in a 5% CO₂ atmosphere for 72 h.No growth was observed when urine was inoculated on bromocresolpurple lactose agar (Becton Dickinson, Meylan, France). Nonhemolyticsmall greyish colonies could be seen on blood agar; the strainpresented two colony types after 5 days of incubation. Microscopicexamination showed nonmotile, non-acid-fast, non-spore-forming,gram-positive organisms. Some bacteria were poorly stained withthe Gram stain, and a few exhibited branching. Growth was observed,although it was slower, under anaerobic conditions and on chocolateagar. Green hemolysis was observed with culture on chocolateagar. Bacteria grown on the latter medium were tested for catalasereactivity, which was absent. Acid was produced from glucose,maltose, ribose, xylose, and trehalose, but not from mannose,mannitol, sorbitol, or amidon. Slight acid production from raffinosewas observed. Esculin and gelatin were not hydrolyzed. Nitratewas not reduced to nitrite. Hippurate was hydrolyzed. Pyrazinamidaseand ${alpha}$ -glucosidase activities were detected. Inversely, no pyrrolidonylarylamidase, alkaline phosphatase, ${alpha}$ -galactosidase, ß-glucuronidase,ß-galactosidase, N-acetyl-ß-glucosaminidase,urease, leucine arylamidase, or arginine dihydrolase activitywas detected (Table 1). The whole-cell fatty acid compositionof the Marseille isolate demonstrated relatively large amountsof hexadecanoic acid (C_16:0; 41.5%), cis-octadec-9-enoic-acid(C_18:19c; 24.2%), and octadecanoic acid (C_18:0; 8.7%). The Marseilleisolate shared 95% homology with A. suis, 93% homology withA. schaalii, 91 to 92% homology with Arcanobacterium spp., and87 to 90% homology with Actinomyces species (Table 2). The shortest16S rRNA sequence included in the phylogenetic analysis wasthat of Actinomyces turicensis (1310 bp); accordingly, alignmentbegan at base 1 of the 16S rRNA gene of A. turicensis. The Marseilleisolate clustered with the Actinobaculum spp. by phylogeneticanalysis (matrix distance approach) of the 16S rRNA genes ofspecies of the family Actinomycetaceae (Fig. 1). Thus, a bootstrapvalue of 97% in the neighbor-joining tree supported the forkseparating the Actinobaculum spp. (including the Marseille isolate)from their closest relatives: members of the genus Arcanobacterium.More importantly, the isolate was clearly different from itsclosest neighbor (A. suis), with a bootstrap value of 99% (Fig.1). Parsimony and maximum likelihood methods confirmed thatthe Marseille isolate clustered within the Actinobaculum spp.and that its lineage was clearly different from that of A. suis.The isolates recovered in October and December 2001 shared 100%sequence homology with the type strain, recovered in May 2001from urine specimens from the same patient.

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TABLE 1. Comparison of the "A. massiliae" phenotype with those of other members of the family Actinomycetaceae, showing its relatedness to other Actinobaculum spp.^a

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TABLE 2. Comparison of 16S rRNA sequence homology of "A. massiliae" with those of other members of the family Actinomycetaceae and Corynebacterium amycolatum

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FIG. 1. Phylogenetic tree showing relationship of "A. massiliae" sp. nov. with other members of the family Actinomycetaceae. The tree was constructed by using the neighbor-joining method, based on the nearly complete sequence (1,310 nucleotides) of the 16S rRNA gene. Bootstrap values resulting from 100 replications are presented at the branch points. Denitrobacterium detoxifians was used as the outgroup.

DISCUSSION

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Discussion

In this paper, we described a new species, "A. massiliae," recoveredfrom the urine of an 81-year-old woman with a chronic urinarytract infection. The same strain was recovered three times overa 7-month period, highlighting the persistence of the infectiondespite antibiotic treatment. Interestingly, this strain wasnot recovered from bromocresol purple lactose agar (Becton Dickinson),which is the medium routinely used in the Marseille laboratory,even after prolonged incubation. Our use of sheep blood agarand prolonged incubation, which permitted strain recovery, wasmotivated by the presence of 10⁶ leukocytes/ml in the patient'surine. This is the second report of a human infection due toActinobaculum species: the A. schaalii type strain was isolatedfrom a 64-year-old man with chronic pyelonephritis (3). Of interest,A. suis has been isolated from sows with cystitis (6). It shouldbe stressed that chronic urinary tract infection due to Actinobaculumspecies might be underdiagnosed because these organisms cannotbe isolated by routine procedures. In addition, the phenotypicheterogeneity of the colonies after 5 days of incubation maylead to an erroneous interpretation of sample contaminationwith two different species of gram-positive bacilli. The infectiondescribed in the present study was cured only with doxycycline,which may be the treatment of choice for such infections. Asrecurrence occurred after therapy with amoxicillin and trimethoprim-sulfamethoxazoleand as resistance to rifampin was observed after exposure tothat drug, close follow-up with stringently controlled urinecultures seems mandatory to confirm a cure.

The phenotypic and biochemical patterns suggested that the Marseilleisolate was related to A. schaalii. Indeed, tests for ureaseand ß-galactosidase activities were found to be negative,and acid was produced from glucose, maltose, ribose, and xylose.However, as for A. suis, acid was produced from glycogen. Inaddition, the Marseille isolate showed only 95% 16S rRNA sequencehomology with A. suis and showed even lower degrees of homologywith all other known species of the family Actinomycetaceae.Our phylogenetic analysis demonstrated that the Marseille isolateshares some degree of evolutionary descent with members of thegenus Actinobaculum. In conclusion, on the basis of phenotypic,genotypic, and phylogenetic assessments, we show that the Marseilleisolate is a previously unrecognized organism within the genusActinobaculum, and we propose placement of the organism in thetaxon "Actinobaculum massiliae."

Description of "A. massiliae" sp. nov. Massilia is the old Greek and Roman name for Marseille, wherethe organism was isolated. The organism consists of straightto slightly curved rods, some of which exhibit branching. Ithas nonmotile, non-acid-fast, gram-positive (easily decolorized)cells. Colonies on sheep blood agar are 1 mm in diameter after72 h of incubation at 37°C in a 5% CO₂ atmosphere, and thereis no hemolysis. It is facultatively aerobic. Esculin and gelatinwere not hydrolyzed. Nitrate was not reduced to nitrite. Acidwas produced from glucose, maltose, ribose, glycogen, and xylose.The type strain of "A. massiliae," which was isolated from theurine of an 81-year-old woman with cystitis, has been depositedin the Collection de l'Institut Pasteur, Paris, France, as strainCIP 107404^T.

ACKNOWLEDGMENTS

We thank Christiane Bibard and Veronique Brice for help.

FOOTNOTES

* Corresponding author. Mailing address: Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Université de la Méditerranée, 27, Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Phone: (00) 33.491.32.43.75. Fax: (00) 33.491.83.03.90. E-mail: didier.raoult{at}medecine.univ-mrs.fr.

REFERENCES

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