Isolation of Lautropia Mirabilis from Sputa of a Cystic Fibrosis Patient SUSAN M

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JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1997, p. 1024–1026 Vol. 35, No. 4 0095-1137/97/$04.00ϩ0 Copyright ᭧ 1997, American Society for Microbiology

Isolation of Lautropia mirabilis from Sputa of a Cystic Fibrosis Patient SUSAN M. BEN DEKHIL,1 MARGARET M. PEEL,2 VIRGINIA A. LENNOX,3 1 1 ERKO STACKEBRANDT, † AND LINDSAY I. SLY * Australian Collection of Microorganisms, Centre for Bacterial Diversity and Identiﬁcation, Department of Microbiology, The University of Queensland, Brisbane,1 Microbiological Diagnostic Unit, Department of Microbiology, The University of Melbourne, Melbourne,2 and Department of Microbiology, Royal North Shore Hospital, Sydney,3 Australia

Received 24 September 1996/Returned for modiﬁcation 1 November 1996/Accepted 6 January 1997

An aggregate-forming coccus, isolated twice as the predominant microorganism in sputa from a cystic fibrosis patient on consecutive days, was shown to belong to the species Lautropia mirabilis on the bases of similarities of 16S rRNA gene sequences and phenotype. These isolates of L. mirabilis appear to be the first reported from a patient with cystic fibrosis and outside of Denmark.

An aggregate-forming gram-negative coccoid bacterium was logical similarity to the gram-negative agreggate-forming coc- isolated on two separate occasions in 1991 at the Royal North cus Morococcus cerebrosus ACM 858 (3). However, the aggre- Shore Hospital, Sydney, Australia, as the predominant bacte- gate formations of the two bacteria are easily distinguishable rium in sputum samples taken on consecutive days from a (Fig. 1). Thus, at the time of isolation the strain could not be 35-year-old female patient with cystic fibrosis. The patient had assigned to any recognized species. The strain was accessioned been admitted to the hospital for intravenous antibiotic ther- into the Australian Collection of Microorganisms (Depart- apy for a chest infection in the upper lobe of both lungs. Her ment of Microbiology, University of Queensland) as strain medical history showed that she was diagnosed with cystic ACM 3763, and molecular characterization was undertaken to fibrosis at 2 years of age when she presented with a failure to thrive. She was treated with the pancreatic enzyme supplement pancrelipase (Cotazym-S-Forte; Organon Australia Pty. Ltd.) and remained healthy until she stopped taking the supplement at 22 years of age. She subsequently developed steatorrhea and insulin-dependent diabetes mellitis. At 27 years of age she recommenced treatment with the pancreatic enzyme supplement and the steatorrhea settled. Microscopic examination of a Gram-stained smear of her sputum taken on admission to the hospital showed numerous pus cells, gram-negative rods, and gram-positive cocci. A heavy growth of Haemophilus influenzae was cultured, along with a light growth of both Staphylococcus aureus and Pseudomonas aeruginosa. Burkholderia cepacia was not isolated. The patient was commenced on imipenem treatment (500 mg, 4 times daily) which was continued for 20 days. No maintenance anti- biotics were given when she was finally discharged, and the patient did not present for a follow-up examination. The aggregate-forming, gram-negative coccoid bacterium was first isolated on blood agar as the predominant microorganism from a sputum sample taken after 14 days of hospital- ization and 2 weeks of antibiotic treatment. A Gram-stain of the sputum showed moderate numbers of pus cells and gram- positive cocci only. However, the gram-negative coccus, along with a moderate background of pseudomonads, yeasts, and fungi, was cultured after 4 days of incubation. The same organism was isolated from a sputum sample taken the next day, and as the patient was not responding adequately to the imipenem treatment, further investigation of the first isolate was undertaken. Initial observation revealed superficial morpho-

* Corresponding author. Mailing address: Centre for Bacterial Di- versity and Identiﬁcation, Department of Microbiology, The Univer- sity of Queensland, Brisbane, Qld 4072, Australia. Phone: 61 7 3365 2396. Fax: 61 7 3365 1566. E-mail: [email protected]. FIG. 1. Scanning electron micrographs showing the surface appearance of † Present address: Deutsche Sammlung von Mikroorganismen und colonies of L. mirabilis ACM 3763 (A) and M. cerebrosus ACM 858 (ϭATCC Zellkulturen GmbH, Braunschweig, Germany. 33486) (B).

1024 VOL. 35, 1997 NOTES 1025

TABLE 1. Phenotypic comparison of case isolate ACM 3763, M. cerebrosus, and L. mirabilisa

M. cerebrosus ACM 858 Characteristic Case isolate ACM 3763 L. mirabilis (ϭATCC 33486) Cell morphology Gram reaction ϪϪϪ Cell shape Polymorphic cocci in clusters Polymorphic cocci in clusters Polymorphic cocci in clusters Cell size (␮m) Ͻ1 1–2 1–2 (occasionally Ͼ5)

Motility Ϫϩϩ

Polysaccharide production on sucrose agar ϩϩϩ

Growth on: Nutrient agar ϩϩϩ Blood agar ϩϩϩ Chocolate agar ϩϩϩ Sucrose agar ϩϩϩ MacConkey agar ϪϪϪ

Biochemical proﬁle Oxidase ϩϩϩ Catalase ϩϩϩ Hemolysis (horse blood) ϩϩϪ Nitrate to nitrite ϩ/Ϫϩ ϩ Nitrate to gas ϩ/ϪϪ ϩ

Production of: b ϪϪϪ H2S from TSI Phenylalanine deaminase ϪϪϪ Urease (Christensen’s) Ϫϩϩ

Decarboxylase test forc: Arginine ϪϪϪ Lysine ϪϪϪ Ornithine ϩϪϪ

Hydrolysis of: Esculin Ϫϩϩ/Ϫ Gelatin ϪϪϪ Starch ϪϪϪ Tween 80 ϪϪϪ

Acid fromd: Adonitol ϪϪϪ Arabinose ϪϪϪ Dulcitol ϪϪϪ Fructose ϩϩϩ Glucose ϩϩϩ Inositol ϪϪϪ Inulin ϪϪϪ Lactose ϪϪϪ Maltose ϩϩϩ Mannitol ϪϪϩ/Ϫ Rafﬁnose ϪϪϪ Rhamnose ϪϪϪ Salicin ϪϪϪ Sorbitol ϪϪϪ Starch ϪϪϪ Sucrose ϩϩϩ Trehalose ϪϪϪ Xylose ϪϪϪ

aData are compiled from this study and references 2 (for L. mirabilis) and 3. ϩ, positive; Ϫ, negative; ϩ/Ϫ, usually positive but sometimes negative. b TSI, triple sugar iron agar. c Moeller’s decarboxylase-dihydrolase test. d Carbohydrates in peptone base with bromcresol purple indicator. determine its phylogenetic position by comparison of its 16S PCR products were puriﬁed by means of the Prep-A-Gene kit rRNA gene (rDNA) sequence with those of known species. (Bio-Rad, Hercules, Calif.), used as described by the manufac- The extraction of genomic DNA and the ampliﬁcation of the turer. The Taq DyeDeoxy Terminator Cycle Sequencing Kit 16S rDNA was performed as described previously (1a). The (Perkin-Elmer/Applied Biosystems, Foster City, Calif.) was 1026 NOTES J. CLIN.MICROBIOL.

TABLE 2. Antimicrobial susceptibilities of case isolate in Tables 1 and 2 are in close agreement with those of L. ACM 3763, M. cerebrosus, and L. mirabilis mirabilis except for hemolysis on horse blood agar and gas Susceptibility by disk diffusion production from nitrate. The isolate was motile on isolation, but several attempts in two laboratories to repeat the demon- Case isolate Antimicrobial M. cerebrosus stration of motility were unsuccessful. ACM 3763 agent L. mirabilis L. mirabilis was described (2) to include polymorphic gram- c Disk Zone Disk Zone result negative cocci from the oral cavity and upper respiratory tract Resultb Result concn sizea concn size which met Ørskov’s (5) description of “Sarcina mirabilis,” for Ampicillin 2 ␮g8 S 2␮g10S S which no strains are extant. Strains assigned to L. mirabilis Chloramphenicol 10 ␮g11S 10␮g12S NT were isolated from the oral cavity on Bordet-Gengou plates, Erythromycin 15 ␮g9 S 15␮g10S S from a frontal sinus drain of a patient recovering from a strep- Gentamicin 10 ␮g14S 10␮g11S S tococcal sinusitis (2), and from the gingival margins of healthy Methicillin 5 ␮g0 R 5␮g0R NT persons (6). Gerner-Smidt et al. considered that although no Nalidixic acid 30 ␮g13S 30␮g12S NT evidence for pathogenicity exists, polysaccharide production Penicillin 10 U 9 S 1.5 U 10 S S from sucrose by this oral bacterium is compatible with a pos- Tetracycline 10 ␮g12S 10␮g12S NT ␮ sible role in the formation of dental plaque (2). There is also Imipenem NT 10 g12S NT no evidence at this stage that L. mirabilis is clinically significant aRadius of inhibition zone (in millimeters). in cystic fibrosis. However, its occurrence in substantial num- b S, susceptible; R, resistant; NT, not tested. bers in the sputum of the patient in this study suggests that it c As reported in reference 2. may colonize the respiratory tract and lungs of persons with cystic fibrosis. It is hoped that this paper will alert clinical microbiologists to this possibility and that an increased aware- used to directly sequence the PCR products, according to the ness of this newly described organism will assist with the eval- protocol provided by the manufacturer, and the reaction mix- uation of its clinical significance. tures were sequenced automatically on an Applied Biosys- Nucleotide sequence accession number. The rDNA se- tems model 373A DNA sequencer. The 16S rDNA sequence quence of L. mirabilis ACM 3763 determined in this study has was aligned manually against representative bacterial 16S been deposited in the EMBL Database, Cambridge, United rDNA sequences contained in the Ribosomal Database Kingdom, under the accession number X97652. Project (4). Positions where length and sequence variations made alignment uncertain were omitted from the analysis. Part of this research was funded by a University of Queensland Pairwise sequence similarities showed that the isolate be- Special Project Grant to L.I.S. and E.S., and this is gratefully acknowl- longed to the beta-subclass of the class Proteobacteria and was edged. most closely related to a gram-negative aggregate-forming coc- S.M.B.D. carried out the sequencing as part of a research project for a Master of Scientific Studies and thanks Matthias Dorsch and Fred cus isolated from the oral cavity of humans in Denmark and Rainey for training in PCR and sequencing techniques. Michael No- which was subsequently described as the type strain of Lau- lan, Royal Brisbane Hospital, is thanked for a gift of imipenem disks. tropia mirabilis in 1994 (2). Strain ACM 3763 was further characterized morphologi- REFERENCES cally, and its biochemical profile was determined by a range of 1. Ben Dekhil, S. M., E. Stackebrandt, and L. I. Sly. Unpublished data. conventional tests (2, 3) to allow a comparison with the de- 1a.Dorsch, M., and E. Stackebrandt. 1992. Some modifications in the procedure of direct sequencing of PCR amplified 16S rDNA. J. Microbiol. Methods 16: scription of L. mirabilis. These results are shown in Table 1, 271–279. along with those previously determined for M. cerebrosus (3) 2. Gerner-Smidt, P., H. Keiser-Nielsen, M. Dorsch, E. Stackebrandt, J. Ursing, and published for L. mirabilis (2). Table 2 shows their antibio- J. Blom, A. C. Christensen, J. J. Christensen, W. Frederiksen, S. Hoffmann, grams, as determined by the disk diffusion technique. W. 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