Sharma et al. BMC Microbiology 2013, 13:97 http://www.biomedcentral.com/1471-2180/13/97 RESEARCH ARTICLE Open Access Phenotypic and genotypic properties of Microbacterium yannicii, a recently described multidrug resistant bacterium isolated from a lung transplanted patient with cystic fibrosis in France Poonam Sharma1, Seydina M Diene1, Sandrine Thibeaut1, Fadi Bittar1, Véronique Roux1, Carine Gomez2, Martine Reynaud-Gaubert1,2 and Jean-Marc Rolain1* Abstract Background: Cystic fibrosis (CF) lung microbiota consists of diverse species which are pathogens or opportunists or have unknown pathogenicity. Here we report the full characterization of a recently described multidrug resistant bacterium, Microbacterium yannicii, isolated from a CF patient who previously underwent lung transplantation. Results: Our strain PS01 (CSUR-P191) is an aerobic, rod shaped, non-motile, yellow pigmented, gram positive, oxidase negative and catalase positive bacterial isolate. Full length 16S rRNA gene sequence showed 98.8% similarity with Microbacterium yannicii G72T type strain, which was previously isolated from Arabidopsis thaliana. The genome size is 3.95Mb, with an average G+C content of 69.5%. In silico DNA-DNA hybridization analysis between our Microbacterium yannicii PS01isolate in comparison with Microbacterium testaceum StLB037 and Microbacterium laevaniformans OR221 genomes revealed very weak relationship with only 28% and 25% genome coverage, respectively. Our strain, as compared to the type strain, was resistant to erythromycin because of the presence of a new erm 43 gene encoding a 23S rRNA N-6-methyltransferase in its genome which was not detected in the reference strain. Interestingly, our patient received azithromycin 250 mg daily for bronchiolitis obliterans syndrome for more than one year before the isolation of this bacterium. Conclusions: Although significance of isolating this bacterium remains uncertain in terms of clinical evolution, this bacterium could be considered as an opportunistic human pathogen as previously reported for other species in this genus, especially in immunocompromised patients. Keywords: Cystic fibrosis, Antibiotic resistance, Multidrug resistant bacteria, Microbacterium yannicii Background regulator gene [1]. Although CF is a multisystemic disease, Cystic fibrosis (CF) is one of the most common inherited the clinical picture is generally dominated by pulmonary autosomal recessive disease in the Caucasian population. involvement, the main cause of morbidity and mortality in It is due to mutations in the product of the gene encoding this disease. Lung disease is characterized by recurrent the CF transmembrane conductance regulator (CFTR), and alternative cycles of airway infection and inflam- resulting in chloride channel dysfunction conductance mation, leading to bronchiectasis and subsequently to respiratory failure where lung transplantation may consti- * Correspondence: [email protected] tute the ultimate therapeutic option [2]. Infections in CF 1Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergents (URMITE), CNRS-IRD, UMR 6236, Méditerranée Infection, Faculté de Médecine patients are considered to be polymicrobial [3]. The pa- et de Pharmacie, Aix-Marseille Université, 27 Bd Jean Moulin, Marseille Cedex thogens which are traditionally involved include Pseudo- 05 13385, France monas aeruginosa, Staphylococcus aureus, Haemophilus Full list of author information is available at the end of the article © 2013 Sharma et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sharma et al. BMC Microbiology 2013, 13:97 Page 2 of 11 http://www.biomedcentral.com/1471-2180/13/97 influenzae and Burkholderia cepacia complex [4-7]. Many Pseudomonas aeruginosa and Burkholderia multivorans studies have shown that the community of microbes colonization along with appearance of Aspergillus fumi- present in the airway of CF patients is more diverse and gatus. During hospitalization in the ICU, probabilistic complex than previously thought [3,8-10]. Many new, antibiotherapy consisted of an association of ceftazidime, emerging and/or multidrug resistant bacteria have been tobramycin and inhaled colistin. After an initial im- recently reported in CF patients using different technolo- provement, despite she still required oxygenotherapy de- gies including new culture media and molecular methods vice and intermittent noninvasive ventilation support, [3,8,11,12]. In this study, we report the isolation and full her respiratory function worsened on January 2011. A description of Microbacterium yannicii isolated from the sputum sample was collected on January 7th, in which sputum sample from a lung transplanted CF adult patient multiresistant Pseudomonas aeruginosa and Burkhol- for which we have recently published the genome se- deria multivorans were isolated on chocolate Poly ViteX quence [13]. Microbacterium yannicii G72T the reference agar (bioMérieux, Marcy l’Etoile, France) and cepacia type strain isolated from surface sterilized roots of agar (AES laboratory, Combourg, France), respectively. Arabidopsis thaliana was used for comparison [14]. The An atypical gram positive strain was isolated at 105 genus Microbacterium was first proposed in 1919 [15]. CFU/ml on Columbia CNA agar plate. A treatment with Microbacterium sp. belongs to the family Microbacteria- ceftazidime, temocillin and inhaled colistin was started ceae [16,17], order Actinomycetales, class Actinobacteria again. Her respiratory function continued to deteriorate [17] which comprises mainly aerobic Gram positive bac- and she died after 2 months in a septic clinical condition. teria with high G+C content and a peptidoglycan defined by a B-type cross linkage [18]. Based on phylogenetic pro- Results perties and chemotaxonomic features, the genera Micro- Phenotypic features bacterium and Aureobacterium were unified to form the The gram positive strain was isolated on Columbia redefined genus Microbacterium in 1998 [19]. From mid colistin-nalidixic acid CNA agar with 5% sheep blood 1990s, the presence of Microbacterium was recognized in (bioMérieux), after 24 hours of incubation at 37°C with human clinical specimens [20-22]. However, to the best of 5% CO2 (Figure 1A,1B,1C). It also grew on COS me- our knowledge, bacteria of this genus have never been dium at 29°C after 24 hours. The colonies are 0.1- reported in clinical samples from CF patients. Here, we 0.2 mm in diameter. The isolate was an aerobic, yellow present a full description of phenotypic and genomic pigmented (Figure 1A), rod-shaped, non-motile, oxidase properties of this new bacterium isolated from a CF spu- negative and catalase positive bacterium. This strain was tum sample. able to grow in microaerophillic atmosphere but not in anaerobic atmosphere. It also grew very weakly at a salt Case report concentration of up to 10% after 48 hours of incubation. A 23-year-old woman who has been lung transplanted As the spectrum for Microbacterium yannicii was not for CF (heterozygote delta F508/1717-1G genotype) was available in the Bruker database at the time of our strain admitted in emergency in November 2010 in our me- isolation, we were not able to identify correctly and after dical department for acute respiratory failure in the the addition of Microbacterium yannicii G72 type strain context of uncontrolled CF-related diabetes with keto- spectrum in our local database, our strain was identified acidosis coma. She required rapidly mechanical venti- as Microbacterium yannicii with a low score (Score 1.3). lation support, adapted metabolic adjustment, diabetes Hence, we proceeded with 16SrRNA sequencing for management, and nutritional supporting care. Her med- precise identification. Table 1 outlines the results of ical history included long term colonization by multi the commercial phenotypic tests done which includes drug resistant Pseudomonas aeruginosa and Burkhol- apiCoryne, apiCH-50 and apiZYM (BioMerieux, Marcy deria multivorans. She had undergone bilateral lung l’Etiole, France) tests to distinguish the CF clinical isolate transplantation when she was 19 years old, and 2 years from five type strains of the genus Microbacterium in- later, she developed progressive chronic lung allograft cluding Microbacterium yannicii G72T DSM 23203, dysfunction (CLAD) with a bronchiolitis obliterans syn- Microbacterium trichothecenolyticum DSM 8608, Micro- drome (BOS) stage 3 since the last 6 months and a bacterium flavescens DSM 20643 and Microbacterium respiratory insufficiency requiring oxygen supplement hominis DSM 12509. In apiZYM, the enzymatic reaction 2 months before the admission. The immunosuppressive for β-glucuronidase was positive for CF Microbacterium regimen on admission consisted of tacrolimus (trough yannicii PS01 as well as Microbacterium yannicii G72T level around 8 to 10 ng/ml), mycophenolate mofetil (DSM 23203). Although some of the biochemical tests (500 mg twice daily) and azithromycin 250 mg daily for for our strain yielded results similar to those reported BOS for more than one year. The worsening of respi- for M. yannicii G72 type strain [14], however, we found ratory function was associated with the persistence of at least nine differences between our isolate and the type Sharma et al. BMC Microbiology