Journal de Mycologie Médicale 16 (2006) 47–50
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m y c m e d
CAS CLINIQUE/CASE REPORT
Nocardia cyriacigeorgica: first case reported in Mexico Nocardia cyriacigeorgica : le premier cas rapporté au Mexique N.L. Sánchez-Saucedo, H. Sandoval *, N. Ramírez-Durán, K. Sánchez-Herrera Universidad Autónoma Metropolitana-Xochimilco, Calzada del Hueso 1100 col, Villa Quietud, C.P. 04960, México, D.F. México Reçu le 20 juillet 2005 ; accepté le 23 novembre 2005 Disponible sur internet le 20 février 2006
KEYWORDS Mycetoma ; Nocardia cyriacigeorgica ; Nocardiosis
Abstract Nocardia have been reported as opportunistic microorganisms in hospitals; they are aerobic, gram positive, branched and saprophytic microorganisms of the soil. This highly heterogeneous group is associated with infectious diseases of the human skin such as micetoma or nocardiosis, the latter being either pulmonary or cutaneous. Given the large number of new species, traditional molecular biology diagnostic techniques are no longer suitable for the identification of these organisms. Many of these groups are biochemically similar rendering species identification with conventional methods uncertain. This paper describes the first case of isolation of Nocardia cyriacigeorgica reported in Mexico. The strain was obtained from a lung pneumonia infection biopsy on a 2-year old child in the Children’s Hospital of Mexico, “Federico Gómez”. The characterization of this strain was performed using the molecular technique 16S rRNA gene analysis. The isolated strain was identified as N. cyriacigeorgica with 98% of nucleotide homology.
© 2006 Elsevier SAS. Tous droits réservés.
MOTS CLÉS Mycétome ; Nocardia cyriacigeorgica ; Nocardiose
Résumé Les Nocardia ont été rapportés comme agents opportunistes dans les hôpitaux. Ce sont des micro-organismes ramifiés, saprophyte du sol, à métabolisme aérobie strict, à coloration Gram positive. Ce groupe très hétérogène est associé à des maladies humaines infectieuses comme le mycétome ou la nocardiose, cette dernière pouvant être pulmonaire ou cutanée. Les méthodes diagnostiques traditionnelles de biologie moléculaire pour l’identification de ces organismes sont inutiles devant le grand nombre de nouvelles espèces rapportées. Beaucoup de ces espèces sont par ailleurs très semblables du point de vue biochimique. Cette communication est le premier cas d’isolement de Nocardia cyriacigeorgica rapporté au Mexique ; la souche a été obtenue d’une biopsie pulmonaire chez un enfant de deux ans atteint de pneumonie, à l’hôpital des enfants de Mexico « Federico-Gómez ». La caractérisa-
* Corresponding
author. Adresse e-mail :
[email protected] (H. Sandoval).
1156-5233/$ - see front matter © 2006 Elsevier SAS. Tous droits réservés. doi:10.1016/j.mycmed.2005.11.002
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tion de cette souche a été réalisée par séquençage du gène de l’ARNr 16S. On a pu identifier la souche isolée comme N. cyriacigeorgica avec 98 % d’homologie.
© 2006 Elsevier SAS. Tous droits réservés.
Introduction The members of the Nocardia genus are aerobic, gram positives, branched and saprophytic microorganisms of the soil. This group is associated to skin infectious human diseases as a mycetoma or as nocardiosis, this last can be pulmonary or cutaneous [4]. Nocardia has been reported as opportunistic microorganisms in hospitals. Their incidence on organ transplanting surgeries is a risk infection factor, because the immunosuppresing therapy favors the Nocardia infections [6,12,13,16]. Nocardia also can be found on AIDS [7], diabetic or cancer patients. Using the 16S rRNA gene sequencing technique Kageyama [11], have reported 303 Nocardia human infectious diseases found in Japan from 1992 to 2001. The frequency distributions of these were: N. farcinica (81); N. asteroides (72); N. brasilensis (66); N. nova (3); N. cyriacigeorgica (31); N. otitidiscaviarum (14); N. transvaliensis (3); N. beijingensis (2) and N. pseudobrasilensis (1). N. cyriacigeorgica, N. transvaliensis and N. pseudobrasiliensis, were first reported in Japan as associated infections to the immunosuppressive drug therapy, used on cancer, diabetes and AIDS diseases. Among the Actinomycetales, Nocardia is a highly heterogeneous group. The traditional diagnostic methods for these organisms identification are unclear in front to the new species reported by molecular biology techniques. Currently the Nocardia genus has 48 reported species on different taxonomic status. Since the 2000, the Approved List of Bacterial Name of the International Committee of Systematic Bacteriology has reported 16 new species [1]. This paper is the first case of isolation of N. cyriacigeorgica reported in Mexico. The strain was obtained from a lung pneumonia infection biopsy on a two years old child from the Children’s Hospital of Mexico “Federico Gómez”. Former strain isolation of N. cyriacigeorgica reported cases are: the new species proposal by Yassin [20], obtained from the bronchial secretions of a chronic bronchitis patient. And as part of an invasive infection of a cerebral wound of an inmunosuppressed patient, reported by Fux [9]. Species identification was performed using the DNA analysis of the 16S ribosomal gene, on both papers.
Materials and methods The initial isolation of the bacteria from a pulmonary biopsy was in blood agar medium, afterwards the strain was conserved in BHI agar (Brain Heart Infusion, Bioxon) under the code “strain 43M”. The observed colonies were white, with irregular edges, dusty appear and flattened in shape. These have shown an abundant and fast growth at 37 °C during 24 hours. The biomass for the tests was prepared utilizing BHI broth, incubated at 37 °C in an orbital incuba-
tor. At 48 hours the biomass was washed with saline solution and prepared for the tests. The preliminary tests were hydrolysis of xanthin, hypoxanthine and tyrosine at 0.5%, and casein at 1%, [18]. An antibiotic susceptibility test was performed using the disc diffusion technique (Kirby-Bauer). The antibiotics tested were polimyxin (300 UI), rifampicine (30 μg), piperacillin (100 μg), carbenicillin (100 μg), bacitracin (10 UI), cefotaxime (30 μg), vancomycin (10 μg), erythromycin (15 μg), ceftaxidime (30 μg), amikacyn (30 μg), minociclyne (30 UI) and cefalotine (30 μg). Biochemical tests were performed using the bioMérieux identification system API-20E, a colony from BHI agar was dispersed in 5 ml of isotonic saline solution. Once a homogeneous suspension was obtained, the galleries were inoculated and incubated at 37 °C during 24 hours. The determination of diaminopimelic acid (DAP) was done by hydrolysis of 10 mg of dry biomass performed by 200 μl of HCl 6N at 100 °C, during 20 hours. The hydrolyzed product was injected in a Beckman 6300. Aminoacid Automated Analyzer. The DAP enantiomer determination was performed by thin layer chromatography. The determination of fatty acids follow the Minnikin [14] technique, 10 mg of dry biomass was methanolyzed, the methylic esters were injected to a Gas Chromatograph Varian GC– CP 3380. It has a 170 °C programmed 1079 injector and a DB–5 phenyl silicon 0.32 mm X 30 mm X0.5 um thick layer capillary column. The mycolic acids were determined by double dimension thin layer chromatography in silica gel plates (Sigma) DNA extraction was performed following the instructions of the “Wizard Genomic DNA Purification Kit” (Promega A 1120). Amplification of the rRNA 16S [5] was carried out using the method of the polymerase chain reaction (PCR). The sequences of utilized primers were: 8f: AGAGTTTGATCMTGGCTCAG and 1492r: TACGGYTACCTTGTTACGACTT (Sigma Genosys). The reaction was carried out using Taq DNA Polymerase in Storage Buffer B (Promega M1661). The utilized parameters of reaction were: a 5 minutes cycle of pre-denaturation at 94 °C; denaturation 30 seconds at 94 °C; annealing 20 seconds at 52 °C; extension 90 seconds at 72 °C. After 34 cycles a final cycle of post-extension for 7 minutes at 72 °C. The amplified fragments were checked by an agarose gel electrophoresis at 1%. For the PCR 16S reaction products purification, the Microcon PCR Filter Units system (Millipore C 7480), was utilized. The products of the amplification reaction of the 16S gene were observed trough a 1% electrophoretic agarose gel runaway. The obtained PCR 16S gene product was sequentiated and compared with the corresponding GenBank registered sequences.
Nocardia cyriacigeorgica: first case reported in Mexico
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Results
Table 1 Susceptibility to antibiotics showed by strain 43M Sensibilité aux antibiotiques de la souche 43M.
The strain is gram positive, highly branched large filaments with further fragmentation to rods and cocobacilli. Colonies are white with dusty appearance, wrinkle surface, irregular edges that exhibit an abundant growth within 24 hours. The xanthine, hypoxanthine, tyrosine and casein hydrolysis, were negatives. The API–20 E identification system results were only positives for glucose and rhamnose. The strain is highly resistant to antibiotics, only susceptible to the amikacin and the minocycline with intermediate susceptible to the cefamandole (Table 1). The cytochemical analysis indicates presence of meso– diaminepimelic acid (meso-DAP), in the cell wall. The fatty acids analysis results reports the presence of tuberculoestearic acid and mycolic acids. The 16S rRNA gene sequencing gives a 98% homology towards N. cyriacigeorgica, with a 744/756 ratio of nucleotides identity.
Antibiotic susceptibility Polimixin(300UI) Rifampicin (30 μg) Piperacillin (100 μg Carbencillin (100 μg Bacitracin (10 UI) Cefotaxime (30 μg) Vancomycin, (10 μg) Erythromycin (15 μg) Ceftaxidime (30 μg) Amikacin (30 μg) Mynocycline (30 UI) Cefalotine. (30 μg) Ciprofloxacin (30 μg) Cefamandole (30 μg)
Discussion Until now it does not exist a fast identification system for pathogenic Actinomycetes, because the Substrates Assimilation Galleries, the enzymatic tests [2,3,15] and the fluorogenic substrates [10], described as routinely tests for the taxonomic identification of different species of Actinomycetes, are not adequately incorporated to the customary clinical laboratory routines.
R R R R R I R R R S S I I I
R : resistance ; S : susceptible ; I : intermediate.
Generally Nocardia species are partially acid resistant however our strain is not acid fast. According to results obtained by Yassin [20], our strain could be considered atypical because exhibits a poor metabolic activity and is not acid fast. Because the studied strain can be misidentified by similarity, respect to the members of the highly heterogeneous Group VI of N. asteroides strains, from which have emerged new species [17], only by the molecular technique of the 16S rRNA gene analysis applied in this study, was possible to identify the isolated strain as N. cyriacigeorgica with a
Figure 1 Phylogenetic tree of some Nocardia species showing clearly the position of strain 43M in the N. cyriacigeorgica cluster. Figure 1 Arbre phylogénétique de quelques espèces de Nocardia montrant précisément la position de la souche 43M dans le cluster N. cyriacigeorgica.
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98% of nucleotide homology (Fig. 1), being the first report of this species in México. According to Wallace et al [19], the Nocardia group heterogeneity has consent a new taxon formation that can derive on new species. Some authors have been considered to N. asteroides, not only as group “sensu stricto”, but as a complex cluster of a variety of pathogenic bacteria. All those have been identified by the test combinations and the 16S rRNA gene sequencing [8]. N. cyriacigeorgica has been associated to immunodeficiency organ infections diseases. According to the atypical characteristics of N. cyriacigeorgica, is not possible to identify this bacteria by conventional biochemical tests, therefore the only way is through the molecular identification techniques.
[9]
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[11]
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