Species Spectrum of Nocardia spp . Isolated from Suspected Tuberculosis Patients

Traditionally Nocardia asteroides was considered the predominant species of causing nocardiosis. The improved identification of isolates using molecular techniques have shown that the genus exhibits considerable taxonomic complexity and phenotypic base identification can be ambiguous. The aim of this study was to assess the species distribution of Nocardia strains mostly recovered from patients suspected of having tuberculosis, during three years period (2009-2012). The clinical isolates were identified to species level using conventional tests and genotypic methods using single and multi locus sequence analysis (MLSA) of 16S rRNA, gyrB and secA1 genes. Nocardiosis was diagnosed in 46 patients. The most frequent underlying condition were organ transplantation (6 patients; 13%), cancer (6 patients; 13%), human immunodeficiency virus (HIV) (6 patients; 13%), non-infectious chronic lung disease (5 patients; 10.8%) and tuberculosis (4 patients; 8.7%). Nocardia species was recovered from 46 different clinical specimens, the most common of which was bronchoalveolar lavage (BAL) (43.5%). Eleven different Nocardia species were identified: N. asteroides (n = 12), N. cyriacigeorgica (n = 9), N. farcinica (n = 7), N. wallacei (n = 6), N. carnea (n = 3), N. otitidiscaviarum (n = 3), N. abscessus (n = 1), N. arthritidis (n = 1), N. kruczakiae (n = 1), N. nova (n = 2) and N. veterana (n = 1). In conclusion, infection caused by Nocardia species appears to be more common than generally appreciated. The current study provides further evidence that Nocardia species are capable of causing a wide range of human diseases in healthy and immunocompromised patients. MLSA is a reliable method for accurate species identification of Nocardia The GenBank accession numbers of investigated clinical isolates of Nocardia determined in this work are as follows: KC262083-KC262128, KC262130-KC262175 and KC296657-KC296702 for the 16S rRNA, gyrB and secA1, respectively. Corresponding author. A. Hashemi-Shahraki et al. 848 isolates and would be more feasible for routine use in clinical laboratories.


Introduction
Members of the genus Nocardia are aerobic, filamentous branching bacilli, gram positive and Partial acid-fast, which include more than 90 different species (http://www.bacterio.cict.fr/n/nocardia.html).The species of Nocardia normally are saprophytes of soil and thought to have a role in the decay of organic plant material.However, Nocardia have increasingly been isolated as infectious agents in immune-suppressed patients and sometimes in healthy individuals and causing localized or disseminated infections, ranging from pulmonary and wound infections to brain abscesses, and bacteremia [1].Since the most common manifestation of nocardial infection is pneumonia, patients with fever, weight loss, cough, pleuritic chest pain, and dyspnea might be confused with other chronic lung infections such as TB or invasive fungal infection [2] [3].While taxonomy within the genus Nocardia is changing rapidly as the recognition and description of new species continues, it is not easy to identify Nocardia isolates to species level by conventional methods [1] [4].In Iran, where tuberculosis is prevalent and considers as one of the most challenges of public health, the role of Nocardia as causative agent of pulmonary and other form of infections has been neglected.Due to little information available from Iran, the aim of the present study was to clarify the species spectrum of the isolates presumptively assigned to the genus Nocardia, recovered from different clinical samples of patients by morphological tests.For definite and reliable identification, the clinical isolates were subjected to sequence base identification by using MLSA of 16S rRNA [5], subunit A of the SecA preprotein translocase (secA1) and the subunit of a type II DNA topoisomerase (gyrB) [6].

Nocardia Strains
In a three-year study conducted from 2009 to 2012, 22 and 10 different isolates of Nocardia recovered from patients whom were suspected to have mycobacterium related complications in Infectious and Tropical Diseases Research Center (Khuzestan, Ahvaz, Iran) and Samyar Medical Laboratory (Tehran, Iran) respectively.At the same time 14 different clinical isolates of Nocardia from other parts of Iran (other than Tehran and Khuzestan provinces) referred to Infectious and Tropical Diseases Research Center (Khuzestan, Ahvaz, Iran) for identification.The patients' history, which the isolates were belonged, is summarized in Table 1.Most of the patients with pulmonary disease diagnosed as tuberculosis cases and put on anti-tuberculosis drugs including isoniazid and rifampicin for six months, which had either not responded to treatment or had poor, responded with subsequent relapse.

Phenotypic Identification
All Nocardia strains from respiratory samples were recovered on Löwenstein-Jensen (LJ) medium with strains from other samples, were isolated on blood agar medium.The strains were examined for growth rate, macroscopic and microscopic morphological features, growth at different temperature and a battery of biochemical tests including growth in lysozyme, growth at 45˚C, arylsulfatase (14 days), urease activity and hydrolysis of adenine, casein, esculin, hypoxanthine, tyrosine and xanthine and utilization of asetamide as the sole carbon source according to standard procedures [7].

Molecular Identification Using 16S rRNA, gyrB and secA1 Genes Sequencing
Genomic DNA was extracted from bacterial cultures using DNA isolation kit (Qiagen, CA, USA) according to the manufacturer's instructions and stored in −20˚C until use.
Nearly full length of the 16S rRNA genes from isolates were amplified using primers pA (5'-AGAG TTTGATCCTGGCTCAG-3') and pI (5'-TGCACACAGGCCACAAGGGA-3') as described previously [5].The sequences of amplified PCR products of 16S rRNA gene for each isolate were determined by using an ABI PRISM® 7700 Sequence Detection System (Applied Biosystems, CA, USA) following the standard protocol of the supplier.

Analysis of Sequence Data
The obtained sequences for each isolate from different loci were aligned separately and compared with all existing relevant sequences of Nocardia retrieved from GenBank database by using jPhydit program [8].Percentages of similarity between sequences of each gene were determined by comparing sequences search to an in-house database of 16S rRNA, gyrB and secA1 genes sequences.
Phylogenetic trees were obtained from DNA sequences by using the NJ method K2P distance correction model with 1000 bootstrap replications supported by the MEGA 4.1 software [9].To better elucidate the relatedness of the clinical isolates with valid species of Nocardia, the respective sequences of 16S rRNA, gyrB and secA1 genes were concatenated in single, 2346 bp long, strands using the jPhydit program [8].The sequences were then aligned and analyzed in the MEGA 4.1 beta program [9] with the NJ method bootstrapped with 1000 replicates.

Results
The etiologic role of the isolates might be inferred from the growing Nocardia isolates were microscopically observed in most of clinical specimens of each patient.Furthermore, in the case of specimens from non-sterile site such as sputum, repeated sampling yielded the same microorganisms in pure culture, which confirmed clinical significance of the isolates.Populations demographics have been inadequately considered because of many isolates were referred to our laboratory for identification, so it was impossible to establish the incidence rate of nocardiosis in our study.The individual characteristics of the 46 patients with nocardiosis are shown in Table 1.Nocardiosis was diagnosed in 25 (54.3%)women.Age ranged from 18 to 88 years.Most of the patients had at least one significant underlying condition.The most frequent were organ transplantation (6 patients; 13%), cancer (6 patients; 13%), HIV (6 patients; 13%), noninfectious chronic lung disease (5 patients; 10.8%) and Tuberculosis (4 patients; 8.7%).Nocardia species was recovered from 46 different clinical specimens, the most common of which were BAL (43.5%).The most common symptoms were fever (69.5%) and cough (47.8%).Chest X-ray findings included infiltrates in 17.4% of cases and cavitations in 19.6%.Nocardiosis was pulmonary in 27 of the 46 patients (58.7%), disseminated in 7 (15.2%)and central nervous system disease (brain abscess) in 4 (8.7%).

Species Identification by Phenotype Tests
The taxonomic status of ten clinical isolate, provisionally assigned as Streptomyces spp. or fungi based on macroscopic morphology but using of growth in lysozyme and modified Kinyoun staining assigned all of the isolates grouped to the genus Nocardia.According to growth characteristics and biochemical tests, the Nocardia asteroids complex like was the most common frequently encountered Nocardia species (45.6%) among clinical isolates (Table 1), but the remaining strains were identified to the genus level only.

Species Identification by Sequencing Tests
In the current study, 46 clinical strains of Nocardia with documented nocardiosis were diagnosed from 2009 to 2012.During the study, sequences of three genes were analyzed separately and then a combined database used to create a single alignment dataset.Comparative sequence analysis of 16S rRNA, gyrB and secA1 genes provided the basis for reliable identification of each isolates.The result of the sequence based identification was presented in Table 2.

Analysis of gyrB and secA1
All clinical isolates of Nocardia were clearly differentiated and forming distinct branches in phylogenetic tree of gyrB and secA1 (data not shown).The range of nucleotide diversity varied for each of the loci: 0.0 to 100 bp for gyrB and 0.0 to 75 bp for secA1.The clinical isolates of N. asteroids, N. cyriacigeorgica, N. carnea, N. farcinica and N. nova were divided into different genotypes which also supported by 16S rRNA analysis.In the case of isolates belongs to N. wallacei significant discrepancy was detected between 16S rRNA genotyping and those of observed using gyrB and secA1 (Table 1).

MLSA Based Identification
Among all 46 strains, the diversity of the concatenated 16S rRNA-gyrB-secA1 sequences ranged from 0 to 246 nucleotide differences occurring between strains.The NJ tree of the concatenated sequences (Figure 2) clearly showed clinical isolates clusters with bootstrap values of 99% -100% with that of corresponding type strain.For all of the strains, species identification was assigned because their MLSA species cluster contained a single type strain.Beyond delineating species clusters, the overall structure of the NJ tree obtained from concatenated sequences of 16S rRNA-gyrB-secA1 correlated well with those of obtained by any of each locus.

Discussion
Because of the frequent reports and significance of Nocardia infections, there is an increasing need for rapid characterization of clinically isolated Nocaradia especially in clinical setting with high tuberculosis rate.Beyond Gram and modified-acid-fast staining, species identification of Nocardia relies on morphological and conventional biochemical reactions, which are cumbersome and not definitive [1] [10].Although 16S rRNA gene sequencing, remains first choice for bacterial identification and different molecular targets represent promising alternatives [1] [11], however the reliability of species delineation on the basis of the sequence of a single gene suffers from different factors such as stochastic genetic variation, horizontal gene transfer and recombination [12].To address this problem, MLSA has been provided very promising results for prokaryotic taxonomy and species identification [12] [13].Recently, McTaggart et al. (2010) have been suggested that five-locus (16S rRNA, gyrB, secA1, hsp65, and rpoB) MLSA is a reliable method of elucidating taxonomic data however, threelocus (16S rRNA-gyrB-secA1) MLSA scheme is nearly as reliable and correctly identifying 98.5% isolates and would be more feasible for routine use in a clinical reference microbiology laboratory for species identification of Nocardia [6].In the current study, 46 clinical isolates of Nocardia were subjected to identification using three-locus (16S rRNA-gyrB-secA1) MLSA scheme.Different genotypes of N. asteroids, N. carnea, N. cyriacigeorgica, N. farcinica, N. otitidiscaviarum and N. nova have been reported based on the 16S rRNA gene [10].Interspecies micro-heterogeneity was also detected based upon SNP analysis of the gyrB and secA1 genes, which were relatively more polymorphic than the 16S rRNA sequences (Table 2).However, the importance of interspecies variation among the isolates from clinical point of view remained to be answered due to limited number of strains.The association between nocardiosis and underlying diseases has been well-documented [4] [14]- [16].In our study, most of the cases had associated underlying diseases and corticosteroid therapy.Current study like other reports revealed that pulmonary and disseminated nocardial disease is observed mostly in immune-compromised patients, whereas primary cutaneous nocardiosis is usually an infection of immune-compe- tent hosts [16]- [19].In recent years, nocardiosis has been increasingly reported in Iran [20]- [23], however in most of them identification of the clinical isolates were determined on the basis of microscopic examination, culturing and sometimes phenotypic tests.Twelve of the isolates (26%] were identified as N. asteroids as the most frequent encounter species in clinical samples by molecular methods.To best of our knowledge, this is the first report of identification of N. asteroids by microbiological methods from Iran as frequently encountered species in clinical samples.During past years, several cases of nocardiosis by N. asteroids from localized to disseminated disease, have been reported from other parts of the world [14]- [16] [24].The second most dominant species in our clinical isolates was N. cyriacigeorgica (9 isolates, 19.5%).Infection due to this species was previously reported from Iran based on phenotypic methods and 16S rRNA sequencing [23].Disease due to this species has recently been reported [25] and it represents 10.2% and 18% of the Nocardia isolates in Japan and Thailand [26] and USA [27] respectively and 22% and 32.5% from two different studies from Spain [16] [24].Together our finding from present study and several cases of nocardiosis by N. cyriacigeorgica from other parts of the world confirm major role of the latter species in the clinical settings.The isolation of N. farcinica is highly variable among countries.In current study, seven isolates identified as N. farcinica as first report of infections caused by this species from Iran.It has been reported to constitute 23.8%, 26.7%, 35.4% 44%, 60.3% and 33.3% and 21.6% of Nocardia isolates in France [14], Japan [28], Thailand [29], Belgium [30] [31], Germany [32] and Spain [16] respectively.Six of the clinical isolates were identified as N. wallacae as the first report of infections caused by the later species in Iran.Few cases of nocardiosis by N. wallacae have been reported [29].To best of our knowledge, this is the first study to report of large number of clinical isolates of N. wallacae.Our data provide evidence that N. wallacae capable of establishing infection in immune-compromised humans.One isolate from bone marrow biopsy of a patient suffering from hemophilia syndrome identified as N. abscessus.Infections by N. abscessus have been reported from different regions such as Japan [33], Germany [34], Argentine [35] and Spain [16] [24].Three isolates were identified as N. otitidiscaviarum.Importantly, two isolates recovered from blood samples of two bone marrow recipients and one from subcutaneous abscesses of healthy patient.Thus, clinicians should be aware that N. otitidiscaviarum tends to infect both immune-compromised and immunecompetent individuals.This study is the first to report the isolation of N. arthritidis, N. carnea, N. kruczakiae, N. nova and N. veterana in Iran.Our data provides more evidences that N. arthritidis, N. kruczakiae and N. veterana as rare pathogens capable of establishing infections in concordant with previous reports [26] [36] [37].

Conclusion
In conclusion, N. asteroides was the most common pathogen causing nocardiosis in Iran, followed by N. cyriacigeorgica, N. farcinica and N. wallacei.In addition, patients with nocardiosis caused by rare species, including N. abscessus, N. arthritidis, N. carnea, N. kruczakiae, N. nova, N. otitidiscaviarum and N. veterana were also found.This nationwide study found that nocardiosis can be caused by different species of Nocardia and laboratory algorithms based on phenotypic identification of Nocardia strains unable to assign the isolates to species level.Precise and reliable identification of Nocardia to the species level currently requires sequence based methods and preferably MLSA of 16S rRNA, gyrB and secA1.

Figure 1 .
Figure 1.16S rRNA sequence-based phylogenetic tree of clinical isolates of Nocardia with those of closely related species which computed by the NJ analyses and K2P model.The support of each branch, as determined from 1000 bootstrap samples, is indicated by percentages at each node.Bar 0.005 substitutions per nucleotide position.

Figure 2 .
Figure 2. Phylogenetic tree of the genus Nocardia computed from the concatenation of 16S rRNA, gyrB and secA1 sequences by the NJ method and K2P model as the substitution model.Percentages at nodes represent bootstrap levels supported by 1000 resampled datasets.Bar 0.01 substitutions per nucleotide position.

Table 1 .
Characteristics and outcome of 46 patients with Nocardiosis.

Table 2 .
Details of identification of clinical isolates of Nocardia by sequence analysis.