First isolation of Nocardia grenadensis from a clinical sample

Correspondence Marı́a Ercibengoa maria.ercibengoaarana@ osakidetza.net Biomedical Research Center Network for Respiratory Diseases (CIBERES), San Sebastián, Spain Special Bacteriology Reference Laboratory, Bacterial Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, Atlanta, GA, USA Microbiology Department, Hospital Universitario Donostia-IIS-Biodonostia, San Sebastián, Spain


Introduction
Bioprospecting and activity directed toward the selection of micro-organisms with economic value is under permanent development.In this regard, there has been significant work on the study of the diversity and biology of the genus Nocardia in natural habitats (Albuquerque de Barros et al., 2003).The genus Nocardia has an important role in the biotechnology and pharmaceutical fields due to the production of antimicrobials and enzymes for medical and industrial use (Mukesh, 2014).
Although less than half of the validly named Nocardia species have been characterized as human pathogens, new pathogenic species causing clinical infections are continually being recognized and reported (Ambrosioni et al., 2010;Jannat-Khah et al., 2010).The development of molecular techniques and increasing access to diagnostic laboratories has greatly eased the identification of Nocardia species.Among them, sequencing of different constitutive genes, especially the 16S rRNA gene, has become one of the most widely accepted methods for accurate species identification (CLSI, 2008).
To date, Nocardia grenadensis has been described on two occasions, both of them from environmental sources Abbreviation: COPD, chronic obstructive airways disease.
The GenBank/EMBL/DDBJ accession number for the sequence of the 1440 bp fragment of the 16S rRNA gene from the Nocardia isolate is KT254140.(Ka ¨mpfer et al., 2012;Wilson, 2013).The aim of the present work was to describe the first isolation of N. grenadensis, identified by 16S rRNA gene sequencing, from the sputum of a patient from the north of Spain suspected of having tuberculosis.

Case Report
N. grenadensis was isolated from a bronchial secretion sample obtained after spontaneous expectoration from the sputum of a 75-year-old man with a history of previous prostate adenocarcinoma, arterial hypertension, hyperlypidaemic disorder and osteoarthritis.The patient had also a lung mass in the lung left upper lobe with a pseudonodule in the first left rib.The patient was a non-smoker, did not have any chronic obstructive pulmonary disease (COPD) and had no recent history of contact with a tuberculosis patient nor any travel abroad in the previous year.

Investigations
In April 2012, the patient attended his primary care physician because of persistent cough and whitish expectoration, sometimes with haemoptysis.In November 2012, the patient continued with cough and expectoration with haemoptysis, and a chest X-ray showed a lung mass in the left upper lobe which was interpreted as a pseudonodule in the first left rib.In December 2012, sputum samples were collected for microbiological cultures (bacterial and tuberculosis) and pathological analysis.A culture positive for N. grenadensis and pathology results negative for neoplastic cells, showing abundant macrophages and squamous metaplasia cells, were was obtained.Both acidfast stain and culture were negative for Mycobacterium.
In January 2013, the patient attended his general practitioner still with cough.The investigation of Nocardia in a new sputum collected in February 2013 was negative.The chest radiograph performed showed no specific abnormalities, although the patient's symptoms persisted for w6 months.The pseudonodule observed in the first chest X-ray was considered as an overlying rib.Finally, without antimicrobial treatments (to our knowledge), the symptoms disappeared.
N. grenadensis showed a good growth of white colonies of 1-2 mm size and matt appearance after 48 h incubation at 35 uC on blood agar.The colony appearance was circular with raised elevation and filamentous edges.The catalase test results were positive.
The isolate was submitted to the Special Bacteriology Reference Laboratory at the Centers for Disease Control and Prevention (USA) for identification.A 1440 bp fragment of the 16S rRNA gene was amplified by PCR and sequenced according to methods described previously (Lasker et al., 2011).The resulting sequence was submitted to GenBank.Related gene sequences in GenBank were identified using BLASTN (https://www.ncbi.nlm.nih.gov/blast/).The best match was found to be N. grenadensis GW5-5797 T , which shared 100 % similarity with the isolate sequence (1440/1440 bp).

Outcome and Follow-up
The patient continued with dry cough and expectoration with haemoptysis for at least 6 months beyond the Nocardia isolation, a period during which the patient had a follow-up at radiological, pathological and microbiological levels, including three more sputum cultures, without any pathogenic micro-organism detected in any of them.No antibiotic treatment was prescribed during this time period.In June 2013, the patient was discharged from the Pneumology Department by absence of bronchopulmonary pathology.

Discussion
Respiratory infection is the most frequent clinical manifestation of nocardiosis, especially in COPD patients because infection normally takes place via the respiratory tract.However, Nocardia can also be found infecting the central nervous system, skin and other locations (Brown-Elliott et al., 2006).The risk of nocardiosis in immunocompromised patients is 140-to 340-fold higher than in the non-immunosuppressed population (Filice, 2005).
The identification of Nocardia isolates to the species level has traditionally been based on the assessment of growth characteristics, antibiotic resistance and biochemical testing (Brown-Elliott et al., 2006).However, with the growing taxonomy of the genus, the utility of traditional biochemical methods for the accurate identification of some Nocardia species is no longer reliable.Currently, the analysis of the sequence of certain genes is considered the best method for correct Nocardia species identification and could be a satisfactory alternative to DNA-DNA hybridization as the gold standard for defining new species (Clarridge, 2004).In one study that analysed the utility of sequencing five different genes (16S rRNA, secA1, gyrB, hsp65 and rpoB) to identify different Nocardia species, it was highlighted that the identification achieved using the combined sequences differed from identifications obtained by analysing individual gene sequences (McTaggart et al., 2010).
Our isolate was identified as N. grenadensis by 16S rRNA sequencing.However, when we analysed the secA1 gene sequence, the GenBank best match (BLASTN) was the secA1 gene of Nocardia sienata with 96.8 % similarity.This result, which initially could be regarded as a discrepant identification, was due to the absence of the secA1 sequence of N. grenadensis in GenBank.It always has to be taken into account that when a gene sequence is being analysed it might not be included in the database searched, especially if a 100 % similarity is not obtained and, as in the case herein described, the genes under study may belong to a relatively newly described species.N. grenadensis was originally isolated in 2012 from a sand sample collected at 5 m depth from the Caribbean Sea near Grenada (Ka ¨mpfer et al., 2012).A N. grenadensis isolate was also identified 1 year later during the characterization of organisms collected from different soils in the Great Smoky Mountains National Park North Carolina (USA) (Wilson, 2013).The patient described here did not have a history of travel in the year previous to the N. grenadensis isolation.This suggests that, considering the different regions where N. grenadensis has been isolated, it is a species that may be ubiquitous.
The clinical significance of N. grenadensis in the sputum of this non-COPD patient is uncertain.Symptoms of persistent cough with whitish expectoration, including haemoptysis, in the absence of other identified causes suggested the possible involvement of N. grenadensis in a bronchial lung infection.However, the absence of altered radiology and blood parameters of infection together with the apparent spontaneous resolution of symptoms could not exclude the possibility that this microbiological finding represented a transient colonization.
In conclusion, the present work shows that gene sequencing offers a rapid and accurate way for identification of even very uncommon Nocardia species.To the best of our knowledge, this is the first report in the literature of the isolation of N. grenadensis in humans.