Genus Pseudonocardia: What we know about its biological properties, abilities and current application in biotechnology.

The genus Pseudonocardia belongs to a group of Actinomycetes, and is a member of the family Pseudonocardiacea. The members of this genus are aerobic, Gram-positive, non-motile bacteria that are commonly found in soil, plant and environment. Although this genus has a low clinical significance; however, it has an important role in biotechnology due to the production of secondary metabolites, some of which have anti-bacterial, anti-fungal and anti-tumour effects. The use of phenotypic tests, such as gelatinase activity, starch hydrolysis, catalase and oxidase tests, as well as molecular methods, such as polymerase chain reaction, are necessary for Pseudonocardia identification at the genus and species levels.

Antimicrobial activity of Actinobacteria isolated from dry land soil in Yazd, Iran.

Historically, many important secondary metabolites including antibiotics used in clinic are purified from the cultural broths of Actinobacteria, which were inhabited in soil. Yazd is located in the center of Iran, the south of the Dasht-e Kavir and the west of the Dasht-e Lut; accordingly it has a hot, dry climate with long summers. In the present study, 18 strains of Actinobacteria isolated from 60 soil samples from Yazd-Iran. Pure isolates were screened for antibacterial activity against the ATCC strains by using two methods: single line streak method and spot inoculation method. ATCC strains include four antibiotic resistant ATCC strains (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae and, Acinetobacter baumannii) and three antibiotic sensitive strains (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli) and, Bacillus subtilis. Seven isolates exhibited antimicrobial activity against the ATCC strains (38.8%). Identification of type I and type II polyketide synthases (pksI, pksII) and nonribosomal peptide synthetase (NRPS) genes were done for these 7 isolates and all of 7 strains, possessed at least one of these genes. The results of this study confirm that soil Actinobacteria bear a great ability to produce antibacterial compounds against resistant and sensitive test organisms.

Mycobacterium ahvazicum sp. nov., the nineteenth species of the Mycobacterium simiae complex.

Four slowly growing mycobacteria isolates were isolated from the respiratory tract and soft tissue biopsies collected in four unrelated patients in Iran. Conventional phenotypic tests indicated that these four isolates were identical to Mycobacterium lentiflavum while 16S rRNA gene sequencing yielded a unique sequence separated from that of M. lentiflavum. One representative strain AFP-003T was characterized as comprising a 6,121,237-bp chromosome (66.24% guanosine-cytosine content) encoding for 5,758 protein-coding genes, 50 tRNA and one complete rRNA operon. A total of 2,876 proteins were found to be associated with the mobilome, including 195 phage proteins. A total of 1,235 proteins were found to be associated with virulence and 96 with toxin/antitoxin systems. The genome of AFP-003T has the genetic potential to produce secondary metabolites, with 39 genes found to be associated with polyketide synthases and non-ribosomal peptide syntases and 11 genes encoding for bacteriocins. Two regions encoding putative prophages and three OriC regions separated by the dnaA gene were predicted. Strain AFP-003T genome exhibits 86% average nucleotide identity with Mycobacterium genavense genome. Genetic and genomic data indicate that strain AFP-003T is representative of a novel Mycobacterium species that we named Mycobacterium ahvazicum, the nineteenth species of the expanding Mycobacterium simiae complex.

Mycobacterium persicum sp. nov., a novel species closely related to Mycobacterium kansasii and Mycobacterium gastri.

Four strains isolated in Iran from pulmonary specimens of unrelated patients are proposed as representative of a novel Mycobacterium species. Similarity, at the phenotypic level, with Mycobacterium kansasii is remarkable with the photochromogenic yellow pigmentation of the colonies being the salient feature. They differ, however, genotypically from this species and present unique sequences in 16S rRNA, hsp65 and rpoB genes. The average nucleotide identity and the genome-to-genome distance fully support the status of an independent species. The name proposed for this species is Mycobacterium persicum sp. nov. with AFPC-000227T (=DSM 104278T=CIP 111197T) as the type strain.

Comparison of restriction enzyme pattern analysis and full gene sequencing of 16S rRNA gene for Nocardia species identification, the first report of Nocardia transvalensis isolated of sputum from Iran, and review of the literature.

Nocardial infections occur in different organs of the body and are common in immune disorder diseases of individuals. The aim of this study was to assess Nocardia species identification by phenotypic tests and molecular techniques applied to nocardiosis in Iranian patients. In the current study, various clinical samples were collected and cultured on conventional media and using the paraffin baiting method. Various phenotypic tests were performed. For accurate identification at the species level, restriction fragment length polymorphisms (RFLP) in the hsp65 and partial 16S rRNA genes and full gene sequencing of the 16S rRNA gene were used. Twenty-seven Nocardia spp. were isolated and analysis of phenotypic tests results showed Nocardia asteroides complex, Nocardia otitidiscaviarum, Nocardia nova, and Nocardia spp. New RFLP patterns of Nocardia strains with hsp65 and partial 16S rRNA genes were obtained. Full gene sequencing of the 16S rRNA gene identified Nocardia cyriacigeorgica, N. otitidiscaviarum, Nocardia farcinica, Nocardia transvalensis, and N. nova. Nocardia infections are rarely reported and this genus is the cause of various illnesses. Accurate identification of Nocardia spp. is important for epidemiology studies and treatment. It should also be noted that some species may have similar RFLP patterns; therefore, full gene sequencing of the 16S rRNA gene is necessary for confirmation.

Rapid Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis using TaqMan Allelic Discrimination.

OBJECTIVES: Multidrug-resistant tuberculosis (MDR-TB) is a global problem that many countries are challenged with. Rapid and accurate detection of MDR-TB is critical for appropriate treatment and controlling of TB. The aim of the present study was to evaluate the TaqMan allelic discrimination without minor groove binder (MGB) as a rapid, efficient, and low-cost method for detection of drug resistant strains of Mycobacterium tuberculosis. METHODS: A total of 112 M. tuberculosis isolates from cases with diagnosed TB were subjected to drug susceptibility testing (DST), using the proportion method. Resistant isolates were tested for characterization of mutations in the rpoB and KatG genes by TaqMan genotyping. RESULTS: Of 112 M. tuberculosis isolates for which DST was performed, three, one, and two isolates were MDR, rifampin (RIF) resistant, and isoniazid (INH) resistant, respectively. According to the threshold cycle (Ct) and curve pattern of mutants, TaqMan probes detect all of the mutations in the analyzed genes (katG 315, AGC→ACC, rpoB 531, TCG→TTG, and rpoB 531, TCG→TGG). CONCLUSION: The present study suggests that drug-resistant strains of M. tuberculosis can be detected by pattern's curve or Ct with TaqMan probes without MGB in real-time polymerase chain reaction (PCR).

Microbiological Analysis of Hemodialysis Water in a Developing Country.

Microbiological control of hemodialysis fluid is important for the prevention of hemodialysis-associated illness. Bacterial populations inhabiting a distribution system for hemodialysis water were studied over a 4 month period in five hospitals (one in Tehran, and the others at Alborz). All the samples from the four hospitals at Alborz had colony counts of ≥100 CFU/ml, which at different points of sampling were higher than the maximum recommended values. A total of 80 samples taken at different points in each hospital's hemodialysis distribution system were collected, and 229 planktonic bacteria isolated on R2A medium. No growth was detected by culturing the samples on Blood agar or Mueller-Hinton agar, according to routine procedures currently used in the five hospitals. A representative of isolates from each of 45 different morphotypes were identified using 16S RNA sequencing. A diverse bacterial community, containing predominantly gram-positive members of Kocuria, Arthrobacter and Staphylococcus and Mycobacterium, was detected. Bacteria from the genera Acinetobacter, Burkholderia, Halomonas, Herbaspirillum, Pseudomonas, and Sphingomonas were identified, which has been described in the build-up of biofilms. Some of the species reported here may represent a health risk to patients receiving hemodialysis treatment. In conclusion, it is recommended that standard protocols for evaluation of microbial contamination be used for regular monitoring and identification of culturable bacteria.

In Vitro Antimicrobial Susceptibility of Nontuberculous Mycobacteria in Iran.

Many species of nontuberculous mycobacteria (NTM) have long been identified as important causes of human disease, the incidence of which is rising. Several reports have suggested increasing trend of both in vitro and in vivo resistance to available treatment regimes. The aim of this study was to evaluate antibiotic susceptibility of clinically relevant NTM isolates using standard microbroth dilution test. Antimicrobial susceptibility testing was performed following National Committee for Clinical Laboratory Standards methods for NTM isolates, including 85 Mycobacterium fortuitum, 39 Mycobacterium chelonae, and 30 Mycobacterium abscessus subsp. abscessus as rapidly growing mycobacteria and 48 Mycobacterium simiae and 40 Mycobacterium kansasii as slowly growing mycobacteria. All isolates were recovered from various types of clinical samples and identified by multilocus sequence analysis. Trimethoprim-sulfamethoxazole (TMP-SMZ), amikacin, tobramycin, clarithromycin, moxifloxacin, linezolid, and imipenem showed better activity against M. fortuitum rather than meropenem, ciprofloxacin, cefoxitin, and doxycycline. Amikacin was active against 93% of M. abscessus subsp. abscessus. Linezolid, clarithromycin, cefoxitin, ciprofloxacin, imipenem, moxifloxacin, tobramycin, TMP-SMZ, doxycycline, and meropenem showed some activities on M. abscessus subsp. abscessus as well. The majority of M. abscessus subsp. abscessus and M. chelonae strains were multidrug resistant. Among the 40 isolates of M. kansasii, all were susceptible to ethambutol, isoniazid, clarithromycin, moxifloxacin, and linezolid. These isolates were also resistant to doxycycline and 50% were resistant to rifampicin and ciprofloxacin. M. simiae was resistant to clarithromycin, doxycycline, isoniazid, and TMP-SMZ, and the majority of isolates showed high levels of resistance to linezolid, ethambutol, ciprofloxacin, streptomycin, and rifampicin. The majority of M. simiae isolates were multidrug resistant. Our data confirm the need for performing of standard susceptibility testing of any clinically important NTM isolate.

Genetic diversity and antimicrobial susceptibility of Nocardia species among patients with nocardiosis.

The aim of this multicenter study was to determine the genetic diversity and antibiotic susceptibility of clinically isolated Nocardia species. One hundred twenty-seven patients with nocardiosis were randomly selected from 5 provinces of Iran. Molecular diagnosis of Nocardia species was performed using multilocus sequence analysis of gyrase B of the ß subunit of DNA topoisomerase (gyrB), and 16S rRNA and subunit A of SecA preproteintranslocase (secA1). Antimicrobial susceptibility testing was performed following the Clinical and Laboratory Standards Institute recommendations. Thirty-five N. cyriacigeorgica, 30 N. asteroides, 26 N. farcinica, 12 N. otitidiscaviarum, and 10 N. abscessus cultures were studied. All isolates were susceptible to linezolid. All isolates of N. cyriacigeorgica, N. asteroides, N. abscessus, and N. otitidiscaviarum were susceptible to trimethoprim-sulfamethoxazole, while 8% of N. farcinica isolates were resistant to this drug. All N. otitidiscaviarum isolates were highly resistant to imipenem, but N. cyriacigeorgica, N. asteroides, N. farcinica, and N. abscessus were only moderate resistant. The susceptibility patterns vary with different species of Nocardia. Resistance to trimethoprim-sulfamethoxazole in Iran is low and this drug should be first line therapy, unless drug susceptibility testing shows resistance. Linezolid also covers Nocardia well and could be a second line agent.

Molecular Methods for Identification of Acinetobacter Species by Partial Sequencing of the rpoB and 16S rRNA Genes.

BACKGROUND: Acinetobacter spp. is a diverse group of Gram-negative bacteria which are ubiquitous in soil and water, and an important cause of nosocomial infections. The purpose of this study was to identify a collection of Acinetobacter spp. clinical isolates accurately and to investigate their antibiotic susceptibility patterns. MATERIALS AND METHODS: A total of 197 non-duplicate clinical isolates of Acinetobacter spp. isolates identified using conventional biochemical tests. The molecular technique of PCR-RFLP and sequence analysis of rpoB and 16S rRNA genes was applied for species identification. Antimicrobial susceptibility test was performed with a disk diffusion assay. RESULTS: Based on 16S rRNA and rpoB genes analysis separately, most of clinical isolates can be identified with high bootstrap values. However, the identity of the isolate 555T was uncertain due to high similarity of A. grimontii and A. junii. Identification by concatenation of 16S rRNA and rpoB confirmed the identity of clinical isolates of Acenitobacer to species level confidently. Accordingly, the isolate 555T assigned as A. grimontii due to 100% similarity to A. grimontii. Moreover, this isolate showed 98.64% to A. junii. Besides, the identity of the isolates 218T and 364T was confirmed as Genomic species 3 and A. calcoaceticus respectively. So, the majority of Acinetobacter spp. isolates, were identified as: A. baumannii (131 isolates, 66%), A. calcoaceticus (9 isolates, 4.5%), and A. genomosp 16 (8 isolates, 4%). The rest of identified species showed the lower frequencies. In susceptibility test, 105 isolates (53%), presented high antibiotic resistance of 90% to ceftriaxone, piperacillin, piperacillin tazobactam, amikacin, and 81% to ciprofloxacin. CONCLUSION: Sequence analysis of the 16S rRNA and rpoB spacer simultaneously was able to do identification of Acinetobacter spp. to species level. A.baumannii was identified as the most prevalent species with high antibiotic resistance. Other species showed lower frequencies ranged from 4 to 9 strains.

"Multidrug-resistant tuberculosis" may be nontuberculous mycobacteria.

INTRODUCTION: Multidrug resistant tuberculosis (MDR-TB) presents a great challenge to public health, especially for developing countries. Some nontuberculous mycobacteria (NTM) cause the similar clinical and radiological characteristics with tuberculosis. We aimed to identify the frequency of NTM infections among subjects who were suspected to have MDR-TB due to lack of response to anti-TB treatment. METHODS: This retrospective study evaluated patients with suspected MDR-TB due to lack of sputum conversion after 2-3 month therapy with first line anti-TB treatment from 2009 through 2014. Cultures for mycobacteria were performed and identification was done to species level by phenotypic and molecular tests. The outcome of the patients with NTM disease and related risk factors for poor outcome were evaluated. RESULTS: Out of 117 consecutive strains isolated from suspected MDR-TB subjects, 35 (30%) strains were identified as NTM by using conventional and molecular approaches. Of these patients with positive NTM cultures, 32 (27%) patients met ATS/IDSA diagnostic criteria. Out of 32, 29 (90%) individuals with confirmed NTM diseases had underlying disorders including 8 subjects with malignancy, 5 with organ transplantations, and 4 with the human immunodeficiency virus. No known underlying disorder was found in 3 (9%) subjects. Treatment outcomes were available for 27 subjects, 17 (63%) of whom were cured and 10 (37%) had poor outcome including 6 (60%) who failed and 4 (40%) who died during treatment. CONCLUSION: The high costs to the patient and society should lead health care providers to consider NTM in all patients suspected of having TB.

Nocardia isolation from clinical samples with the paraffin baiting technique.

BACKGROUND: The genus Nocardia is a cause of infection in the lungs, skin, brain, cerebrospinal fluid, eyes, joints and kidneys. Nocardia isolation from polymicrobial specimens is difficult due to its slow growth. Several methods have been reported for Nocardia isolation from clinical samples. In the current study, we used three methods: paraffin baiting technique, paraffin agar, and conventional media for Nocardia isolation from various clinical specimens from Iranian patients. METHODS: In this study, we examined 517 samples from various clinical specimens such as: sputum of patients with suspected tuberculosis, bronchoalveolar lavage, sputum of patients with cystic fibrosis, tracheal aspirate, cutaneous and subcutaneous abscesses, cerebrospinal fluid, dental abscess, mycetoma, wound, bone marrow biopsy, and gastric lavage. All collected specimens were cultured on carbon-free broth tubes (paraffin baiting technique), paraffin agar, Sabouraud dextrose agar, and Sabouraud dextrose agar with cycloheximide and were incubated at 35°C for one month. RESULTS: Seven Nocardia spp. were isolated with paraffin baiting technique, compared with 5 positive results with the paraffin agar technique and 3 positive results with Sabouraud dextrose agar with and without cycloheximide. The prevalence of nocardial infections in our specimens was 5.28%. CONCLUSION: In the present study, the use of the paraffin baiting technique appeared to be more effective than other methods for Nocardia isolation from various clinical specimens.

Mycobacterium celeriflavum sp. nov., a rapidly growing scotochromogenic bacterium isolated from clinical specimens.

Six strains of a rapidly growing scotochromogenic mycobacterium were isolated from pulmonary specimens of independent patients. Biochemical and cultural tests were not suitable for their identification. The mycolic acid pattern analysed by HPLC was different from that of any other mycobacterium. Genotypic characterization, targeting seven housekeeping genes, revealed the presence of microheterogeneity in all of them. Different species were more closely related to the test strains in various regions: the type strain of Mycobacterium moriokaense showed 99.0 % 16S rRNA gene sequence similarity, and 91.5-96.5 % similarity for the remaining six regions. The whole genome sequences of the proposed type strain and that of M. moriokaense presented an average nucleotide identity (ANI) of 82.9 %. Phylogenetic analysis produced poorly robust trees in most genes with the exception of rpoB and sodA where Mycobacterium flavescens and Mycobacterium novocastrense were the closest species. This phylogenetic relatedness was confirmed by the tree inferred from five concatenated genes, which was very robust. The polyphasic characterization of the test strains, supported by the ANI value, demonstrates that they belong to a previously unreported species, for which the name Mycobacterium celeriflavum sp. nov. is proposed. The type strain is AFPC-000207(T) ( = DSM 46765(T) = JCM 18439(T)).

DNA extraction from nocardia species for special genes analysis using PCR.

BACKGROUND: Nocardia species have a complex cell wall structure similar to that of mycobacteria, and the extraction of DNA from this bacterium is extremely difficult. Currently, to identify Nocardia species particularly, it is essential to utilize molecular techniques. AIMS: In the present study, we investigated STET (sodium chloride-TRIS-EDTA-triton) buffer for the extraction of high-quality genomic DNA from 20 clinical and environmental isolates. MATERIALS AND METHODS: The extracted DNA was evaluated for portion of the 16S rRNA, 65-kDa heat-shock protein and 16S rRNA genes via polymerase chain reaction. RESULTS: The extracted DNA had high molecular mass, and its concentration and purity was suitable when tested in 1% agarose gel, and using UV spectrophotometry. Amplification of three different genes was successfully performed. CONCLUSION: This paper reveals an inexpensive, reproducible and efficient method of DNA extraction from Nocardia species, which is appropriate for accurate identification of this bacterium via polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism.

Heterogeneity of Iranian clinical isolates of Mycobacterium fortuitum.

BACKGROUND AND OBJECTIVES: The increase of infections caused by nontuberculous mycobacteria (NTM) is receiving increasing attention worldwide. Mycobacterium fortuitum is encountered with increasing frequency in clinical laboratories of Iran. MATERIALS AND METHODS: Sequence variation of 48 M. fortuitum clinical isolates, were investigated by sequence analysis of the 16S-23S Internal Transcribed Spacer. RESULTS: Twelve different sequence types (sequevar) were identified by sequence analysis of ITS region. Seven previously described sequevar including MfoA, MfoB, MfoC, MfoD, MfoE, MfoF and MfoG identified. Five novel sequevar namely MfoH, MfoI, MfoJ, MfoK and MfoL that were distinctly different from the previously described sequevar were detected among different clinical strains of M. fortuitum, from Iran. CONCLUSION: This study showed that the ITS region possesses high discriminatory power between the clinical isolates up to the clonal level. The results also suggest the possibility of the existence of predominant clone of M. fortuitum in affected patients in Iran. The data also point to the conclusion that a large variety of M. fortuitum clone can produce disease although certain clones seem to be predominant.

Mycobacterium simiae: a possible emerging pathogen in Iran.

Mycobacterium simiae has been reported worldwide, particularly from the Middle East. This organism has been recognized as a causative agent of pulmonary and disseminated infections. In this study, we used molecular methods to detect this organism from patients who were suspected of having tuberculosis. A total of 117 isolates of mycobacteria were evaluated from different regions of Iran. Isolates were identified using phenotypic methods and gene sequencing of 16S rRNA, rpoB, hsp65, and ITS. Of the 117 isolates, 12 M. simiae isolates (10.2%) were identified from different clinical samples, including bronchoalveolar lavage and sputum (n = 8), blood (n = 3), and lymph node biopsy (n = 1). Three isolates (3/12, 25%) were recovered from blood samples of HIV cases when the CD4(+) count was less than 50/µl. There was no significant relationship between infection and age or gender. Infection with nontuberculous mycobacteria (NTM), including M. simiae, is the major problem among immunocompromised patients. The results of this study illustrated the importance of molecular methods for accurate and rapid detection of NTM infections in the treatment of nonresponding patients with suspected tuberculosis.

Species spectrum of nontuberculous mycobacteria isolated from suspected tuberculosis patients, identification by multi locus sequence analysis.

Identification of Mycobacterium species is difficult due to a complex and rapidly changing taxonomy, the failure of 16S rRNA to discriminate many closely related species and the unreliability of phenotypic testing. We investigated a collection of nontuberculous mycobacteria (NTM) strains isolated from suspected tuberculosis patients at Tuberculosis Reference Centre (Ahvaz, Iran) and Masoud Laboratory (Tehran, Iran) during 2008-2012 to evaluate the species spectrum of NTM isolates. Based on phenotypic tests, the isolates were identified up to species or complex level; however they were heterogonous by hsp65-PCR restriction fragment length polymorphism analysis (PRA) method. Representative isolates from each hsp65-PRA pattern, were subjected to identification using single locus and multi locus sequence analysis (MLSA) based on 16S rRNA, rpoB, hsp65 and 16S-23S internal transcribes spacer (ITS) fragments to determine their taxonomic affiliations. All 92 NTM isolates from different clinical specimens were considered as etiological agents causing disease according to American Thoracic Society (ATS) guideline. Phenotypic evaluation alone assigned 66 (72%) isolates to a species or complex level and consequently 76 (82%) isolates showed previously reported hsp65-PRA patterns. Although sequence base identification using single locus such as 16S rRNA, rpoB, hsp65 or ITS identified the isolates up to species level, MLSA correctly identified 16 different species of NTM from clinical isolates. In summary, four-locus MLSA is a reliable method for elucidating taxonomic data and reliable species identification of Mycobacterium isolates and therefore, would be more feasible for routine use in Tuberculosis (TB) reference laboratory.

Mycobacterium iranicum sp. nov., a rapidly growing scotochromogenic species isolated from clinical specimens on three different continents.

The isolation and characterization of a novel, rapidly growing, scotochromogenic mycobacterial species is reported. Eight independent strains were isolated from clinical specimens from six different countries of the world, two in Iran, two in Italy and one in each of following countries: Greece, The Netherlands, Sweden and the USA. Interestingly, two of the strains were isolated from cerebrospinal fluid. The strains were characterized by rapid growth and presented orange-pigmented scotochromogenic colonies. DNA-based analysis revealed unique sequences in the four regions investigated: the 16S rRNA gene, the rRNA gene internal transcribed spacer 1 and the genes encoding the 65 kDa heat-shock protein and the beta-subunit of RNA polymerase. The phylogenetic analysis placed the strains among the rapidly growing mycobacteria, being most closely related to Mycobacterium gilvum. The genotypic and phenotypic data both strongly supported the inclusion of the strains investigated here as members of a novel species within the genus Mycobacterium; the name Mycobacterium iranicum sp. nov. is proposed to indicate the isolation in Iran of the first recognized strains. The type strain is M05(T) ( = DSM 45541(T) = CCUG 62053(T) = JCM 17461(T)).