Molecular characterization of nontuberculous mycobacteria in hospital waters: a two-year surveillance study in Tehran, Iran.

Microbiological control of hospital waters as one of the main sources of nontuberculous mycobacteria (NTM) is important for the prevention of NTM-associated illness. This study aimed to investigate the prevalence of NTM in the hospital water systems of Tehran, Iran. A total of 218 samples from different hospital waters (i.e., tap water and medical devices such as humidifying cup of oxygen manometer, dialysis devices, nebulizers, and dental units) were included in this study. Phenotypic and molecular tests were used to identify the isolated organisms to species level. Of 218, 85 (39.0%) samples at 37 °C and 87 (40.0%) samples at 25 °C were identified as NTM. Using hsp65-sequencing method, Mycobacterium lentiflavum was the most frequently encountered, followed by M. gordonae and M. paragordonae. No significant difference was seen in frequency and species in mycobacteria isolated at 37 °C and 25 °C temperatures. Humidifying cup of oxygen manometer had the most contaminated water among the investigated water distribution systems in hospitals. Isolation of NTM from hospital water sources is a serious public health problem in Iran and merits further attention by health authorities. Establishment of microbiological monitoring systems for hospital waters and expanding the number of facilitated laboratories are strongly recommended.

Mycobacterium angelicum sp. nov., a non-chromogenic, slow-growing species isolated from fish and related to Mycobacterium szulgai.

The name 'Mycobacterium angelicum' dates back to 2003 when it was suggested for a slowly growing mycobacterium isolated from freshwater angelfish. This name is revived here and the novel species is proposed on the basis of the polyphasic characterization of four strains including the original one. The four strains presented 100 % 16S rRNA gene sequence similarity with Mycobacterium szulgai but clearly differed from M. szulgai for the milky white aspect of the colonies. The sequence similarity with the type strain of M. szulgai ranged, in eight additionally investigated genetic targets, from 78.9 to 94.3 %, an evident contrast with the close relatedness that emerged at the level of 16S rRNA gene. The average nucleotide identity between the genomes of M. szulgai DSM 44166T and strain 126/5/03T (type strain of the novel species) was 92.92 %, and supported the status of independent species. The confirmation of the name Mycobacterium angelicum sp. nov. is proposed, with strain 126/5/03T ( = CIP 109313T = DSM 45057T) as the type strain.

Diversity and antibacterial activity of endophytic bacteria associated with medicinal plant, Scrophularia striata.

To search endophytic bacteria diversity and evaluate their antibacterial activity, healthy medicinal plant of Scrophularia striata was chosen in this study. One hundred endophytic bacteria were isolated from surface-sterilized tissues (root, stem and leaf) of S. striata. Using sequence analysis targeting 16S rRNA gene, eight genera, including Agrococcus, Arthrobacter, Bacillus, Chryseobacterium, Delftia, Kocuria, Pseudomonas and Sphingomonas were identified. Antibacterial activity of endophytic bacteria was examined against some test bacteria, employing agar well diffusion method. Out of 31 endophytic bacterial isolates, 24(77.42%) isolates showed significant antimicrobial activity against Bacillus cereus, 17(54.84%) isolates exhibited maximum activity against Staphylococcus aureus, 14(45.16%) isolates against Escherichia coli and 5(16.13%) isolates showed positive activity against Proteus mirabilis.The results obtained in this study suggested that the medicinal plant, S. striatais is a potent source of endophytic bacteria with antibacterial activity and offers promise for discovery of more impressive biological compounds.

First Molecular Characterization and Seasonality of Larvae of Trichostrongylid Nematodes in Arrested Development in the Abomasum of Iranian Naturally Infected Sheep.

PURPOSE: The aim of this study was to determine molecularly the species of larvae of trichostrongylid nematodes in arrested development in the abomasum of sheep from Iran and their seasonality. METHODS: A total of 240 sheep abomasa were randomly collected at different seasons from Ilam abattoir in 2017-2018. The adult nematodes were isolated and identified. The mucosa of the abomasum was removed and digested using a pepsin solution. DNA from different parts of the abomasum was extracted using a SinaPureTM kit. Polymerase chain reaction (PCR) amplification was performed; the ITS2-rRNA gene was amplified using specific primers. Amplified PCR products were purified and sequenced by Bioneer Corporation (South Korea). The obtained ITS2 sequences were aligned using BLAST analysis and submitted to GenBank. RESULTS: Morphologically, five species of adult nematodes of four genera including Haemonchus contortus, Marshallagia marshalli, Marshallagia occidentalis, Parabronema skrjabini, and Teladorsagia circumcincta were identified. Marshallagia marshalli (65.4%, 120.2 ± 28.5) had the highest prevalence during autumn. Molecularly, arrested development of larvae of M. marshalli and T. circumcincta were demonstrated in mucosa of the abomasum in autumn and early winter using PCR of the ITS-2 region of rRNA. CONCLUSION: The results evidenced the first molecular characterization of the larvae of trichostrongylid nematodes from abomasum of sheep from Iran.

Molecular study on parasitic nematodes infection in the abomasum of sheep in Ilam, Iran.

Parasitic nematodes of ovine abomasum are of economic and hygienic importance throughout the world and Iran. This study was aimed to evaluate molecular identity and species diversity of parasitic nematodes in the abomasum of slaughtered sheep in Ilam, Iran. In this study, a total number of 240 of abomasa were randomly collected from the slaughtered sheep at industrial slaughterhouses in Ilam in all seasons between 2017 and 2018. The abomasum content and abomasal mucosa were removed and washed. The collected nematodes were morphologically identified. The genomic DNA was extracted and a 300 bp-fragment-length from internal transcribed spacer 2 ribosomal ribonucleic acid (ITS2-rRNA) gene was amplified. Overall prevalence was 66.70% (160/240). Five species of four genera of nematodes including Marshallagia marshalli (43.70%), Ostertagia circumcincta (15.50%), Parabronema skrjabini (5.00%), M. occidentalis (2.50%), and Haemonchus contortus (0.04%) were identified. Ostertagia circumcincta and H. contortus were found to be different in two nucleotides. There was no nucleotide difference between M. marshalli and M. occidentalis. This study revealed a significant prevalence of parasitic nematodes in sheep abomasum and species diversity of Trichostrongylid nematodes in the region.

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.

Detection of highly ciprofloxacin resistance acinetobacter baumannii isolated from patients with burn wound infections in presence and absence of efflux pump inhibitor.

The emergence of fluoroquinolone resistance among A. baumannii isolates is now of particular concern. Phenotypic and genotypic characteristics of resistance to ciprofloxacin among 50 Acinetobacter baumannii isolated from burn wound infections of Tehran were evaluated by E-test and broth microdilution in presence and absence of efflux pump inhibitor phenylalanine- arginine ß-naphthylamide (PAßN) and PCR-sequencing methods. All isolates were then typed by REP-PCR fingerprinting to find the clonal relationship between resistant isolates. Our results indicated that resistance to ciprofloxacin among A. baumannii isolated from burn infections in Tehran are high with resistance rate of 100% and ciprofloxacin resistant isolates have a mutation of Serine 83 →Leucine in the quinolone resistance determining region (QRDR) of DNA gyrase subunit A (GyrA). 38% of the isolates showed MIC ranges of 64 to ≥512µg/ml and were considered as highly resistant. We could not detect Par C mutations and plasmid-mediated quinolone resistance A (qnrA) among ciprofloxacin resistant isolates. When we used the efflux pump inhibitor PAbN, MIC of ciprofloxacin was reduced two-to four folds. REP-type A (25/50; 50%), B (20/50; 30%) and C (10/50; 20%) were the most common REP-types among A. baumannii isolates. It seems that mutation in GyrA is the main mechanism of resistant to ciprofloxacin among A. baumannii isolates from burn infections and presence of efflux pumps is just secondary target for ciprofloxacin resistant among A. baumannii in Iran. Regarding with limitation of REP-types detected in this study, we found good correlation between resistance to ciprofloxacin and REP-types A-C.

Emergence of SCCmec type III with variable antimicrobial resistance profiles and spa types among methicillin-resistant Staphylococcus aureus isolated from healthcare- and community-acquired infections in the west of Iran.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen of public health importance. The prevalence of MRSA and its antimicrobial resistance pattern, as well as SCCmec and spa types, remain unclear both in the community and in the hospitals of the western region of Iran. METHODS: One hundred MRSA isolates were collected from different hospitals in the west of Iran during 2010-2011. Antimicrobial susceptibility testing to 15 antimicrobial agents was carried out by disk agar diffusion (DAD) method in accordance with the Clinical and Laboratory Standards Institute guidelines. Vancomycin minimum inhibitory concentrations (MICs) were evaluated by a broth microdilution method. The Etest was used for the detection of highly gentamicin-resistant MRSA. A combination of single and multiplex PCR was used for the detection of different resistance genes, including beta-lactamase, aminoglycoside modifying enzymes (AMEs), and macrolide-lincosamine, and for SCCmec typing of MRSA isolates. Genotyping of MRSA isolates was performed via spa typing. RESULTS: All tested isolates were susceptible to quinupristin-dalfopristin, linezolid, and vancomycin, but were resistant to penicillin (100%), erythromycin (50%), clindamycin (27%), and gentamicin (18%). MIC50 and MIC90 was 256 µg/ml among gentamicin-resistant MRSA. The most prevalent AME genes among aminoglycoside-resistant isolates were aac(6')-1e-aph(2")-1a (77.8%), aph(3')-IIIa (38.9%), and ant(4')-1a (27.8%). Nearly all tetracycline- and erythromycin-resistant MRSA had ermA and/or ermC but not ermB. Five SCCmec types and subtypes, 13 spa types, and four BURP groups (A-D) were identified. SCCmec types III (45%) and IVc (24%), spa type t701 (30%), and new spa type t12311 (15%) were the most prevalent among MRSA isolates. CONCLUSIONS: This study showed the emergence of MRSA with SCCmec type III and with spa types t12311, t10740, t1234, t1991, and t2651 with different phenotypic and genotypic antimicrobial resistance in the west of Iran. We found different SCCmec and spa types distributed among nosocomial and non-nosocomial MRSA in the west of Iran.

Report of two cases of Mycobacterium europaeum from Iran.

Herein, we report repeated isolation of Mycobacterium europaeum from the sputum samples of an Iranian human immunodeficiency virus-infected patient and a cystic fibrosis patient with chronic pulmonary disease. To our knowledge, this is the first isolation of M. europaeum from human clinical specimens in Asia to be reported.

Molecular Identification of Rare Clinical Mycobacteria by Application of 16S-23S Spacer Region Sequencing.

OBJECTIVES: In addition to several molecular methods and in particular 16S rDNA analysis, the application of a more discriminatory genetic marker, i.e., 16S-23S internal transcribed spacer gene sequence has had a great impact on identification and classification of mycobacteria. In the current study we aimed to apply this sequencing power to conclusive identification of some Iranian clinical strains of mycobacteria. MATERIALS AND METHODS: The test strains consisted of nineteen mycobacterial isolates which were initially identified by the use of conventional phenotypic techniques and molecular methods and subjected to further definitive identification using the 16S-23S internal transcribed spacer gene sequencing. RESULTS: Out of 19 studied strains, 7 isolates were found to be rapidly growing and 12 isolates as slowly growing mycobacteria. With the exception of one isolate, i.e., the isolate HNTM87, which yielded a distinct ITS sequence incomparable with all previously identified mycobacteria, the remaining isolates produced the sequences similar to the established mycobacteria and were clearly identified and differentiated from closely related taxa. A phylogenetic tree based on maximum parsimony analysis of 16S-23S internal transcribed spacer gene sequences constructed showing the relatedness of Iranian clinical isolates with the closely related type species of mycobacteria. CONCLUSION: This study showed that the 16S-23S internal transcribed spacer gene of the genus Mycobacterium exhibits a high variation which is of value for discriminating closely related taxa and could be used independently or in combination with 16S rDNA sequencing to delineate the true identity of rare mycobacterial species.

Mycobacterium stomatepiae sp. nov., a slowly growing, non-chromogenic species isolated from fish.

Slowly growing, non-chromogenic mycobacteria were isolated from striped barombi mbo cichlids (Stomatepia mariae) maintained at the London Zoo Aquarium, UK. The isolates could be differentiated from other slowly growing, non-pigmented mycobacteria by a combination of phenotypic features including their inability to grow at 37 degrees C, positive tests for heat-stable catalase, tellurite reduction and arylsulfatase activity, and the absence of urease activity, Tween 80 hydrolysis, nitrate reductase, iron uptake and semiquantitative catalase. The almost full-length 16S rRNA gene sequence, together with partial sequences from the 65 kDa heat-shock protein (hsp65) and the beta-subunit of the bacterial RNA polymerase (rpoB) genes and the 16S-23S internal transcribed spacer 1 (ITS 1) region were identical for all three novel strains, but distinct from those of all known mycobacterial species. Phylogenetic analysis based on 16S rRNA gene sequences placed the novel isolates within the slowly growing mycobacteria group in close proximity to Mycobacterium florentinum. Based on genotypic and phenotypic findings, it is proposed that these isolates represent a novel species of the genus Mycobacterium, for which the name Mycobacterium stomatepiae sp. nov. is proposed with strain T11(T) (=DSM 45059(T)=CIP 109275(T)=NCIMB 14252(T)) as the type strain.

Survival and replication of Piscirickettsia salmonis in rainbow trout head kidney macrophages.

Piscirickettsia salmonis is pathogenic for a variety of cultured marine fish species worldwide. The organism has been observed within host macrophages in natural disease outbreaks among coho salmon and European sea bass. In vitro studies, incorporating transmission electron microscopy (TEM) and ferritin loading of lysosomes, have confirmed that P. salmonis is capable of surviving and replicating in rainbow trout macrophages. Certain features of this intracellular survival underline its difference to other intracellular pathogens and suggest that a novel combination of defence mechanisms may be involved. Escape into the macrophage cytoplasm is not used as a means to avoid phago-lysosomal fusion and the organism remains at least partly enclosed within a vacuole membrane. While the piscirickettsial vacuole is often incomplete, survival and replication appear to require occupation of a complete, tightly-apposed, vacuolar membrane which does not fuse with lysosomes. Unlike some mammalian rickettsiae, actin-based motility (ABM) is not used as a means of intercellular spread. It is postulated that the presence of numerous small vesicles within vacuoles, and at gaps in the vacuolar membrane, may result from the blebbing of the piscirickettsial outer membrane seen early in the infection.

Molecular systematics support the revival of Mycobacterium salmoniphilum (ex Ross 1960) sp. nov., nom. rev., a species closely related to Mycobacterium chelonae.

Mycobacterial infections in fish are usually attributed to strains of Mycobacterium marinum, Mycobacterium chelonae and Mycobacterium fortuitum. Bacteria identified as M. chelonae have been isolated numerous times from salmonid fishes. Recently, this bacterium has been associated with salmon mortalities in the aquaculture industry. An M. chelonae-like species from salmon, 'Mycobacterium salmoniphilum', was described in 1960. However, the species name lost standing in nomenclature when it was omitted from the 1980 Approved Lists of Bacterial Names because the species could not be distinguished with confidence from M. fortuitum. In the 1980s, mycobacteria isolated from salmon were characterized as a distinct subspecies, 'Mycobacterium chelonae subsp. piscarium'. Again, the uncertainty of the validity of the species resulted in the subsequent withdrawal of the name. Since then, most studies have considered isolates from salmon to be M. chelonae. Nucleotide sequence analysis of the small-subunit rRNA, hsp65 and rpoB genes was used to examine the taxonomic relatedness of type cultures and authentic isolates in our culture collection available from earlier studies. The M. chelonae-like strains from salmon were phylogenetically distinct from other Mycobacterium strains and members of the M. chelonae complex. Moreover, the cell-wall-bound mycolic acids were not representative of known mycolate patterns for M. chelonae-complex organisms. These results supported the status of the species as a separate taxon and effect the valid publication of the name 'M. salmoniphilum' as Mycobacterium salmoniphilum (ex Ross 1960) sp. nov., nom. rev., with the type strain SCT (=ATCC 13578T =DSM 43276T).