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

Burden of potentially novel species of non tuberculosis mycobacteria in Iranian tuberculosis laboratories

AbstractAims and objectivesNontuberculous mycobacterium (NTM) infections are caused by mycobacteria that are found in water and soil. The chances of missing NTM species are higher in tuberculosis endemic countries like Iran, which are poorly equipped and overburdened with other diseases. The aim of this study is to access the diversity of rare, unusual and frequent species in Iranian tuberculosis laboratories.MethodsFrom 2012–2014 a total of 243 different clinical isolates of NTM were recovered from Mycobacteriology Reference Laboratories across Iran (Ahvaz, Esfahan, Tehran, Gorgan, Kermanshah). The isolates identified by rpoB gene sequencing are useful markers in mycobacterium identification.ResultsBased on rpoB gene sequencing, 20 groups do not belong to any of the officially recognized mycobacteria. A total of 210 isolates of NTM were identified to species level, including M. fortuitum (66), M. kansasii (20), M. simiae (20), M. conceptionense (10), M. peregrinum (9), M. thermoresistibile (9), M gordonae (8), M. lentiflavum (8), M. phlei (6), M. intracellulare (6), M. abscessus subsp. abscessus (5), M. chelonae (5), M. abscessus subsp. bolletii (4), M. avium (4), M. flavescents (4), M. nonchromogenicum (4), M. terrae (4), M. branderi (3), M. iranicum (3), M. mucogenicum (3), M. scrofulaceum (3), M. smegmatis (3) and M. triplex (2).Among all isolates, M. fortuitum was identified as the most frequent encounter (27%) of NTM, and M. kansasii and M. simiae were the second most dominant species (8.2%) among the isolates. A total of 33 isolates (13%) were unidentifiable to species level. M. fortutium was the most frequent species in Ahvaz, Esfahan, Tehran and Gorgan, but M. simiae was the most frequent species in Kermanshah. Thirty-three isolates (20 groups) presented unique genetic features. Further molecular tests should be carried out to reliable identification of these isolates to species level.ConclusionsThe present study provides evidence for the importance of NTM identification in the clinical setting and the presence of diverse species in clinical samples as a causative agent of disease.The presence of diverse species of NTM and unidentifiable strains in a clinical setting in Iran emphasizes the use of sequence analysis of genes for reliable identification

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