Rapid DNA extraction of bacterial genome using laundry detergents and assessment of the efficiency of DNA in downstream process using polymerase chain reaction

Genomic DNA extraction from bacterial cells involves processes normally performed in most biological laboratories. Therefore, various methods have been offered, manually and kit, but these methods may be time consuming and costly. In this paper, genomic DNA extraction of Pseudomonas aeruginosa was investigated using some laundry detergent brands available in Iran. Afterwards, efficiency of the detergents was compared with manually standard methods and kits. To evaluate the efficiency of the genomic DNA in the processes in which DNA is used as a template, the polymerase chain reaction (PCR) tests and enzyme digestion of PCR product were used. The results show that the detergents could be used to extract genomic DNA. Among the brands studied, five-enzyme Taj and three-enzyme Saftlan had the best performance compared to standard methods.Key words: Bacterial genome, DNA extraction, laundry powder, detergent

Evaluation of polymyxin b susceptibility profile and detection of drug resistance genes among Acinetobacter baumannii clinical isolates in tehran, iran during 2015-2016

Acinetobacter baumannii is an important opportunistic pathogen, responsible for approximately 10 of all gram-negative nosocomial infection. The aim of this study was to determine aminoglycoside and quinolone resistance genes and their antimicrobial susceptibility profile in the clinically A. baumannii. In this cross-sectional study, a total of 100 nonduplicative A. baumannii isolates were collected from different clinical samples. Antimicrobial susceptibility test was performed by disk diffusion method. QnrA, anrB, qnrS, aac(3)-IIa, and aac(6')-Ib genes were identified using PCR method. The results of antibiotic susceptibility test showed that polymyxin B was the most effective antimicrobial against A. baumannii. 97, 95 and 82 of isolates were resistant to cefepime, ceftriaxone, and amikacin, respectively. The molecular distribution of aac(3)-IIa, aac(6')-Ib, and qnrA genes were 45, 50, and 50 of isolates, respectively. However, qnrB and qnrS genes could not be detected in any strain. This study showed that polymyxin B was the best drug against A. baumannii clinical isolates. This data is also valid for polymyxin E (colistin), which is mostly used in clinics. There is a high level of resistance genes among clinical A. baumannii isolates. This high prevalence rate highlights the necessity for the development of rapid diagnostic assays and continuous monitoring of antibiotic resistance. © 2018, Mediterranean Journal of Hematology and Infectious Diseases

Molecular typing of Brucella species isolated from humans and animals using polymerase chain reaction-restriction fragment length polymorphism technique

Background: Brucellosis is a zoonotic disease that causes major economic and public health problems. It is one of the most important diseases in humans and domestic animals. Hence, the exact identification of Brucella spp. is important for strategies of treatment and control. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is one of the molecular techniques characterized by amplification of deoxyribonucleic acid (DNA) sequence and restriction enzyme digestion. Objectives: This study aimed at identifying genetic polymorphisms of omp2a genes among 90 Brucella isolated from humans and animals, using the PCR-RFLP method. Methods: Ninety Brucella spp. isolated from humans and animals in two different regions of Iran were used in this study. Biochemical tests and the Brucella omp2a (1100 bp) gene-PCR was used for identification of Brucella isolates. Polymerase Chain Reaction products were digested by restriction endonuclease enzyme pstI and gene sequencing analysis was carried out for molecular typing of Brucella strains. Therefore, genetic relatedness was revealed by a dendrogram. Results: Analysis of the 90 Brucella strains by biochemical tests, PCR, and PCR-RFLP methods with PstI enzyme and omp2a sequencing showed four unique RFLP Profiles (P1-P4). Seventy-nine (87.8) of the Brucella isolates belonged to B. melitensis strain 20236. From 30 animal isolates, nine (30) belonged to B. melitensis biovare1 and two (6.6) to B. abortus strain. According to the RFLP dendrogram, group 1 and 2 had higher genetic relatedness similarity. Conclusions: The results showed B. melitensis strain 20236 was the predominant strain among human and animal Brucella isolates. Likewise, according to dendrogram results, the PCR-RFLP technique was not able to separate human and animal species of B. melitensis from B. abortus. © 2018, Archives of Clinical Infectious Diseases

Molecular Detection and Evaluation of ML Resistance M. Pneumoniae Associated with Mutation in 23S RNA Gene among Iranian Patients with Respiratory Infections

Background: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia. The global increased resistance of M. pneumoniae strains to macrolide (ML) has become a worrisome health problem. The widespread use of these medications has led to increased rate of reported ML-resistant M. pneumoniae (MRMP) throughout the world. This study was aimed to evaluate the resistance of M. pneumoniae against erythromycin due to mutations in the 23S rRNA gene of patients with respiratory infections in Iran. Methods: In this study, 100 samples of throat swab from a patient with respiratory problems were collected. After the cultured of all samples in M. pneumonia-specific PPLO medium, PCR technique was performed with specific primers. Afterwards, the broth micro-dilution MIC assay was employed. Finally, the PCR product of the 23S rRNA gene was sequenced to detect mutations of domain V in 23S rRNA gene of MRMP. Results: It was found that 17 cases (17) were positive for mycoplasma genus and six cases (6) positive for M. pneumoniae species. Also, analysis of the sequence of 23S rRNA gene, revealed that one of the samples had mutations at positions A2431G and G2491A. All positive samples M. pneumoniae with 23S rRNA gene were sensitive to erythromycin. Conclusion: These use of these antibiotics should be limited to prevent the emergence of MRMP in Iran

Diversity of virulence genes in Brucella melitensis and Brucella abortus detected from patients with rheumatoid arthritis

The presence of Brucella melitensis and Brucella abortus genomes were investigated in the synovial fluid (SF) samples from 90 patients with rheumatoid arthritis (RA). DNA extraction and PCR assay were performed for simultaneous identification and discrimination of B. melitensis and B. abortus from the SF using three specific primers. After gel electrophoresis, the PCR products were confirmed by DNA sequencing. The cbg, omp31, manA, virB, and znuA virulence genes typing were performed by multiplex-PCR. Of the 90 samples, 14 were positive for B. melitensis (n = 9; 10) and B. abortus (n = 5; 5.5). The virulotyping of positive samples revealed the presence of all five virulence genes in B. melitensis. The virB, cbg, and otn31 were detected in all five samples of B. abortus. In addition, zhuA and manA were detected in three (60) and four (80) samples, respectively, of the B. abortus-positive samples. Moreover, a total of 94.2 and 89.2 of the 14 positive samples were also found positive for manA and znuA, respectively. Our findings revealed that the Brucella spp. genomes can be detected in the SF of RA patients by the PCR-based method. We thus suggest that physicians should consider the Brucella spp. as indicators of potential RA for the timely diagnosis and treatment of RA

Determining the patterns of antimicrobial susceptibility and the distribution of blaCTX-M genes in strains of Acinetobacter Baumannii isolated from clinical samples

Background: Nowadays, multidrug resistant (MDR) Acinetobacter baumannii (A. baumannii) strains are as one of the problematic opportunistic pathogens, especially in intensive care units in the world. The purpose of current study was to define the antibiotic susceptibility patterns and detect the prevalence of producing strains of extended-spectrum β-lactamase (ESBL) and the appearance of betalactamase CTX-M-II enzymes in A. baumannii strains. Methods: This study was conducted in 3 major hospitals in Tehran, Iran, on 500 clinical samples during one year. After the identification of isolates in species, test sensitivity was carried out to 4 antibiotics by using the disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines; also minimum inhibitory concentrations (MIC) was determined for cefepime, cefotaxime and ceftazidime; and finally, to identify the producing strains of ESBL, phenotypic method of combined-disk was used and then, isolates were considered for presence of blaCTX-M-II gene by polymerase chain reaction (PCR) assay. Findings: 130 Acinetobacter species were isolated from the patients. The majority of isolates was from blood specimens and revealed the highest resistance to cefepime and ceftriaxone. The MIC of cefepime in 91, for ceftazidime in 84, and for cefotaxime in 80 of the studied isolates was more than 128 μg/ml. The results of the combined-disk test demonstrated that 20 of samples were ESBL positive. The PCR results showed that 19 of our isolates had bla CTX-M-II gene. Conclusion: Multidrug resistant (MDR) A. baumannii is widespread in Iran and is considered as hazard risk for hospitalized patients; also by virtue of the results of this survey, there are more significant mechanisms than ESBL bacteria such as secretory pumps and changes in purine resistance which causes more resistance, too, and should be consider

Molecular genotyping of Acinetobacter baumannii species isolated from patients in Tehran, Iran, by repetitive element PCR fingerprinting

Background & objective: Acinetobacter baumannii is an opportunistic pathogen with high pathogenic and antibiotic-resistance potential and is also considered as one of the main nosocomial agents, specifically in the intensive care units (ICUs). It is highly important to use molecular biology methods in the epidemiological studies, determine the source of infection, and understand the relationships and distributional patterns of pathogens. Therefore, the current study aimed to determining the similar molecular types in the A. baumannii species isolated from patients in Tehran, Iran, by the repetitive element PCR fingerprinting (REP-PCR) method. Methods: A total of 350 clinical samples were collected from patients admitted to different hospital in Tehran, assessed to identify Acinetobacter spp., based on the special culture media and biochemical test results. The resistance of isolates was evaluated against 11 different antibiotics. The cefepime and ceftazidime were assessed by the minimum inhibitory concentration (MIC) method, based on serial dilutions. The genome of isolated strains was extracted using the modified boiling method and amplified in REP-PCR technique using specific primers. Results: In the current study, out of 120 isolates of Acinetobacter spp., 100 (76.9) were identified as A. baumannii, mostly from ICUs and infectious diseases wards. The isolates of A. baumannii in the current study mostly showed antimicrobial resistance against cefepime and ceftazidime, and had the highest sensitivity to polymyxin B. About 70 of A. baumannii isolates in the current study were resistant to 3 or more antibiotics. According to dendrogram analyses, the patterns were classified to A-I with the maximum population (36) of group A. All genotypes of Acinetobacter spp. in the current study showed resistance against carbapenems and aminoglycosides. Conclusion: High similarities between the isolates in the current study indicated the high distribution of A. baumannii species in the hospitals of Tehran. © 2018, IRANIAN JOURNAL OF PATHOLOGY

Immunogenic and protective antigens of Brucella as vaccine candidates

Brucella is an intracellular pathogen that causes abortion in domestic animals and undulant fever in humans. Due to the lack of a human vaccine against brucellosis, animal vaccines play an important role in the management of animal and human brucellosis for decades. Strain 19, RB51 and Rev1 are the approved Brucella spp. vaccine strains that are most commonly used to protect livestock against infection and abortion. However, due to some disadvantages of these vaccines, numerous studies have been conducted for the development of effective vaccines that could also be used in other susceptible animals. In this review, we compare different aspects of immunogenic antigens that have been a candidate for the brucellosis vaccine. © 2019 Elsevier Lt

Molecular Detection and Evaluation of ML- Resistance M. Pneumoniae Associated with Mutation in 23S RNA Gene among Iranian Patients with Respiratory Infections

Background: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia. The global increased resistance of M. pneumoniae strains to macrolide (ML) has become a worrisome health problem. The widespread use of these medications has led to increased rate of reported ML-resistant M. pneumoniae (MRMP) throughout the world. This study was aimed to evaluate the resistance of M. pneumoniae against erythromycin due to mutations in the 23S rRNA gene of patients with respiratory infections in Iran. Methods: In this study, 100 samples of throat swab from a patient with respiratory problems were collected. After the cultured of all samples in M. pneumonia-specific PPLO medium, PCR technique was performed with specific primers. Afterwards, the broth micro-dilution MIC assay was employed. Finally, the PCR product of the 23S rRNA gene was sequenced to detect mutations of domain V in 23S rRNA gene of MRMP. Results: It was found that 17 cases (17) were positive for mycoplasma genus and six cases (6) positive for M. pneumoniae species. Also, analysis of the sequence of 23S rRNA gene, revealed that one of the samples had mutations at positions A2431G and G2491A. All positive samples M. pneumoniae with 23S rRNA gene were sensitive to erythromycin. Conclusions: These use of these antibiotics should be limited to prevent the emergence of MRMP in Iran