Mutation in mgrB is the major colistin resistance mechanism in Klebsiella pneumoniae clinical isolates in Tehran, Iran

Colistin is considered as one of a last resort antimicrobial agent against multidrug-resistant Gramnegative bacteria including Escherichia coli and Klebsiella pneumoniae. However, the recent emergence of colistin resistance (ColR) worldwide that severely restricts therapeutic options is a serious threat to global public health. In this study we have investigated the molecular determinants in ColR K. pneumoniae isolates collected from clinical specimens. A total of 98 E. coli and 195 K. pneumoniae clinical isolates were collected from two hospitals from August 2018 to December 2019 in Tehran, Iran. Colistin susceptibility and minimum inhibitory concentrations (MIC) were determined according to the Clinical and Laboratory Standards Institute by disk diffusion method, and microdilution method, respectively. For isolates with colistin MIC >= 4 mu g mL(-1), PCR was performed for the detection of mcr-1 to mcr-4 genes. Moreover, nucleotide sequences of mgrB, phoP, phoQ, pmrA, and pmrB genes were determined by sequencing. Finally, the transcriptional level of pmrK and pmrC genes was evaluated by quantitative reverse transcription PCR (RT-qPCR). None of the E. coli isolates were resistant to colistin while 21 out 195 K. pneumoniae isolates were identified as resistant, 19 of which carried mutation in the mgrB gene. Three different mutations were observed in the pmrB gene in 3 K. pneumoniae isolates. None of the ColR isolates showed alternations in pmrA, phoP, and phoQ genes. Furthermore, none of the plasmid-encoding genes were detected. Transcriptional level of the pmrK gene increased in all ColR isolates meanwhile, pmrC overexpression was detected in 16 out 21 (76.19%) isolates. Eventually, all ColR isolates were susceptible to tigecycline. Our results demonstrated that the alternation of mgrB gene is the main mechanism related to colistin resistance among ColR K. pneumoniae isolates in this study

Methicillin-Resistant Staphylococcus epidermidis in Iran: A Systematic Review and Meta-Analysis

Objective: Methicillin-resistant Staphylococcus epidermidis (MRSE) remains one of the most prevalent drug-resistant bacteria causing health care infections. Limited data are available about how the frequency of MRSE changed in Iran over the past years. The current study aimed at determining the frequency of MRSE in different cities of Iran. Methods: Databases including Web of Sciences, Scopus, Embase, Medline, and Iranian databases were searched to find studies addressing the frequency of MRSE in Iran published from Mar 2006 to Jan 2016. The data were analyzed using comprehensive meta-analysis version 2.2 (Biostat). Of the 139 records identified in the databases, 15 studies met the inclusion criteria. Results: The analyses showed that the frequency of MRSE infections was 73.9% [95% confidence interval (95% CI) 61.4 - 83.4] among culture-positive cases of S. epidermidis in different parts of Iran. The frequency of MRSE was higher in the studies conducted from 2011 to 2015, based on further stratified analyses. Conclusions: The regular surveillance on antimicrobial susceptibility pattern and formulation of definite antibiotic policy may control high rate of MRSE associated infections in Iran. Moreover, rapid and reliable diagnosis of MRSE isolates and regular screening of the personnel and surfaces of hospitals in terms of MRSE are indispensable

Cloning of the Recombinant Cytochrome P450 Cyp141 Protein of Mycobacterium tuberculosis as a Diagnostic Target and Vaccine Candidate

Background: Tuberculosis has been announced as a global emergency by World Health Organization and the second infectious agent of mortality worldwide. The general policy in the development of new vaccines is to develop some vaccines with higher efficiency not only for infants but also for adults compared with the Bacillus Calmette-Guerin vaccine. Recently, cytochrome P450 cyp141 has been introduced as a new target for detecting Mycobacterium tuberculosis from clinical samples. Objectives: The aim of this study was to clone this gene in order to pave the way for more evaluation. Materials and Methods: M. tuberculosis H37Rv DNA was extracted by a standard phenol-chlorophorm protocol. After designing the specific primers, P450 cyp141 gene was replicated by PCR. The purified PCR products were then subcloned into the pTZ57R/T plasmid vector. After extraction, enzyme digestion, and recombinant pTZ57R/T-cyp141 plasmid vector sequencing, the aforementioned products were cloned into a pET-26b plasmid vector. Then, the recombinant pET26b-cyp141 plasmid molecules were transformed to Escherichia coli strain BL21 (DE3) using the transformation method. Next, the recombinant pET26b-cyp141 plasmids were purified and evaluated by the enzyme digestion analysis. Results: The cloning of P450 cyp141 gene was confirmed by the enzyme digestion and sequencing of the recombinant pTZ57R/T-cyp141 and pET26b-cyp141 plasmid vectors. Conclusions: The results of this study demonstrated that the P450 cyp141 gene was successfully cloned into a pET26b plasmid vector as an expression vector. In this paper, for the first time in Iran, this gene was cloned for more purposes, including the expression and purification of the recombinant cytochrome P450 cyp141 protein

Expression and Purification of the Recombinant Cytochrome P450 CYP141 Protein of Mycobacterium Tuberculosis as a Diagnostic Tool and Vaccine Production

Background: Tuberculosis (TB) is regarded as a health problem worldwide, particularly in developing countries. Mycobacterium tuberculosis (M. tuberculosis) is the cause of this disease. Approximately two billion people worldwide are infected by M. tuberculosis and annually about two million individuals die in consequence. Forty million people are estimated to die because of M. tuberculosis over the next 25 years if the measures for controlling this infection are not extensively developed. In the vaccination field, Bacillus Calmette-Guerin (BCG) is still the most effective vaccine but it shows no efficacy in adult pulmonary patients. One of the other problems regarding TB is its appropriate diagnosis. Objectives: In this experimental study, the recombinant cytochrome P450 CYP141 protein of M. tuberculosis was expressed and purified to be used as a vaccine candidate and diagnostic purpose in subsequent investigations. Materials and Methods: The optimization of the cytochrome P450 CYP141 protein expression was evaluated in different conditions. Then, this protein was purified with a resin column of nickel-nitrilotriacetic acid and investigated via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting. Results: The highest expression of the cytochrome P450 CYP141 protein was obtained by the addition of 1 mM of isopropyl beta-D-1-thiogalactopyranoside (IPTG) to the bacterial culture grown to an optical density at 600 nm (OD600) of 0.6, 16 hours after induction. This protein was subsequently purified with a purification of higher than 80%. The results of Western Blotting indicated that the purified protein was specifically detected. Conclusions: In this experimental study, for the first time in Iran the expression and purification of this recombinant protein was done successfully. This recombinant protein could be used as a vaccine candidate and diagnostic purpose in subsequent investigations

First report of coexistence of AmpC beta-lactamase genes in Klebsiella pneumoniae strains isolated from burn patients

Klebsiella spp. are among the most frequently isolated bacteria from burn wounds. These organisms are among the most important opportunistic pathogens, causing hospital-acquired and healthcare-associated infections worldwide. Limited information is available about prevalence of AmpC-producing Klebsiella pneumoniae from burn patients. Therefore, the aim of this study was to determine the characterization of AmpC beta-lactamase among K. pneumoniae isolated from burn patients. Samples were collected from wound specimens of patients with burn injury from a burn hospital in Tehran during 18 months (March 2015 to August 2016). For phenotypic detection of AmpC beta-lactamase, disk diffusion method with cefoxitin was used for screening, AmpC disk test and boronic acid inhibitor-based method were used as confirmatory tests. Polymerase chain reaction (PCR) was performed to screen all isolates with AmpC genes including ACCM, DHAM, EBCM, FOXM, MOXM, and CITM. Finally, PCR products were validated using sequencing. During this study, 102 isolates of K. pneumoniae were collected. Among these isolates, 52.9% suspected as AmpC producer by disk agar diffusion cefoxitin screening method. By confirmatory phenotypic methods, 19.6% of isolates considered as AmpC producer. Molecular analysis revealed 43.1% of cefoxitin-resistant isolates harbored at least one of the AmpC genes including CITM (22.5%), EBCM (21.5%), DHAM (7.8%), and FOXM (0.98%). In addition, 5.8% of isolates harbored two AmpC genes and 2.9% harbored three AmpC genes. In conclusion, K. pneumoniae is becoming a serious problem in burn patients. Accurate and precise methods and guidelines should be designed for detection of antibiotic-resistant mechanisms. Our data showed the high rate of AmpC beta-lactamase among K. pneumoniae isolated from burn patients, which limit the treatment options. Therefore, the results of this study can provide evidence to help for appropriate treatment of burn patients

Exploring the interplay between Fusobacterium nucleatum with the expression of microRNA, and inflammatory mediators in colorectal cancer

BackgroundFusobacterium nucleatum has been recognized as an important key bacterium in the cause and spread of colorectal carcinogenesis. Nevertheless, the clinical relevance of F. nucleatum in colorectal cancer (CRC) and its effect on immune factors and the tumor microenvironment have not been fully elucidated.Materials and methodsThe frequency of F. nucleatum was measured in 100 paired tumor and normal tissue specimens by TaqMan quantification Real-Time Polymerase Chain Reaction (qPCR). The mRNA expression levels of cytokines (IL-6, IL-10, IL-12β, IL-17, TNF-α, TLR-2, and TLR-4), and miRNAs (miR-21, miR-31) were examined. Eventually, any potential correlations between the molecular and clinicopathological features of the neoplastic samples and the abundance of F. nucleatum were analyzed.ResultsThe relative frequency of F. nucleatum was significantly increased in cancerous tissue compared to adjacent non-tumor tissues. Furthermore, the high level of F. nucleatum was significantly associated with histological grade III and IV CRC tissues (P = 0.027 and P = 0.022, respectively) and perineural invasion-positive patients (P = 0.037). In addition, the expression levels of IL-6, IL-17, TNF-α,IL-12β, TLR-2, and TLR-4 as well as miR-21 and miR-31 showed a significant increase in the cancer group. A notable correlation was also observed between the high status of F. nucleatum and the expression of IL-6, TNF-α and miR-21.ConclusionOur results emphasize the importance of F. nucleatum and changes in the expression of genes involved in CRC. Studying the microbial profile and gene expression changes in CRC patients may be a promising approach to improve screening methods and provide therapeutic strategies

High frequency of methicillin-resistant Staphylococcus aureus (MRSA) with SCCmec type III and spa type t030 in Karaj’s teaching hospitals, Iran

Methicillin-resistant Staphylococcus aureus (MRSA) has been one of the most important antibiotic-resistant pathogen in many parts of the world over the past decades. This cross-sectional study was conducted to investigate MRSA isolated between July 2013 and July 2014 in Karaj, Iran. All tested isolates were collected in teaching hospitals from personnel, patients, and surfaces and each MRSA was analyzed by SCCmec and spa typing. Antibiotic susceptibility testing was accomplished by disk diffusion method. Out of 49 MRSA isolates from the Karaj’s teaching hospitals, 82%, 10%, and 6% of the isolates were SCCmec types III, II, and I, respectively. The main spa type in this study was spa t030 with frequency as high as 75.5% from intensive care unit (ICU) of the hospitals and high rate of resistance to rifampicin (53%) was found in MRSA isolates. In conclusion, high frequency of spa t030 with SCCmec type III and MRSA phenotype illustrated circulating of one of the antibiotic-resistant strains in ICU of Karaj’s teaching hospitals and emphasizes the need for ongoing molecular surveillance, antibiotic susceptibility monitoring, and infection control

Detection of tetracycline resistance genes, aminoglycoside modifying enzymes, and coagulase gene typing of clinical isolates of Staphylococcus aureus in the Southwest of Iran

Objective(s): The aim of the present study was to determine the aminoglycoside modifying enzymes (AMEs) encoded genes, tetracycline resistance genes, and the coa based typing of Staphylococcus aureus isolates in the Southwest of Iran. Materials and Methods: Antimicrobial susceptibility of isolates was carried out by agar disk diffusion methods. Two sets of multiplex PCR mixture were used for detection of AME genes and tet genes.  All of the isolates were typed with the coagulase gene typing method. Of the 121 isolates, 29.75% and 47.93% were resistant to at least one aminoglycosides and tetracyclines, respectively. Results: The aac(6')-Ie-aph(2'') was the most frequent gene (97.22%), and aph (3')-IIIa and ant (4')-Ia genes were detected in 61.11% and 11.11% of aminoglycoside resistant isolates, respectively. The tetK and tetM genes were detected in 82.75% and 56.9% of tetracycline resistant isolates, respectively. Overall 31.4% of isolates were MRSA. Totally 17 distinct coa gene RFLP patterns, numbered C1 to C17, were observed.  The C5 was the most frequent coa type with 31 isolates. Conclusion: The aac(6')-Ie-aph(2'') and aph (3')-IIIa genes were the most important genes contributing to aminoglycosides resistance, while resistance to tetracyclines was mediated by tetK and tetM genes. Interestingly all S. aureus with C5 as the most prevalent coa-type were resistant to at least one of the aminoglycoside antibiotics and tetracycline simultaneously. Moreover, 30 out of 31 isolates with this coa type were MRSA, indicating the importance of the C5 coa-type in MRSA strains and also in isolates that were resistant to aminoglycosides and tetracycline