Unveiling the impact of antibiotic stress on biofilm formation and expression of toxin-antitoxin system genes in Clostridium difficile clinical isolates.

OBJECTIVES: The study investigates how antibiotics affect biofilm formation and toxin gene expression in Clostridium difficile, which is essential for its survival and persistence. METHODS: The study confirmed 25 strains of C. difficile and assessed biofilm formation. The MIC of metronidazole and vancomycin was determined through agar dilution, and the impact of sub-MIC levels on biofilm formation and eradication was investigated. Additionally, Real-time PCR was used to analyze the expression levels of target genes related to antibiotic treatment. RESULTS: We found that certain genes, such as the ImmA/IrrE system, were associated with increased biofilm formation in isolates. Sub-MIC antibiotic levels influenced gene expression related to biofilm activities, particularly emphasizing the importance of toxin-antitoxin systems. The results suggest that antibiotics at sub-MIC levels may play a signaling role in promoting biofilm formation and gene expression in C. difficile. CONCLUSION: Our study suggests that toxin and antitoxin genes may impact C. difficile biofilm formation, while antibiotics could signal biofilm strengthening and gene expression increase.

Investigating the Effectiveness of mqsR-Peptide Nucleic Acid as a Novel Solution for the Eradication of Persister Cells in Clinical Isolates of Escherichia coli.

BACKGROUND: Bacterial persisters are non- or slow-growing phenotypic variants that may be responsible for recalcitrance and relapse of persistent infections and antibiotic failure. In Escherichia coli, mqsRA is a well-known type II toxin-antitoxin system associated with persister cell formation. This study aimed to investigate the efficiency of an antisense peptide nucleic acid (PNA) targeting mqsRA in eliminating E. coli persisters. METHODS: The study included 600 non-duplicated urine samples from adult patients with suspected urinary tract infections. The isolates were subjected to antimicrobial susceptibility testing and bacterial persister cells assay. The presence of mqsRA in the isolates was evaluated by polymerase chain reaction. Finally, expression of the mqsR and mqsA genes was assessed after exposure to normal conditions, stress, and different concentrations of mqsR-PNA (1 - 35 µM). RESULTS: The mqsR gene was significantly overexpressed under stress conditions, which was compensated by the PNA treatment. Complete inhibition of E. coli persister cells was achieved after overnight treatment with the anti-mqsR-PNA at concentrations ≥ 15 µM. CONCLUSIONS: The growth of E. coli persister cells can be inhibited by the anti-mqsR-PNA. Further studies are required to evaluate the effectiveness of this antisense PNA in both preclinical and clinical settings.

Antibody-Antibiotic Conjugates: A Comprehensive Review on Their Therapeutic Potentials Against BacterialInfections.

INTRODUCTION: Antibodies are significant agents in the immune system and have proven to be effective in treating bacterial infections. With the advancement of antibody engineering in recent decades, antibody therapy has evolved widely. AIM: This review aimed to investigate a new method as a therapeutic platform for the treatment of bacterial infections and explore the novel features of this method in conferring pathogen specificity to broad-spectrum antibiotics. MATERIAL AND METHODS: A literature review was conducted addressing the following topics about antibody-antibiotic conjugates (AACs): (1) structure and mechanism of action; (2) clinical effectiveness; (3) advantages and disadvantages. RESULT: Antibody conjugates are designed to build upon the progress made in the development of monoclonal antibodies for the treatment of diseases. Despite the growing emergence of antibiotic resistance among pathogenic bacteria worldwide, novel antimicrobials have not been sufficiently expanded to combat the global crisis of antibiotic resistance. A recently developed strategy for the treatment of infectious diseases is the use of AACs, which are specifically activated only in host cells. CONCLUSION: A novel therapeutic AAC employs an antibody to deliver the antibiotic to the bacteria. The AACs can release potent antibacterial components that unconjugated forms may not exhibit with an appropriate therapeutic index. This review highlights how this science has guided the design principles of an impressive AAC and discusses how the AAC model promises to enhance the antibiotic effect against bacterial infections.

Tetracyclines resistance in Mycoplasma and Ureaplasma urogenital isolates derived from human: a systematic review and meta-analysis.

BACKGROUND: Urogenital Mycoplasma infections are considered an important public health problem, owing to the presence of antibiotic resistance or decreased susceptibility, the treatment options are limited. OBJECTIVE: Therefore, this meta-analysis aimed to estimate resistance rates of genital Mycoplasmas to tetracyclines (tetracycline, doxycycline, and minocycline). METHODS: We searched the relevant published studies in PubMed, Scopus, and Embase until 3, March 2022. All statistical analyses were carried out using the statistical package R. RESULTS: The 26 studies included in the analysis were performed in 15 countries. In the metadata, the proportions of tetracycline, doxycycline, and minocycline resistance in Mycoplasma and Ureaplasma urogenital isolates were reported 14.2% (95% CI 8.2-23.2%), 5% (95% CI 3-8.1%), and 11.9% (95% CI 6.3-21.5%), respectively. According to the meta-regression, the tetracycline and minocycline resistance rate decreased over time. Although, the doxycycline resistance rate increased over time. There was a statistically significant difference in the tetracyclines resistance rates between different continents/countries (P < 0.05). CONCLUSION: The prevalence rate and antibiotic susceptibility profiles vary geographically. Therefore, rigorous or improved antimicrobial stewardship, contact tracing, and enhanced intensive surveillance systems are necessitated for preventing the emergence and further spreading of tetracyclines resistance in genital Mycoplasmas.

Molecular characterizations of antibiotic resistance, biofilm formation, and virulence determinants of Pseudomonas aeruginosa isolated from burn wound infection.

BACKGROUND: Burn injuries result in disruption of the skin barrier against opportunistic infections. Pseudomonas aeruginosa is one of the main infectious agents colonizing burn wounds and making severe infections. Biofilm production and other virulence factors along with antibiotic resistance limit appropriate treatment options and time. MATERIALS AND METHODS: Wound samples were collected from hospitalized burn patients. P. aeruginosa isolates and related virulence factors identified by the standard biochemical and molecular methods. Antibiotic resistance patterns were determined by the disc diffusion method and ß-lactamase genes were detected by polymerase chain reaction (PCR) assay. To determine the genetic relatedness amongst the isolates, enterobacterial repetitive intergenic consensus (ERIC)-PCR was also performed. RESULTS: Forty P. aeruginosa isolates were identified. All of these isolates were biofilm producers. Carbapenem resistance was detected in 40% of the isolates, and blaTEM (37/5%), blaVIM (30%), and blaCTX-M (20%) were the most common ß-lactamase genes. The highest resistance was detected to cefotaxime, ceftazidime, meropenem, imipenem and piperacillin, and 16 (40%) isolates were resistant to these antibiotics. The minimum inhibitory concentrations (MIC) of colistin was lower than 2 µg/mL and no resistance was observed. Isolates were categorized to 17 MDR, 13 mono-drug resistance, and 10 susceptible isolates. High genetic diversity was also observed among the isolates (28 ERIC types) and most carbapenem-resistant isolates were classified into four main types. CONCLUSION: Antibiotic resistance, particularly carbapenem resistance was considerable among the P. aeruginosa isolates colonizing burn wounds. Combining carbapenem resistance with biofilm production and virulence factors would result in severe and difficult-to-treat infections.

Tigecycline antibacterial activity, clinical effectiveness, and mechanisms and epidemiology of resistance: narrative review.

Tigecycline is unique glycylcycline class of semisynthetic antimicrobial agents developed for the treatment of polymicrobial infections caused by multidrug-resistant Gram-positive and Gram-negative pathogens. Tigecycline evades the main tetracycline resistance genetic mechanisms, such as tetracycline-specific efflux pump acquisition and ribosomal protection, via the addition of a glycyclamide moiety to the 9-position of minocycline. The use of the parenteral form of tigecycline is approved for complicated skin and skin structure infections (excluding diabetes foot infection), complicated intra-abdominal infections, and community-acquired bacterial pneumonia in adults. New evidence also suggests the effectiveness of tigecycline for the treatment of severe Clostridioides difficile infections. Tigecycline showed in vitro susceptibility to Coxiella spp., Rickettsia spp., and multidrug-resistant Neisseria gonnorrhoeae strains which indicate the possible use of tigecycline in the treatment of infections caused by these pathogens. Except for intrinsic, or often reported resistance in some Gram-negatives, tigecycline is effective against a wide range of multidrug-resistant nosocomial pathogens. Herein, we summarize the currently available data on tigecycline pharmacokinetics and pharmacodynamics, its mechanism of action, the epidemiology of tigecycline resistance, and its clinical effectiveness.

Heteroresistance to clarithromycin and metronidazole in patients with a Helicobacter pylori infection: a systematic review and meta-analysis.

BACKGROUND: Antimicrobial resistance of H. pylori can lead to treatment failure. Importantly, several studies have reported on heteroresistance, i.e. the presence of resistant and susceptible H. pylori populations in the same sample and/or a difference in the susceptibility patterns between biopsy samples. This meta-analysis aims to provide comprehensive data on the prevalence of metronidazole and clarithromycin heteroresistance and the approaches to their detection. MATERIAL AND METHODS: A systematic review was performed after the search of MEDLINE, Scopus and Web of Science. The study outcomes were the weighted pooled prevalence of heteroresistance to clarithromycin and metronidazole in H. pylori positive samples and/or isolates with a subanalysis by continent. RESULTS: A total of 22 studies that had investigated 3852 H. pylori positive patients were included in the meta-analysis. Heteroresistance to clarithromycin was reported in 20 studies, with a weighted pooled prevalence of 6.8% (95% CI 5.1-8.6; 3654 H. pylori positive patients; the substantial heterogeneity I2 = 55.6%). Heteroresistance to metronidazole was reported in 12 studies, with a weighted pooled prevalence of 13.8% (95% CI 8.9-18.6; 1670 H. pylori positive patients; the substantial heterogeneity I2 = 60.9%). The weighted pooled prevalence of clarithromycin heteroresistance was similar in Asia and Europe (p = 0.174584), however, metronidazole heteroresistance was detected more often in Europe (p < 0.00001). Clarithromycin heteroresistance was detected more often by phenotype rather than by using genotyping methods (12 vs 8 studies), whereas heteroresistance to metronidazole was detected only by phenotype. CONCLUSION: The prevalence of heteroresistance to clarithromycin and/or metronidazole is not negligible and can be detected in approximately 7 and 14% of H. pylori positive samples, respectively. These findings highlight the need to raise the awareness of gastroenterologists and microbiologists to the heteroresistance to clarithromycin and metronidazole in patients with a H. pylori infection.

Antibacterial activity of recently approved antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) strains: A systematic review and meta-analysis.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infections are considered an important public health problem, and treatment options are limited. Accordingly, in this meta-analysis, we analyzed published studies to survey in vitro activity of recently approved antibiotics against MRSA isolates. METHODS: We searched electronic databases; PubMed, Scopus, and Web of Science to identify relevant studies (until November 30, 2020) that have focused on the in vitro activity of telavancin, dalbavancin, oritavancin, and tedizolid against MRSA isolates. Statistical analyses were conducted using STATA software (version 14.0). RESULTS: Thirty-eight studies were included in this meta-analysis. Overall in vitro activity of tedizolid on 12,204 MRSA isolates was 0.250 and 0.5 µg/mL for MIC50 and MIC90, (minimum inhibitory concentration at which 50% and 90% of isolates were inhibited, respectively), respectively. The overall antibacterial activity of dalbavancin on 28539 MRSA isolates was 0.060 and 0.120 µg/mL for MIC50 and MIC90, respectively. The overall antibacterial activity of oritavancin on 420 MRSA isolates was 0.045 and 0.120 µg/mL for MIC50 and MIC90, respectively. The overall antibacterial activity of telavancin on 7353 MRSA isolates was 0.032 and 0.060 µg/mL for MIC50 and MIC90, respectively. The pooled prevalence of tedizolid, telavancin, and dalbavancin susceptibility was 100% (95% CI: 100-100). CONCLUSION: Telavancin, dalbavancin, oritavancin, and tedizolid had potent in vitro activity against MRSA isolates. The low MICs and high susceptibility rates of these antibiotics recommend a hopeful direction to introduce useful antibiotics in treating MRSA infections in the future.

Global status of Azithromycin and Erythromycin Resistance Rates in Neisseria gonorrhoeae: A Systematic Review and Meta-analysis.

Background: The widespread development of antibiotic resistance or decreased susceptibility in Neisseria gonorrhoeae (NG) infection is a global and significant human public health issue. Objectives: Therefore, this meta-analysis aimed to estimate worldwide resistance rates of NG to the azithromycin and erythromycin according to years, regions, and antimicrobial susceptibility testing (AST). Methods: We systematically searched the published studies in PubMed, Scopus, and Embase from 1988 to 2021. All analyses were conducted using Stata software. Results: The 134 reports included in the meta-analysis were performed in 51 countries and examined 165,172 NG isolates. Most of the included studies were from Asia (50 studies) and Europe (46 studies). In the metadata, the global prevalence over the past 30 years were 6% for azithromycin and 48% for erythromycin. There was substantial change in the prevalence of macrolides NG resistance over time (P <0.01). In this metadata, among 58 countries reporting resistance data for azithromycin, 17 (29.3%) countries reported that >5% of specimens had azithromycin resistance. Conclusions: The implications of this study emphasize the rigorous or improved antimicrobial stewardship, early diagnosis, contact tracing, and enhanced intensive global surveillance system are crucial for control of further spreading of gonococcal emergence of antimicrobial resistance (AMR).

The potential use of theranostic bacteria in cancer.

Conventional chemotherapy approaches have not been fully successful in the treatment of cancer, due to limitations imposed by the pathophysiology of solid tumors, leading to nonspecific drug uptake by healthy cells, poor bioavailability, and toxicity. Thus, novel therapeutic modalities for more efficient cancer treatment are urgently required. Living bacteria can be used as a theranostic approach for the simultaneous diagnosis and therapy of tumors. Herein, we summarize the currently available literature focused on the advantages and challenges for the use of theranostic bacteria in cancer therapy.

Antibiotic heteroresistance in Mycobacterium tuberculosis isolates: a systematic review and meta-analysis.

BACKGROUND: Mycobacterium tuberculosis (MTB) is responsible for tuberculosis; that continues to be a public health threat across the globe. Furthermore, increasing heteroresistance (HR)-the presence of resistant and susceptible isolates among MTB strains- has been reported from around the world. This phenomenon can lead to full resistance development and treatment failure. METHODS: We systematically searched the relevant studies in PubMed, Scopus, and Embase (Until October 21, 2020). The study outcomes revealed the weighted pooled prevalence of antibiotic HR in MTB isolates with subgroup analysis by year, quality of study, and heteroresistance detection method. RESULTS: A total of 38 studies which had investigated MTB isolates were included in the meta-analysis. Geographically, the highest number of studies were reported from Asia (n = 24), followed by Africa (n = 5). Nineteen studies reported HR to isoniazid, with a weighted pooled prevalence of 5% (95% CI 0-12) among 11,761 MTB isolates. Also, there is no important trend for the subgroup analysis by the study period (2001-2014 vs 2015-2017 vs 2018-2020). HR to rifampin was reported in 17 studies, with a weighted pooled prevalence of 7% (95% CI 2-14) among 3782 MTB isolates. HR to fluoroquinolone and ethambutol were reported in 12 and 4 studies, respectively, with weighted pooled prevalence of 10% and 1% among 2153 and 1509 MTB isolates, correspondingly. CONCLUSION: Based on our analysis, HR in MTB isolates with different frequency rate is present worldwide. Thus, the selection of appropriate and reliable methods for HR detection is crucial for TB eradication.

Clonal Lineage Analysis of Shigella flexneri Isolates Circulating in Ahvaz, Iran.

BACKGROUND: Shigellosis is a significant public health challenge particularly in developing countries, and the spread of antibiotic resistance genes through integron structures has become an important problem in the treatment of Shigellosis. The aim of this study was to investigate the genetic association of Shigella flexneri antibiotic resistant clones collected from Ahvaz between 2013 and 2015 by pulse field gel electrophoresis (PFGE) method. METHODS: A total of 45 S. flexneri isolates, which were resistant to ampicillin and cotrimoxazole, were obtained from patients with Shigellosis referred to Ahvaz hospitals during 2013 - 2015. PCR was performed to evaluate the frequency of Sul1, int1, blaOXA, and int2 genes. In addition, pulse field gel electrophoresis method was used to investigate the genetic relationship between 40 S. flexneri isolates. RESULTS: PCR results showed that the highest frequency was related to the sul1 gene with 80% (36 isolates) and the lowest frequency was related to class 2 integron with 15.5% (7 isolates); 31.11% (14 isolates) of the isolates were sul1 and int1. Also, 13.33% (6 isolates) had blaOXA and int1 genes, simultaneously. But none of the isolates had class 1 integrons and class 2 integrons at the same time. PFGE results showed 25 different pulsotype patterns, of which 16 isolates had their own unique pattern and were divided into 16 pulsotypes, and 27 isolates were divided into 9 pulsotypes. CONCLUSIONS: int2 and sul1 resistance genes had an upward trend from 2013 to 2015 and the results of PFGE indicated a different origin of S. flexneri clones.

Simultaneous Molecular Detection of Common Bacterial Enteropathogens in Children with Diarrhea by Multiplex-PCR Assay.

BACKGROUND: Diarrhea remains a major threat to children in low- and middle-income countries, which is the second cause of death among children in the world. The aim of the present study was to develop and evaluate a multiplex-PCR assay for direct detection of common bacterial enteropathogens in fecal specimens. METHODS: One hundred and three stool specimens were collected from children under 5 years of age with gastroenteritis during a six-month period in Ilam, Iran. The multiplex PCR assay simultaneously detected Shigella spp., Campylobacter jejuni, Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), and Salmonella enterica in stool samples. RESULTS: Our results demonstrated that the prevalence of Shigella spp. Campylobacter jejuni, EPEC, ETEC, and Salmonella enterica were 21.35%, 10.67%, 1.94%, 0.97% and 0%, respectively. In addition, Shigella spp. with Campylobacter jejuni and EPEC with Campylobacter jejuni coinfection were observed in sample 11 (10.67%). The analytical sensitivity of the multiplex PCR assay was estimated to be 0.01 ng/µL of genomic DNA from culture. The analytical specificity was determined to be 100% by using common and standard enteropathogenic bacterial strains. CONCLUSIONS: The molecular method developed in the study was rapid, sensitive, and specific for detection of common bacterial enteropathogens.

Assessment of Zinc Nanoparticle Effect and Expression of Zinc Uptake Gene in Drug Resistance Acinetobacter baumannii Strain FMHLN5.

BACKGROUND: Drug resistance bacteria pose an increasing threat to public health. Antimicrobial agents often lack efficacy toward recently developed drug resistance bacteria. Nanoparticles are one of the most effective treatment agents. The aim of this study was to evaluate the expression of zinc uptake regulator gene with zinc nanoparticle stress in drug resistant Acinetobacter baumannii strain FMHLN5. METHODS: The antimicrobial susceptibility technique was evaluated through disk diffusion methods. ZnO nanoparticles (ZnO-NPs) were synthesized using an acetate precursor-based sol-gel route. In vitro, the FMHLN5 strain susceptibility to ZnO-NPs has been tested by the agar wells diffusion method, using varying sizes (20 to 520 nm) of ZnO NPs. The purities and sizes of Nano-ZnO was determined by X-ray diffraction (XRD) and scanning electron microscope (SEM). The expression of the zinc-uptake gene was investigated through the qRT-PCR technique. RESULTS: The results revealed that the ZnO NPs against clinical FMHLN5 strain were useful at different sizes. The expression of the zinc-uptake gene was observed. CONCLUSIONS: The effect of ZnO NPs was strong, as reflected by inhibition zones at different sizes. Thus, ZnO NPs potentially induced bactericidal effect for fighting FMHLN5 strain.

Neutrophil-to-Lymphocyte Ratio as a Poor Prognostic Factor in Iranian COVID-19 Patients.

BACKGROUND: We aimed to accumulate evidence that suggests the potential role of neutrophil-to-lymphocyte ratio (NLR) in determining the prognostic factor for COVID-19 patients. METHODS: A cohort of COVID-19 hospitalized patients at the Ilam University of Medical Sciences was analyzed. Logistic regression models were performed to identify the potential role of NLR in determining the prognostic factor for COVID-19 patients. RESULTS: The total number of in-hospital mortality was 43/328 (13.1%). Multivariate analysis identified that there was a 26% higher risk of in-hospital death for each unit increase in NLR (Odds ratio [OR] = 1.08; 95% confidence interval [95% CI], 1.01 to 1.14; p = 0.0147). Multivariate analysis identified that there was an 8% higher risk of in-hospital death for each unit increase in NLR (Odds ratio [OR] = 1.08; 95% confidence interval [95% CI], 1.01 to 1.14; p = 0.0147). Compared with patients in the NLR < 5 group, the NLR of patients in the NLR ≥ 5 group had a 16-fold higher risk of mortality (OR = 16.04; 95% CI, 1.14 to 224.95; p = 0.0395) after adjustment for potential confounders. CONCLUSIONS: NLR is an independent risk factor of mortality COVID-19 patients.

The characterization of bacterial communities of oropharynx microbiota in healthy children by combining culture techniques and sequencing of the 16S rRNA gene.

The high incidence of bacterial respiratory infections has led to a focus on evaluating the human respiratory microbiome. Studies based on culture-based and molecular methods have shown an increase in the bacterial community that includes the bacterial phyla Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria in the oropharynx of healthy individuals. Therefore, recognizing this microbial compound and subsequently identifying those carriers of specific pathogens can be of great help in predicting future infections and their control. In this prospective study, we sought to characterize the bacterial communities of the respiratory microbiome in healthy children aged between 3 and 6 years old by combining both cultural techniques and sequencing of the 16S rRNA gene. Seventy-seven oropharynx samples using Dacron swabs were collected from 77 healthy children in the kindergartens of Ilam, Iran. Bacterial identification was performed by phenotypic methods and in house developed PCR-based sequencing (the V1-V9 hypervariable region of the bacterial 16S ribosomal RNA gene). In total, 346 bacterial isolates were characterized based on phenotypic and sequencing-based molecular methods. The 3 most predominant phyla were Firmicutes (74%), Proteobacteria (22%), and Actinobacteria (4%). At the level of the genus, Staphylococci (coagulase-positive and coagulase-negative) and Streptococci were dominant. Also, the most commonly identified potentially pathogenic colonisers were S. aureus (75%), Enterobacteriaceae spp. (40.1%), and A. baumannii (15.6%). The present study identified 3 phyla and 9 family of bacteria in the oropharyngeal microbiome. Remarkably, the presence of potential pathogenic bacteria in the nasopharynx of healthy children can predispose them to infectious diseases, and also frequent exposure to human respiratory bacterial pathogens are further risk factors.

The increasing antimicrobial resistance of Helicobacter pylori in Iran: A systematic review and meta-analysis.

BACKGROUND: Antimicrobial resistance of Helicobacter pylori can result in eradication failure. Metadata on the antimicrobial resistance of H pylori in Iran could help to formulate H pylori eradication strategies in Iran. METHODS: A systematic review was performed after searching in MEDLINE, Scopus, Embase, Web of Science, and the Cochrane Library. A meta-analysis was performed, and a comparison of the rates between children and adults; time periods (1999-2010, 2011-2016, 2017-2019); and the methods used was carried out. RESULTS: A total of 66 studies investigating 5936 H pylori isolates were analyzed. The weighted pooled resistance (WPR) rates were as follows: clarithromycin 21% (95% CI 16-26), metronidazole 62% (95% 57-67), clarithromycin in combination with metronidazole 16% (95% CI 10-23), ciprofloxacin 24% (95% CI 15-33), levofloxacin 18% (95% CI 9-30), erythromycin 29% (95% CI 12-50), furazolidone 13% (95% CI 4-27), tetracycline 8% (95% CI 5-13), and amoxicillin 15% (95% CI 9-22). During the three time periods, there was an increased resistance to amoxicillin, clarithromycin, ciprofloxacin, furazolidone, and tetracycline (P Ë‚ .05). Furazolidone and a clarithromycin/metronidazole combination had the higher resistance rates in children (P Ë‚ .05). CONCLUSION: An increasing rate of resistance to amoxicillin, clarithromycin, ciprofloxacin, furazolidone, and tetracycline in Iranian H pylori isolates was identified. In children, the resistance to furazolidone and a combination of clarithromycin and metronidazole is higher compared to adults. As a stable, high resistance to metronidazole was found in children and adults in all Iranian provinces, we suggest that metronidazole should not be included in the Iranian H pylori eradication scheme.

Multiple-Locus Variable-Number Tandem-Repeat Analysis Genotyping of Brucella Isolates from Iran.

BACKGROUND: Brucellosis is considered a main health concern in humans and animals. Neither familiar molecular methods nor the classical biotyping techniques are acceptable for subtyping Brucella spp. Loci containing variable number tandem repeats (VNTRs) have recently demonstrated their practicality in typing isolates from human and animal origin despite the excessive genetic homogeneity in the genus Brucella. METHODS: The genotypic characteristics of sixty-six Brucella melitensis and thirty-four Brucella abortus isolates from veterinary samples and human brucellosis cases in Iran during 2014 - 2018. They were analyzed using multiple-locus variable-number tandem-repeat analysis (MLVA) which consisted of sixteen primer pairs and designed and classified as belonging to one of the three panels: panel 1 (MLVA-8: eight loci including Bruce06, Bruce08, Bruce11, Bruce12, Bruce42, Bruce43, Bruce45, and Bruce55), panel 2A (three loci including Bruce18, Bruce19, and Bruce21), and panel 2B (five loci including Bruce04, Bruce07, Bruce09, Bruce16, and Bruce30); MLVA-11 (panels 1 and 2A), and MLVA-16 (panels 1, 2A, and 2B) using BioNumerics software (Version 7.6). RESULTS: Using panel 1, 2A, and 2B (MLVA-16), 59 genotypes with a genetic similarity coefficient ranging from 91 to 100% were obtained from the 100 Brucella spp. isolates. For all isolates, only genotype 36 and genotype 26 were obtained using panels 1 and 2A, respectively. The B. abortus isolates showed variations at 9 different genotypes, while B. melitensis isolates have been dispersed in 50 different genotypes. Bruce16 and Bruce4 showed the highest discriminatory power. CONCLUSIONS: The MLVA-16 assay appeared to be a useful and important molecular genotyping tool that is capable of proving epidemiological linkages in outbreak and trace-back investigations and is helpful in improving the effectiveness of brucellosis control programs.

Rapid Simultaneous Molecular Stool-Based Detection of Toxigenic Clostridioides difficile by Quantitative TaqMan Real-Time PCR Assay.

BACKGROUND: Clostridioides difficile is a major cause of nosocomial infectious diarrhea in hospitalized patients throughout the world. METHODS: A multiplex real-time PCR assay was developed and evaluated in comparison with toxigenic culture (TC) (as gold standard method) for direct detection of toxigenic C. difficile in fecal specimens. The multiplex real-time PCR assay simultaneously detected glutamate dehydrogenase (gluD), toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtB) genes in stool samples. RESULTS: The results of multiplex real-time PCR were compared to those of the TC method in 250 patients suspected of C. difficile infection. The prevalence of positive TC was 13.6%. Forty-two stool samples (16.8%) were determined to be gluD+ using multiplex real-time PCR. These included 35 (83.3%) toxigenic (32 tcdA+, tcdB+ and three tcdB+) and 7 (20.0%) were cdtB+. The multiplex real-time PCR assay had a sensitivity of 91.45%, specificity of 99.54%, and positive and negative predictive values of 97% and 98.6%, respectively, compared to the TC method for diagnosis of C. difficile. The analytical sensitivity of the multiplex real-time PCR assay was estimated to be 102 CFU/g of stools and 0.0200 pg of genomic DNA from culture. The analytical specificity was determined to be 100% by using enteric and non-C. difficile standard bacterial strains. CONCLUSIONS: The molecular method developed in the study was rapid, sensitive, and specific for detection of toxigenic C. difficile. It is applicable to be performed in clinical laboratories and correlated well with the results obtained by TC.

Extraction and purification of the H9N2 virus nucleoprotein: A simple and practical method.

Background: Avian Influenza disease annually entails a significant economic loss to the poultry industry around the world. Influenza virus is a polymorphic virus of the orthomyxoviridae family (single-stranded RNA genome), and nucleoprotein (NP) is the structural and internal protein of the virus. The aim of the work was to purify nucleoprotein for further investigations with a simple, low-cost, fast and practical method. Methods: In this study, H9N2 influenza virus was isolated in specific pathogen-free embryonated chicken eggs by allantoically inoculating 103 to 105 egg-infective doses (EID50) for 9 to 11 days, purified by 10% (W/V) polyethylene glycol (PEG) 6000 with a sucrose gradient of 60% to 30%. The influenza virus proteins were collected and prepared as fractions by preparative electrophoresis. Finally, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot procedures. Results: The protein analysis with SDS-PAGE and silver nitrate staining indicated that the desired samples contained purified nucleoprotein and lacked other viral proteins. The results of the investigation of lyophilized fractions containing nucleoprotein on the SDS-PAGE revealed the absence of viral RNA in nucleoprotein and its high purity. Conclusion: According to this study, purified nucleoprotein can be used to produce nucleoprotein vaccines, as well as to study structural, molecular and diagnostic and therapeutic materials.