scholarly journals Genetic Mimetics of Mycobacterium tuberculosis and Methicillin-Resistant Staphylococcus aureus as Verification Standards for Molecular Diagnostics

2017 ◽  
Vol 55 (12) ◽  
pp. 3384-3394 ◽  
Author(s):  
Edith Erika Machowski ◽  
Bavesh Davandra Kana
Keyword(s):  
Staphylococcus Aureus ◽  
Mycobacterium Tuberculosis ◽  
Molecular Diagnostics ◽  
Nucleic Acid Amplification ◽  
World Health ◽  
Molecular Diagnostic ◽  
Specific Gene ◽  
Methicillin Resistant ◽  
Content Type ◽  
Rifampin Resistance

ABSTRACTMolecular diagnostics have revolutionized the management of health care through enhanced detection of disease or infection and effective enrollment into treatment. In recognition of this, the World Health Organization approved the rollout of nucleic acid amplification technologies for identification ofMycobacterium tuberculosisusing platforms such as GeneXpert MTB/RIF, the GenoType MTBDRplusline probe assay, and, more recently, GeneXpert MTB/RIF Ultra. These assays can simultaneously detect tuberculosis infection and assess rifampin resistance. However, their widespread use in health systems requires verification and quality assurance programs. To enable development of these, we report the construction of genetically modified strains ofMycobacterium smegmatisthat mimic the profile ofMycobacterium tuberculosison both the GeneXpert MTB/RIF and the MTBDRplusline probe diagnostic tests. Using site-specific gene editing, we also created derivatives that faithfully mimic the diagnostic result of rifampin-resistantM. tuberculosis, with mutations at positions 513, 516, 526, 531, and 533 in the rifampin resistance-determining region of therpoBgene. Next, we extended this approach to other diseases and demonstrated that aStaphylococcus aureusgene sequence can be introduced intoM. smegmatisto generate a positive response for the SCCmecprobe in the GeneXpert SA Nasal Complete molecular diagnostic cartridge, designed for identification of methicillin-resistantS. aureus. These biomimetic strains are cost-effective, have low biohazard content, accurately mimic drug resistance, and can be produced with relative ease, thus illustrating their potential for widespread use as verification standards for diagnosis of a variety of diseases.

scholarly journals In VitroEfficacies and Resistance Profiles of Rifampin-Based Combination Regimens for Biofilm-Embedded Methicillin-Resistant Staphylococcus aureus

2013 ◽  
Vol 57 (11) ◽  
pp. 5717-5720 ◽  
Author(s):  
Hung-Jen Tang ◽  
Chi-Chung Chen ◽  
Kuo-Chen Cheng ◽  
Kuan-Ying Wu ◽  
Yi-Chung Lin ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Fusidic Acid ◽  
Antibacterial Activities ◽  
Methicillin Resistant ◽  
Content Type ◽  
Biofilm Model ◽  
Rifampin Resistance ◽  
Combination Regimens ◽  
Resistant Mutation

ABSTRACTTo compare thein vitroantibacterial efficacies and resistance profiles of rifampin-based combinations against methicillin-resistantStaphylococcus aureus(MRSA) in a biofilm model, the antibacterial activities of vancomycin, teicoplanin, daptomycin, minocycline, linezolid, fusidic acid, fosfomycin, and tigecycline alone or in combination with rifampin against biofilm-embedded MRSA were measured. The rifampin-resistant mutation frequencies were evaluated. Of the rifampin-based combinations, rifampin enhances the antibacterial activities of and even synergizes with fusidic acid, tigecycline, and, to a lesser extent, linezolid, fosfomycin, and minocycline against biofilm-embedded MRSA. Such combinations with weaker rifampin resistance induction activities may provide a therapeutic advantage in MRSA biofilm-related infections.

scholarly journals Updating Molecular Diagnostics for Detecting Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus Isolates in Blood Culture Bottles

2019 ◽  
Vol 57 (11) ◽  
Author(s):  
Fred C. Tenover ◽  
Isabella A. Tickler ◽  
Victoria M. Le ◽  
Scott Dewell ◽  
Rodrigo E. Mendes ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Blood Culture ◽  
False Negative ◽  
The United States ◽  
Molecular Diagnostic ◽  
Methicillin Resistant ◽  
Content Type ◽  
Target Nucleic Acid ◽  
Antimicrobial Resistance Genes

ABSTRACT Molecular diagnostic tests can be used to provide rapid identification of staphylococcal species in blood culture bottles to help improve antimicrobial stewardship. However, alterations in the target nucleic acid sequences of the microorganisms or their antimicrobial resistance genes can lead to false-negative results. We determined the whole-genome sequences of 4 blood culture isolates of Staphylococcus aureus and 2 control organisms to understand the genetic basis of genotype-phenotype discrepancies when using the Xpert MRSA/SA BC test (in vitro diagnostic medical device [IVD]). Three methicillin-resistant S. aureus (MRSA) isolates each had a different insertion of a genetic element in the staphylococcal cassette chromosome (SCCmec)-orfX junction region that led to a misclassification as methicillin-susceptible S. aureus (MSSA). One strain contained a deletion in spa, which produced a false S. aureus-negative result. A control strain of S. aureus that harbored an SCCmec element but no mecA (an empty cassette) was correctly called MSSA by the Xpert test. The second control contained an SCCM1 insertion. The updated Xpert MRSA/SA BC test successfully detected both spa and SCCmec variants of MRSA and correctly identified empty-cassette strains of S. aureus as MSSA. Among a sample of 252 MSSA isolates from the United States and Europe, 3.9% contained empty SCCmec cassettes, 1.6% carried SCCM1, <1% had spa deletions, and <1% contained SCCmec variants other than those with SCCM1. These data suggest that genetic variations that may interfere with Xpert MRSA/SA BC test results remain rare. Results for all the isolates were correct when tested with the updated assay.

scholarly journals Evolution and Single-Nucleotide Polymorphisms in Methicillin-Resistant Staphylococcus aureus Strains with Reduced Susceptibility to Vancomycin and Daptomycin, Based on Determination of the Complete Genome

2015 ◽  
Vol 59 (6) ◽  
pp. 3585-3587 ◽  
Author(s):  
Tetsuo Yamaguchi ◽  
Shingo Suzuki ◽  
Sakiko Okamura ◽  
Yuri Miura ◽  
Ayaka Tsukimori ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Single Nucleotide Polymorphisms ◽  
Complete Genome ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Methicillin Resistant ◽  
Content Type ◽  
Rifampin Resistance ◽  
Generation Sequencing

ABSTRACTWe obtained a series of methicillin-resistantStaphylococcus aureusisolates, including both daptomycin-susceptible strain TD1 and daptomycin-resistant strain TD4, from a patient. We determined the complete genome sequences of TD1 and TD4 using next-generation sequencing, and only four single-nucleotide polymorphisms (SNPs) were identified, one each incapB(E58K),rpoB(H481Y),lytN(I16V), andmprF(V351E). We determined that these four SNPs were sufficient to cause the strains to develop daptomycin, vancomycin, and rifampin resistance.

scholarly journals Methicillin-Resistant Staphylococcus aureus in Hospitals: Latest Trends and Treatments Based on Bacteriophages

2019 ◽  
Vol 57 (12) ◽  
Author(s):  
Andrea Álvarez ◽  
Lucía Fernández ◽  
Diana Gutiérrez ◽  
Beatriz Iglesias ◽  
Ana Rodríguez ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Pathogenic Bacteria ◽  
World Health ◽  
Natural Phenomenon ◽  
Methicillin Resistant ◽  
Content Type ◽  
Wide Range ◽  
Mrsa Strains ◽  
Health Organization

ABSTRACT Even though antibiotic resistance in bacteria is a natural phenomenon, the alarming increase in pathogenic bacteria refractory to a wide range of antimicrobials is attracting attention worldwide. Indeed, the World Health Organization (WHO) has recently published a list of priority pathogens for which new antimicrobial alternatives are urgently needed. Among these pathogens, methicillin-resistant Staphylococcus aureus (MRSA) strains are perhaps the best known by the general public. In addition to its potential to acquire antibiotic resistance, S. aureus can produce a large number of virulence factors, such as hemolysins, enterotoxins, and proteases, and exhibits the ability to form biofilms as well as to evolve into different clones that can spread and colonize new environments. This review provides a brief overview of the latest options in antibacterial therapies, mainly focusing on phage therapy. In this regard, the current stage of research about antimicrobial compounds based on bacteriophages and endolysins against MRSA infections is shown and discussed.

scholarly journals Causes and Implications of the Disappearance of Rifampin Resistance in a Rat Model of Methicillin-Resistant Staphylococcus aureus Foreign Body Osteomyelitis

2015 ◽  
Vol 59 (8) ◽  
pp. 4481-4488 ◽  
Author(s):  
Cassandra L. Brinkman ◽  
Harmony L. Tyner ◽  
Suzannah M. Schmidt-Malan ◽  
Jayawant N. Mandrekar ◽  
Robin Patel
Keyword(s):  
Staphylococcus Aureus ◽  
Foreign Body ◽  
Treatment Completion ◽  
Resistant Bacteria ◽  
Methicillin Resistant ◽  
Content Type ◽  
Susceptible Isolate ◽  
Rifampin Resistance

ABSTRACTOrthopedic foreign body-associated infections are often treated with rifampin-based combination antimicrobial therapy. We previously observed that rifampin-resistant and methicillin-resistantStaphylococcus aureus(MRSA) isolates were present 2 days after cessation of rifampin therapy in experimental foreign body osteomyelitis. Unexpectedly, only rifampin-susceptible isolates were detected 14 days after the completion of treatment. We studied two rifampin-resistant isolates recovered 2 days after treatment and one rifampin-susceptible isolate recovered 14 days after treatment. Growing these isolates alonein vitroorin vivodemonstrated no fitness defects; however, in mixed culture, rifampin-susceptible bacteria outcompeted rifampin-resistant bacteria.In vivo, two courses of rifampin treatment (25 mg/kg of body weight every 12 h for 21 days) yielded a greater decrease in bacterial quantity in the bones of treated animals 14 days following treatment than that in animals receiving a single course of treatment (P= 0.0398). In infections established with equal numbers of rifampin-resistant and rifampin-susceptible bacteria, one course of rifampin treatment did not affect bacterial quantities. Rifampin-resistant and rifampin-susceptible isolates were recovered both 2 days and 14 days following treatment completion; however, the proportion of animals with rifampin-resistant isolates was lower at 14 days than that at 2 days following treatment completion (P= 0.024). In untreated animals infected with equal numbers of rifampin-resistant and rifampin-susceptible bacteria for 4 weeks, rifampin-susceptible isolates were exclusively recovered, indicating the outcompetition of rifampin-resistant by rifampin-susceptible isolates. The data presented imply that although there is no apparent fitness defect in rifampin-resistant bacteria when grown alone, they are outcompeted by rifampin-susceptible bacteria when the two are present together. The findings also suggest that selected rifampin resistance may not persist in initially rifampin-susceptible infections following the discontinuation of rifampin.

Molecular Diagnostics for Mycobacterium tuberculosis Infection

2019 ◽  
Vol 70 (1) ◽  
pp. 77-90 ◽  
Author(s):  
Kristen V. Dicks ◽  
Jason E. Stout
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Diagnostics ◽  
Tuberculosis Infection ◽  
Resistance Testing ◽  
Molecular Diagnostic ◽  
Specific Gene ◽  
Successful Implementation ◽  
Mycobacterium Tuberculosis Infection ◽  
Major Barrier ◽  
Imperfect Knowledge

Resistance to antimycobacterial drugs is a major barrier to effective treatment of Mycobacterium tuberculosis infection. Molecular diagnostic techniques based on the association between specific gene mutations and phenotypic resistance to certain drugs offer the opportunity to rapidly ascertain whether drug resistance is present and to alter treatment before further resistance develops. Current barriers to successful implementation of rapid diagnostics include imperfect knowledge regarding the full spectrum of mutations associated with resistance, limited utilization of molecular diagnostics where they are most needed, and the requirement for specialized laboratory facilities to perform molecular testing. Further understanding of genotypic–phenotypic correlates of resistance and streamlined implementation platforms will be necessary to optimize the public health impact of molecular resistance testing for M. tuberculosis.

scholarly journals Prevalence of methicillin-resistant Staphylococcus aureus and pattern of antimicrobial resistance in mastitis milk of cattle in Chitwan, Nepal

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Asmita Shrestha ◽  
Rebanta Kumar Bhattarai ◽  
Himal Luitel ◽  
Surendra Karki ◽  
Hom Bahadur Basnet
Keyword(s):  
Public Health ◽  
Staphylococcus Aureus ◽  
Antimicrobial Resistance ◽  
Subclinical Mastitis ◽  
World Health ◽  
Dairy Herds ◽  
Microbiological Analysis ◽  
Biochemical Tests ◽  
Methicillin Resistant ◽  
Milk Samples

Abstract Background The threat of methicillin-resistant Staphylococcus aureus (MRSA) exists globally and has been listed as a priority pathogen by the World Health Organization. One of the sources of MRSA emergence is livestock and its products, often raised in poor husbandry conditions. There are limited studies in Nepal to understand the prevalence of MRSA in dairy animals and its antimicrobial resistance (AMR) profile. A cross-sectional study was conducted in Chitwan, one of the major milk-producing districts of Nepal, from February 2018 to September 2019 to estimate the prevalence of MRSA in milk samples and its AMR profile. The collected milk samples (n = 460) were screened using the California Mastitis Test (CMT) and positive samples were subjected to microbiological analysis to isolate and identify S. aureus. Polymerase Chain Reaction (PCR) was used to identify the presence of the mecA gene and screen for MRSA. Results In total, 41.5% (191/460) of milk samples were positive in the CMT test. Out of 191 CMT positive milk samples, the biochemical tests showed that the prevalence of S. aureus was 15.2% (29/191). Among the 29 S. aureus isolates, 6.9% (2/29) were identified as MRSA based on the detection of a mecA gene. This indicates that that 1.05% (2/191) of mastitis milk samples had MRSA. The antibiotic sensitivity test showed that 75.9% (22/29) and 48.3% (14/29) S. aureus isolates were found to be sensitive to Cefazolin and Tetracycline respectively (48.3%), whereas 100% of the isolates were resistant to Ampicillin. In total 96.6% (28/29) of S. aureus isolates were multidrug-resistant (MDR). Conclusions This study revealed a high prevalence of S. aureus-mediated subclinical mastitis in dairy herds in Chitwan, Nepal, with a small proportion of it being MRSA carrying a mecA gene. This S. aureus, CoNS, and MRSA contaminated milk poses a public health risk due to the presence of a phenotype that is resistant to very commonly used antibiotics. It is suggested that dairy herds be screened for subclinical mastitis and treatments for the animals be based on antibiotic susceptibility tests to reduce the prevalence of AMR. Furthermore, future studies should focus on the Staphylococcus spp. to explore the antibiotic resistance genes in addition to the mecA gene to ensure public health.

scholarly journals In VitroActivities of Daptomycin-, Vancomycin-, and Teicoplanin-Loaded Polymethylmethacrylate against Methicillin-Susceptible, Methicillin-Resistant, and Vancomycin-Intermediate Strains of Staphylococcus aureus

2011 ◽  
Vol 55 (12) ◽  
pp. 5480-5484 ◽  
Author(s):  
Yuhan Chang ◽  
Wen-Chien Chen ◽  
Pang-Hsin Hsieh ◽  
Dave W. Chen ◽  
Mel S. Lee ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
High Performance ◽  
Low Dose ◽  
Antibacterial Activities ◽  
High Dose ◽  
Bone Cements ◽  
Antibacterial Effects ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACTThe objective of this study was to evaluate the antibacterial effects of polymethylmethacrylate (PMMA) bone cements loaded with daptomycin, vancomycin, and teicoplanin against methicillin-susceptibleStaphylococcus aureus(MSSA), methicillin-resistantStaphylococcus aureus(MRSA), and vancomycin-intermediateStaphylococcus aureus(VISA) strains. Standardized cement specimens made from 40 g PMMA loaded with 1 g (low-dose), 4 g (middle-dose) or 8 g (high-dose) antibiotics were tested for elution characteristics and antibacterial activities. The patterns of release of antibiotics from the cement specimens were evaluated usingin vitrobroth elution assay with high-performance liquid chromatography. The activities of broth elution fluid against differentStaphylococcus aureusstrains (MSSA, MRSA, and VISA) were then determined. The antibacterial activities of all the tested antibiotics were maintained after being mixed with PMMA. The cements loaded with higher dosages of antibiotics showed longer elution periods. Regardless of the antibiotic loading dose, the teicoplanin-loaded cements showed better elution efficacy and provided longer inhibitory periods against MSSA, MRSA, and VISA than cements loaded with the same dose of vancomycin or daptomycin. Regarding the choice of antibiotics for cement loading in the treatment ofStaphylococcus aureusinfection, teicoplanin was superior in terms of antibacterial effects.

scholarly journals Comparison of Borate Bioactive Glass and Calcium Sulfate as Implants for the Local Delivery of Teicoplanin in the Treatment of Methicillin-Resistant Staphylococcus aureus-Induced Osteomyelitis in a Rabbit Model

2015 ◽  
Vol 59 (12) ◽  
pp. 7571-7580 ◽  
Author(s):  
Wei-Tao Jia ◽  
Qiang Fu ◽  
Wen-Hai Huang ◽  
Chang-Qing Zhang ◽  
Mohamed N. Rahaman
Keyword(s):  
Staphylococcus Aureus ◽  
Bioactive Glass ◽  
Calcium Sulfate ◽  
Rabbit Model ◽  
Local Delivery ◽  
Methicillin Resistant ◽  
Content Type ◽  
Borate Bioactive Glass ◽  
Phosphate Buffered Saline

ABSTRACTThere is growing interest in biomaterials that can cure bone infection and also regenerate bone. In this study, two groups of implants composed of 10% (wt/wt) teicoplanin (TEC)-loaded borate bioactive glass (designated TBG) or calcium sulfate (TCS) were created and evaluated for their ability to release TECin vitroand to cure methicillin-resistantStaphylococcus aureus(MRSA)-induced osteomyelitis in a rabbit model. When immersed in phosphate-buffered saline (PBS), both groups of implants provided a sustained release of TEC at a therapeutic level for up to 3 to 4 weeks while they were gradually degraded and converted to hydroxyapatite. The TBG implants showed a longer duration of TEC release and better retention of strength as a function of immersion time in PBS. Infected rabbit tibiae were treated by debridement, followed by implantation of TBG or TCS pellets or intravenous injection with TEC, or were left untreated. Evaluation at 6 weeks postimplantation showed that the animals implanted with TBG or TCS pellets had significantly lower radiological and histological scores, lower rates of MRSA-positive cultures, and lower bacterial loads than those preoperatively and those of animals treated intravenously. The level of bone regeneration was also higher in the defects treated with the TBG pellets. The results showed that local TEC delivery was more effective than intravenous administration for the treatment of MRSA-induced osteomyelitis. Borate glass has the advantages of better mechanical strength, more desirable kinetics of release of TEC, and a higher osteogenic capacity and thus could be an effective alternative to calcium sulfate for local delivery of TEC.

scholarly journals Discordance between Xpert MTB/RIF Assay and Bactec MGIT 960 Culture System for Detection of Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Country with a Low Tuberculosis (TB) Incidence

2015 ◽  
Vol 53 (4) ◽  
pp. 1351-1354 ◽  
Author(s):  
Eiman Mokaddas ◽  
Suhail Ahmad ◽  
Hanaa S. Eldeen ◽  
Noura Al-Mutairi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Culture System ◽  
Content Type ◽  
Rifampin Resistance ◽  
Xpert Assay

Among 452 samples that were positive by the Xpert MTB/RIF (Xpert) assay and MGIT 960 system (MGIT), 440 and 10Mycobacterium tuberculosissamples were detected as rifampin susceptible and rifampin resistant, respectively. Two isolates that were rifampin susceptible by the MGIT system were rifampin resistant by the Xpert assay.rpoBsequencing identified a silent (CTG521TTG) mutation in one isolate and a missense (GAC516TAC) mutation in another. The detection of rifampin resistance is imperfect with both the Xpert assay and MGIT system. Any discordant rifampin resistance results should be confirmed by sequencing of therpoBgene.

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