scholarly journals Accurate Detection of Methicillin-Resistant Staphylococcus aureus in Mixtures by Use of Single-Bacterium Duplex Droplet Digital PCR

2017 ◽  
Vol 55 (10) ◽  
pp. 2946-2955 ◽  
Author(s):  
Jun Luo ◽  
Junhua Li ◽  
Hang Yang ◽  
Junping Yu ◽  
Hongping Wei
Keyword(s):  
Staphylococcus Aureus ◽  
Limit Of Detection ◽  
Direct Method ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Clinical Samples ◽  
Methicillin Resistant ◽  
Content Type ◽  
Accurate Detection ◽  
Nasal Swabs

ABSTRACT Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is needed to screen MRSA carriers and improve treatment. The current widely used duplex PCR methods are not able to differentiate MRSA from coexisting methicillin-susceptible S. aureus (MSSA) or other methicillin-resistant staphylococci. In this study, we aimed to develop a direct method for accurate and rapid detection of MRSA in clinical samples from open environments, such as nasal swabs. The new molecular assay is based on detecting the cooccurrence of nuc and mecA markers in a single bacterial cell by utilizing droplet digital PCR (ddPCR) with the chimeric lysin ClyH for cell lysis. The method consists of (i) dispersion of an intact single bacterium into nanoliter droplets, (ii) temperature-controlled release of genomic DNA (gDNA) by ClyH at 37°C, and (iii) amplification and detection of the markers ( nuc and mecA ) using standard TaqMan chemistries with ddPCR. Results were analyzed based on MRSA index ratios used for indicating the presence of the duplex-positive markers in droplets. The method was able to achieve an absolute limit of detection (LOD) of 2,900 CFU/ml for MRSA in nasal swabs spiked with excess amounts of Escherichia coli , MSSA, and other mecA -positive bacteria within 4 h. Initial testing of 104 nasal swabs showed that the method had 100% agreement with the standard culture method, while the normal duplex qPCR method had only about 87.5% agreement. The single-bacterium duplex ddPCR assay is rapid and powerful for more accurate detection of MRSA directly from clinical specimens.

scholarly journals Evaluation of Antibiotics Active against Methicillin-Resistant Staphylococcus aureus Based on Activity in an Established Biofilm

2016 ◽  
Vol 60 (10) ◽  
pp. 5688-5694 ◽  
Author(s):  
Daniel G. Meeker ◽  
Karen E. Beenken ◽  
Weston B. Mills ◽  
Allister J. Loughran ◽  
Horace J. Spencer ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Limit Of Detection ◽  
Relative Activity ◽  
Methicillin Resistant ◽  
Content Type ◽  
Comparable Activity ◽  
Antibiotic Delivery

ABSTRACTWe usedin vitroandin vivomodels of catheter-associated biofilm formation to compare the relative activity of antibiotics effective against methicillin-resistantStaphylococcus aureus(MRSA) in the specific context of an established biofilm. The results demonstrated that, underin vitroconditions, daptomycin and ceftaroline exhibited comparable activity relative to each other and greater activity than vancomycin, telavancin, oritavancin, dalbavancin, or tigecycline. This was true when assessed using established biofilms formed by the USA300 methicillin-resistant strain LAC and the USA200 methicillin-sensitive strain UAMS-1. Oxacillin exhibited greater activity against UAMS-1 than LAC, as would be expected, since LAC is an MRSA strain. However, the activity of oxacillin was less than that of daptomycin and ceftaroline even against UAMS-1. Among the lipoglycopeptides, telavancin exhibited the greatest overall activity. Specifically, telavancin exhibited greater activity than oritavancin or dalbavancin when tested against biofilms formed by LAC and was the only lipoglycopeptide capable of reducing the number of viable bacteria below the limit of detection. With biofilms formed by UAMS-1, telavancin and dalbavancin exhibited comparable activity relative to each other and greater activity than oritavancin. Importantly, ceftaroline was the only antibiotic that exhibited greater activity than vancomycin when testedin vivoin a murine model of catheter-associated biofilm formation. These results emphasize the need to consider antibiotics other than vancomycin, most notably, ceftaroline, for the treatment of biofilm-associatedS. aureusinfections, including by the matrix-based antibiotic delivery methods often employed for local antibiotic delivery in the treatment of these infections.

scholarly journals Efficacy of Skin and Nasal Povidone-Iodine Preparation against Mupirocin-Resistant Methicillin-Resistant Staphylococcus aureus and S. aureus within the Anterior Nares

2015 ◽  
Vol 59 (5) ◽  
pp. 2765-2773 ◽  
Author(s):  
Michele J. Anderson ◽  
Maren L. David ◽  
Matt Scholz ◽  
Sally J. Bull ◽  
Dan Morse ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Human Skin ◽  
Povidone Iodine ◽  
Human Subjects ◽  
Surgical Site Infections ◽  
Attractive Alternative ◽  
Methicillin Resistant ◽  
Content Type ◽  
Nasal Swabs ◽  
Mrsa Infection

ABSTRACTMupirocin decolonization of nasalStaphylococcus aureusprior to surgery decreases surgical-site infections; however, treatment requires 5 days, compliance is low, and resistance occurs. In 2010, 3M Company introduced povidone-iodine (PVP-I)-based skin and nasal antiseptic (Skin and Nasal Prep [SNP]). SNP has rapid, broad-spectrum antimicrobial activity. We tested SNP's efficacy using full-thickness tissue (porcine mucosal [PM] and human skin) explant models and human subjects. Prior to or following infection with methicillin-resistantStaphylococcus aureus(MRSA) (mupirocin sensitive and resistant), explants were treated with Betadine ophthalmic preparation (Bet), SNP, or mupirocin (Bactroban nasal ointment [BN]) or left untreated. One hour posttreatment, explants were washed with phosphate-buffered saline (PBS) plus 2% mucin. One, 6, or 12 h later, bacteria were recovered and enumerated. Alternatively, following baseline sampling, human subjects applied two consecutive applications of SNP or saline to their anterior nares. One, 6, and 12 h after application of the preparation (postprep), nasal swabs were obtained, andS. aureuswas enumerated. We observed that treatment of infected PM or human skin explants with SNP resulted in >2.0 log10CFU reduction in MRSA, regardless of mupirocin sensitivity, which was significantly different from the values for BN- and Bet-treated explants and untreated controls 1 h, 6 h, and 12 h after being washed with PBS plus mucin. Swabbing the anterior nares of human subjects with SNP significantly reduced residentS. aureuscompared to saline 1, 6, and 12 h postprep. Finally, pretreatment of PM explants with SNP, followed by a mucin rinse prior to infection, completely prevented MRSA infection. We conclude that SNP may be an attractive alternative for reducing the bioburden of anterior nares prior to surgery.

scholarly journals Application of One-Step Reverse Transcription Droplet Digital PCR for Dengue Virus Detection and Quantification in Clinical Specimens

Diagnostics ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 639
Author(s):  
Dumrong Mairiang ◽  
Adisak Songjaeng ◽  
Prachya Hansuealueang ◽  
Yuwares Malila ◽  
Paphavee Lertsethtakarn ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Lower Limit ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Clinical Samples ◽  
Qrt Pcr ◽  
One Step ◽  
Detection And Quantification

Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several “in-house” components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantification results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity.

scholarly journals Evaluation of BD Max StaphSR and BD Max MRSA XT Assays Using ESwab-Collected Specimens

2015 ◽  
Vol 53 (8) ◽  
pp. 2525-2529 ◽  
Author(s):  
Suzane Silbert ◽  
Carly Kubasek ◽  
Faris Galambo ◽  
Elaine Vendrone ◽  
Raymond Widen
Keyword(s):  
Staphylococcus Aureus ◽  
Laboratory Testing ◽  
First Generation ◽  
Enrichment Culture ◽  
Clinical Samples ◽  
Methicillin Resistant ◽  
Content Type ◽  
Pcr Assays

The BD Max MRSA XT and the BD Max StaphSR assays were validated for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in ESwab samples. In addition, the BD Max StaphSR assay was evaluated for its ability to detect and differentiate S. aureus and MRSA in the same sample. A total of 255 ESwab samples collected from the anterior nares of patients were tested by each of three BD Max assays, including the BD Max MRSA first-generation assay. The results were compared to those of direct and enrichment culture. Additionally, a challenge panel comprising 14 control strains was evaluated to determine the ability of these assays to correctly identify MRSA and also appropriately differentiate S. aureus by the BD Max StaphSR assay. Out of 255 clinical samples tested, 161 were negative and 30 were positive for MRSA, and 45 were positive for S. aureus (by BD Max StaphSR) and negative for MRSA by all three PCR assays and culture. Nineteen samples had discrepant results; all of them were retested by additional laboratory testing. All strains from the challenge panel were correctly identified or excluded by the BD Max MRSA XT and BD Max StaphSR assays. The results showed that the BD Max StaphSR and the BD MRSA XT assays have excellent sensitivity (94.3%) and specificity (97.7%) for detecting MRSA. The BD Max StaphSR assay demonstrated excellent sensitivity (96.4%) and specificity (93.6%) for detecting S. aureus .

scholarly journals Multicenter Evaluation of the Xpert MRSA NxG Assay for Detection of Methicillin-Resistant Staphylococcus aureus in Nasal Swabs

2017 ◽  
Vol 56 (1) ◽  
Author(s):  
Melanie L. Yarbrough ◽  
David K. Warren ◽  
Karen Allen ◽  
Dennis Burkholder ◽  
Robert Daum ◽  
...  
Keyword(s):  
Health Care ◽  
Staphylococcus Aureus ◽  
Sensitivity And Specificity ◽  
Direct Detection ◽  
Clinical Performance ◽  
Reference Method ◽  
Predictive Values ◽  
Methicillin Resistant ◽  
Content Type ◽  
Nasal Swabs

ABSTRACT Health care-associated methicillin-resistant Staphylococcus aureus (MRSA) infections are a burden on the health care system. Clinical laboratories play a key role in reducing this burden, as the timely identification of MRSA colonization or infection facilitates infection control practices that are effective at limiting invasive MRSA infections. The Xpert MRSA NxG assay recently received FDA clearance for the direct detection of MRSA from nasal swabs. This multicenter study evaluated the clinical performance characteristics of the Xpert MRSA NxG assay with prospectively collected rayon nasal swabs (n = 1,103) and flocked swab (ESwab) nasal specimens (n = 846). Culture-based identification methods and antimicrobial susceptibility testing were used as the reference standards for comparison. According to the reference method, the positivity rates for MRSA in the population evaluated were 11.1% (122/1,103) for rayon swabs and 11.6% (98/846) for flocked swabs. The overall sensitivity and specificity of the rayon swabs were 91.0% (95% confidence interval [CI], 84.6 to 94.9%) and 96.9% (95% CI, 95.7 to 97.8%), respectively, across eight testing sites. The flocked swab specimens were 92.9% sensitive (95% CI, 86.0 to 96.5%) and 97.6% specific (95% CI, 96.2 to 98.5%) for MRSA detection across six testing sites. The sensitivity and specificity of the combined flocked and rayon swab data were 91.8% (95% CI, 87.4 to 94.8%) and 97.2% (95% CI, 96.3 to 97.9%), respectively. The positive predictive value (PPV) for rayon swabs was 78.7%, versus 83.5% for ESwabs. The negative predictive values (NPVs) for rayon swabs and ESwab specimens were 98.9% and 99.1%, respectively. In conclusion, the Xpert MRSA NxG assay is a sensitive and specific assay for the direct detection of MRSA from nasal swab specimens.

scholarly journals Absence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex CC398 as a Nasal Colonizer of Pigs Raised in an Alternative System

2011 ◽  
Vol 78 (4) ◽  
pp. 1296-1297 ◽  
Author(s):  
Christiane Cuny ◽  
Alexander W. Friedrich ◽  
Wolfgang Witte
Keyword(s):  
Staphylococcus Aureus ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Clonal Complex ◽  
Methicillin Resistant ◽  
Content Type ◽  
Alternative System ◽  
Conventional Farms ◽  
Nasal Swabs ◽  
Mrsa St398

ABSTRACTLivestock-associated methicillin-resistantStaphylococcus aureus(LA-MRSA) ST398 isolated from pigs raised in conventional farms was previously reported. Here we report a study on 25 farms adhering to an alternative system. LA-MRSA ST398 was not detected in nasal swabs from 178 pigs or from 89 humans working and living on these farms.

scholarly journals Mutation-Based Antibiotic Resistance Mechanism in Methicillin-Resistant Staphylococcus aureus Clinical Isolates

2021 ◽  
Vol 14 (5) ◽  
pp. 420
Author(s):  
Tanveer Ali ◽  
Abdul Basit ◽  
Asad Mustafa Karim ◽  
Jung-Hun Lee ◽  
Jeong-Ho Jeon ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Active Site ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Meca Gene ◽  
Resistance Mechanism ◽  
Ligand Docking ◽  
Clinical Samples ◽  
Allosteric Site ◽  
Methicillin Resistant

β-Lactam antibiotics target penicillin-binding proteins and inhibit the synthesis of peptidoglycan, a crucial step in cell wall biosynthesis. Staphylococcus aureus acquires resistance against β-lactam antibiotics by producing a penicillin-binding protein 2a (PBP2a), encoded by the mecA gene. PBP2a participates in peptidoglycan biosynthesis and exhibits a poor affinity towards β-lactam antibiotics. The current study was performed to determine the diversity and the role of missense mutations of PBP2a in the antibiotic resistance mechanism. The methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical samples were identified using phenotypic and genotypic techniques. The highest frequency (60%, 18 out of 30) of MRSA was observed in wound specimens. Sequence variation analysis of the mecA gene showed four amino acid substitutions (i.e., E239K, E239R, G246E, and E447K). The E239R mutation was found to be novel. The protein-ligand docking results showed that the E239R mutation in the allosteric site of PBP2a induces conformational changes in the active site and, thus, hinders its interaction with cefoxitin. Therefore, the present report indicates that mutation in the allosteric site of PBP2a provides a more closed active site conformation than wide-type PBP2a and then causes the high-level resistance to cefoxitin.

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.

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