Development and Evaluation of a Sensitive Bacteriophage-Based MRSA Diagnostic Screen

Engineered luciferase reporter bacteriophages provide specific, sensitive, rapid and low-cost detection of target bacteria and address growing diagnostic needs in multiple industries. Detection of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization and antibiotic susceptibility play a critical supportive role in preventing hospital-acquired infections and facilitating antibiotic stewardship. We describe the development and evaluation of a novel phage-based MRSA diagnostic screen for nasal swab specimens. The screen utilizes two luciferase reporter phages capable of recognizing genetically-diverse Staphylococcus aureus. The beta-lactam antibiotic cefoxitin is included to differentiate between resistant (MRSA) and susceptible organisms. The screen positively identified 97.7% of 390 clinical MRSA isolates at low bacterial concentrations. At higher inoculums, 93.5% of 123 clinical non-MRSA Staphylococcus aureus yielded appropriate negative results. Although cross-reactivity of the phage cocktail was observed with other staphylococcal and bacillus species, these false positives were absent under selective conditions. MRSA remained detectable in the presence of 38 distinct competing species and was accurately identified in 100% of 40 spiked nasal specimens. Thus, this six-hour screen sensitively detected MRSA both in vitro and in human nasal matrix.

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A bacteriocin-based antimicrobial formulation to effectively disrupt the cell viability of methicillin-resistant Staphylococcus aureus (MRSA) biofilms

npj Biofilms and Microbiomes ◽

10.1038/s41522-020-00166-4 ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Christian Kranjec ◽

Kirill V. Ovchinnikov ◽

Torstein Grønseth ◽

Kumar Ebineshan ◽

Aparna Srikantam ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Viability ◽

Cell Damage ◽

Penicillin G ◽

Antibiotic Resistant ◽

Methicillin Resistant ◽

Lactam Antibiotic ◽

Mrsa Strains ◽

Therapeutic Tools

AbstractAntibiotic-resistant and biofilm-associated infections brought about by methicillin-resistant Staphylococcus aureus (MRSA) strains is a pressing issue both inside as well as outside nosocomial environments worldwide. Here, we show that a combination of two bacteriocins with distinct structural and functional characteristics, garvicin KS, and micrococcin P1, showed a synergetic antibacterial activity against biofilms produced in vitro by S. aureus, including several MRSA strains. In addition, this bacteriocin-based antimicrobial combination showed the ability to restore the sensitivity of the highly resilient MRSA strain ATCC 33591 to the β-lactam antibiotic penicillin G. By using a combination of bacterial cell metabolic assays, confocal and scanning electron microscopy, we show that the combination between garvicin KS, micrococcin P1, and penicillin G potently inhibit cell viability within S. aureus biofilms by causing severe cell damage. Together these data indicate that bacteriocins can be valuable therapeutic tools in the fight against biofilm-associated MRSA infections.

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Dalbavancin, Vancomycin and Daptomycin Alone and in Combination with Cefazolin against Resistant Phenotypes of Staphylococcus aureus in a Pharmacokinetic/Pharmacodynamic Model

Antibiotics ◽

10.3390/antibiotics9100696 ◽

2020 ◽

Vol 9 (10) ◽

pp. 696 ◽

Cited By ~ 1

Author(s):

Jacinda C. Abdul-Mutakabbir ◽

Razieh Kebriaei ◽

Kyle C. Stamper ◽

Zain Sheikh ◽

Philip T. Maassen ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Bactericidal Activity ◽

Inhibitory Concentration ◽

Pharmacodynamic Model ◽

In Vitro Tests ◽

Beta Lactam ◽

Fold Reduction ◽

The One ◽

Time Kill

The most efficacious antimicrobial therapy to aid in the successful elimination of resistant S. aureus infections is unknown. In this study, we evaluated varying phenotypes of S. aureus against dalbavancin (DAL), vancomycin (VAN), and daptomycin (DAP) alone and in combination with cefazolin (CFZ). The objective of this study was to observe whether there was a therapeutic improvement in adding a beta-lactam to a glycopeptide, lipopeptide, or a lipoglycopeptide. We completed a series of in vitro tests including minimum inhibitory concentration testing (MIC) of the antimicrobials in combination, time-kill analysis (TKA), and a 168 h (7-day) one-compartment pharmacokinetic/pharmacodynamic (PK/PD) model on two daptomycin non-susceptible (DNS), vancomycin intermediate S. aureus strains (VISA), D712 and 6913. Results from our MIC testing demonstrated a minimum 2-fold and a maximum 32-fold reduction in MIC values for DAL, VAN, and DAP in combination with CFZ, in contrast to either agent used alone. The TKAs completed on four strains paralleled the enhanced activity demonstrated via the combination MICs. In the one-compartment PK/PD models, the combination of DAP plus CFZ or VAN plus CFZ resulted in a significant (p < 0.001) improvement in bactericidal activity and overall reduction in CFU/ml over the 7-day period. While the addition of CFZ to DAL improved time to bactericidal activity, DAL alone demonstrated equal and more sustained overall activity compared to all other treatments. The use of DAL alone, with or without CFZ and the combinations of VAN or DAP with CFZ appear to result in increased bactericidal activity against various recalcitrant S. aureus phenotypes.

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Rapid Consumption of Vancomycin in the Presence of Beta-Lactam Antibiotics Causes Beta-Lactam Antibiotic-Induced Vancomycin-Resistance in Methicillin-resistant Staphylococcus aureus

International Journal of Infectious Diseases ◽

10.1016/j.ijid.2008.05.714 ◽

2008 ◽

Vol 12 ◽

pp. e265

Author(s):

H. Hanaki ◽

C. Yanagisawa ◽

M. Yagisawa ◽

T. Nakae ◽

K. Sunakawa

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistant Staphylococcus Aureus ◽

Vancomycin Resistance ◽

Beta Lactam ◽

Methicillin Resistant ◽

Beta Lactam Antibiotic ◽

Beta Lactam Antibiotics ◽

Lactam Antibiotic

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In vitro activity of beta-lactam antibiotics to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA)

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-011-1333-8 ◽

2011 ◽

Vol 31 (4) ◽

pp. 475-480 ◽

Cited By ~ 8

Author(s):

C. Germel ◽

A. Haag ◽

B. Söderquist

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistant Staphylococcus Aureus ◽

Vitro Activity ◽

In Vitro Activity ◽

Beta Lactam ◽

Methicillin Resistant ◽

Beta Lactam Antibiotics

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Development of a Novel Rapid Immunodiagnostic Kit Based on Flagellar 40 kDa Antigen Epitope for the Detection of Typhoid Fever in Indian Patients

The Scientific World JOURNAL ◽

10.1155/2013/363652 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Rahul Mitra ◽

Surya Bhan ◽

Gopal Nath ◽

Narender Kumar ◽

Ziledar Ali

Keyword(s):

Staphylococcus Aureus ◽

Typhoid Fever ◽

Direct Detection ◽

Febrile Illness ◽

Cross Reactivity ◽

Negative Results ◽

Kala Azar ◽

Primary Health Centres ◽

Test Kit ◽

Specific Igg

To aid the clinical diagnosis of typhoid fever in India, where most hospitals and primary health centres have no facilities for culture, we report on the development of a novel and rapid immunodiagnostic kit for the direct detection ofSalmonellaTyphi—specific IgG antibodies againstS. Typhi flagellar H antigen. The disease often does not show a specific clinical picture, and can be confused with other febrile illness such as malaria, dengue fever andStaphylococcus aureus. To overcome the problem of cross reactivity specific epitope of the flagellar H antigen was immobilised on the testing kit strip eliminating chances of cross reactivity and false positive results thereby increasing the specificity of the test. Since the immunodiagnostic kit, uses the flagellar H antigen from bacteria present in our country, the antibodies present in the serum of patients of our country will have maximum binding affinity, enhancing the sensitivity of our test kit. The immunodiagnostic kit on analysis gave a positive result with clinically diagnosed typhoid positive patient serum and negative results were obtained with the sera of clinically diagnosed malaria, abscess ofStaphylococcus aureusand Visceral leishmaniasis (Kala-azar) patients.

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Protective Signatures of Roselle (Hibiscus sabdariffa L.) Calyx Fractions against Staphylococcus aureus in Drosophila Infection Model

HAYATI Journal of Biosciences ◽

10.4308/hjb.27.4.306 ◽

2020 ◽

Vol 27 (4) ◽

pp. 306

Author(s):

Firzan Nainu ◽

M. Natsir Djide ◽

Subehan Subehan ◽

Sartini Sartini ◽

Tri Puspita Roska ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Early Death ◽

Luciferase Reporter ◽

Infection Model ◽

Hibiscus Sabdariffa ◽

Immune Stimulation ◽

Wild Type ◽

Toll Pathway

The rise of antibiotic-resistant Staphylococcus aureus-related clinical cases is an alarming chronicle for global communities. This research was conducted to examine the antistaphylococcal effect of roselle (Hibiscus sabdariffa L.) calyx fractions in the Drosophila model. In the infection experiment, wild-type and immunodeficient Drosophila were pricked with S. aureus and subsequently subjected to fly survivorship and colony-forming assays, in the presence or absence of roselle calyx fractions. The Involvement of immune stimulation in the host antibacterial protection was assessed in vitro using cell-based luciferase reporter assay and in vivo using RT-qPCR analysis on adult flies. A declining rate of fly survivorship and augmentation of bacterial growth were observable in S. aureus-infected wild-type flies but subject to improvement in the presence of roselle calyx fractions. Cell-based analysis revealed the absence of host immune stimulation via Drosophila Toll pathway and roselle calyx fractions-treated immune-deficient flies lacking for components in the Toll pathway were protected from infection-induced early death phenotype and harbored reduced number of S. aureus colonies. Overall, our data confirmed the in vivo anti-staphylococcal activity of roselle calyx fractions in Drosophila infection model and such protective signature was devoid of host immune stimulation.

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Genetic traceability of Staphylococcus aureus strains isolated from primiparous dairy cows mastitis, humans and environment in the Northeast region of Brazil

Ciência Rural ◽

10.1590/0103-8478cr20200679 ◽

2021 ◽

Vol 51 (4) ◽

Author(s):

Amanda Thaís Ferreira Silva ◽

José Givanildo da Silva ◽

Breno Bezerra Aragão ◽

Núbia Michelle Vieira da Silva ◽

Priscylla Carvalho Vasconcelos ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Dairy Cows ◽

Dairy Herds ◽

Beta Lactam ◽

Beta Lactams ◽

High Genetic Diversity ◽

Northeast Region ◽

Intramammary Infections ◽

D Value

ABSTRACT: This research aimed to investigate the genotypic relatedness of 18 Staphylococcus aureus strains isolated from intramammary infections in primiparous cows and extramammary sites on five dairy herds by rep-PCR using RW3A primers, and by PFGE using the endonuclease SmaI. The isolates were also evaluated in vitro for the susceptibility against beta-lactam antimicrobials drugs (penicillin and oxacillin), considering that beta-lactams are frequently used for treating staphylococcal intrammamary infections. The rep-PCR typing was highly discriminatory (D value= 0.9804) and a total of 15 patterns were detected. The PFGE method was also highly discriminatory (D value= 0.9667) and a total of 13 patterns were observed. A total of 15 out of 18 (83%) isolates were resistant to penicillin and one out of 18 (6%) to oxacillin. In conclusion, these findings confirmed the occurrence of a high genetic diversity of S. aureus strains at the herds and the presence of clonally-related strains only at the same herd, emphasizing a variety of genotypic profiles among the isolates.

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Validation Study of a Receptor-Based Lateral Flow Assay for Detection of Beta-Lactam Antibiotics in Milk

Journal of AOAC International ◽

10.1093/jaoac/92.3.959 ◽

2009 ◽

Vol 92 (3) ◽

pp. 959-974 ◽

Cited By ~ 5

Author(s):

Mohamed Abouzied ◽

Michael Sarzynski ◽

Aaron Walsh ◽

Heather Wood ◽

Mark Mozola

Keyword(s):

Bovine Milk ◽

Operating Parameter ◽

Raw Milk ◽

Cross Reactivity ◽

Penicillin G ◽

Beta Lactam ◽

Milk Samples ◽

Beta Lactam Antibiotics ◽

Cell Counts ◽

Lactam Antibiotic

Abstract Avalidation study designed tomeet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration (FDA), Center for Veterinary Medicine, was conducted for a receptor-based, immunochromatographic method (BetaStar US) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. Results of the Part I (internal) and Part II (independent laboratory) dose-response studies using spiked samples were in very close agreement for all five drugs tested, with differences between the Part I and Part II 90/95 sensitivity values ranging from 0 to 1 ppb. The test was able to detect all five drugs at the approximate 90/95 sensitivity levels when present as incurred residues in milk collected from cows that had been treated with the specific drug. Asixth drug, ceftiofur, was found to be undetectable at levels of 500 ppb (as total ceftiofur metabolites from incurred residues in milk samples). The selectivity of the assay was 100, because no false-positive results were obtained in tests of >1000 control milk samples. The assay was found to be applicable to the testing of frozen raw milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar US assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in rawmilk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels.

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Addition of Ceftaroline to Daptomycin after Emergence of Daptomycin-Nonsusceptible Staphylococcus aureus during Therapy Improves Antibacterial Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00797-12 ◽

2012 ◽

Vol 56 (10) ◽

pp. 5296-5302 ◽

Cited By ~ 76

Author(s):

Warren E. Rose ◽

Lucas T. Schulz ◽

David Andes ◽

Rob Striker ◽

Andrew D. Berti ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Combination Therapy ◽

Membrane Damage ◽

Beta Lactam ◽

Beta Lactams ◽

Bacterial Killing ◽

Content Type ◽

Cell Membrane Damage

ABSTRACTAntistaphylococcal beta-lactams enhance daptomycin activity and have been used successfully in combination for refractory methicillin-resistantStaphylococcus aureus(MRSA) infections. Ceftaroline possesses MRSA activity, but it is unknown if it improves the daptomycin potency comparably to other beta-lactams. We report a complex patient case of endocarditis who was treated with daptomycin in combination with ceftaroline, which resulted in clearance of a daptomycin-nonsusceptible strain. Anin vitropharmacokinetic/pharmacodynamic model of renal failure was used to simulate the development of daptomycin resistance and evaluate the microbiologic effects of daptomycin plus ceftaroline treatment. Combination therapy with daptomycin and ceftaroline restored daptomycin sensitivityin vivoand resulted in clearance of persistent blood cultures. Daptomycin susceptibilityin vitrowas increased in the presence of either ceftaroline or oxacillin. Daptomycin at 6 mg/kg of body weight every 48 h was bactericidal in the model but resulted in regrowth and daptomycin resistance (MIC, 2 to 4 μg/ml) with continued monotherapy. The addition of ceftaroline at 200 mg every 12 h after the emergence of daptomycin resistance enhanced bacterial killing. Importantly, daptomycin plus ceftaroline as the initial combination therapy produced rapid and sustained bactericidal activity and prevented daptomycin resistance. Bothin vivo- andin vitro-derived daptomycin resistance resulted in bacteria with more fluid cell membranes. After ceftaroline was added in the model, fluidity was restored to the level of the initialin vivoisolate. Daptomycin-resistant isolates required high daptomycin exposures (at least 10 mg/kg) to optimize cell membrane damage with daptomycin alone. Ceftaroline combined with daptomycin was effective in eliminating daptomycin-resistant MRSA, and these results further justify the potential use of daptomycin plus beta-lactam therapy for these refractory infections.

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