Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test

The indiscriminate use of first-line drugs contributed to the spread of resistant bacteria, a major concern for both human and veterinary medicine. Methicillin resistance is acquired through the mecA gene, which encodes for the PBP2a protein and lends the resistance to β-lactams. Verifying the corre...

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Autores principales:	Serenella Silvestri, Elisa Rampacci, Valentina Stefanetti, Michele Trotta, Caterina Fani, Lucia Levorato, Chiara Brachelente, Fabrizio Passamonti
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	Staphylococcus pseudintermedius methicillin resistance fluorescent antibody technique penicillin-binding protein 2a antimicrobial drug resistance pyoderma Veterinary medicine SF600-1100
Acceso en línea:	https://doaj.org/article/d8036d52548940279274a6a7738a5dd7
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spelling	oai:doaj.org-article:d8036d52548940279274a6a7738a5dd72021-12-01T18:30:56ZImmunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test2297-176910.3389/fvets.2021.740934https://doaj.org/article/d8036d52548940279274a6a7738a5dd72021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fvets.2021.740934/fullhttps://doaj.org/toc/2297-1769The indiscriminate use of first-line drugs contributed to the spread of resistant bacteria, a major concern for both human and veterinary medicine. Methicillin resistance is acquired through the mecA gene, which encodes for the PBP2a protein and lends the resistance to β-lactams. Verifying the correspondence between gene harboring and protein expression and accelerating methicillin resistance diagnosis is critical to improve the management of antimicrobial administration and to reduce the spread of drug resistances. We tested the applicability of immunofluorescence targeting PBP2a protein to identify a new potential methicillin resistance screening test, ancillary to conventional culture methods. We collected 26 clinical Staphylococcus pseudintermedius (SP) isolates: 25 from canine pyoderma and 1 from dermatitis in a dog owner. SP is one of the most important etiological agents in canine pyoderma and can harbor the mecA gene. We performed PCR for mecA gene detection, broth microdilution (BMD) for phenotypic methicillin resistance, and immunofluorescence targeting PBP2a protein. Compared to the PCR as the gold standard, immunofluorescence showed an apparent prevalence of 34.6% vs. a true prevalence of 53.8%, with 100% specificity, 64.3% sensitivity, and 80.8% diagnostic accuracy. PBP2a expression showed isolate-dependent variability: in some isolates, most of the bacterial cells showed an intense and clearly membranous pattern, while in others only a few of them could be detected. Performing the assay in duplicate improved the diagnostic accuracy. Since the mecA gene is shared among the members of the Staphylococcus genus, the test can be applied to identify methicillin resistance independently from the staphylococcal species, both in human and animal samples. Being a rapid and easy method and providing the unique possibility to study the expression of PBP2a by directly visualizing the morphology, it could represent a new interesting tool for both research and diagnostics. To accelerate methicillin resistance diagnosis, it would be worth further testing of its performance on cytological samples.Serenella SilvestriElisa RampacciValentina StefanettiMichele TrottaCaterina FaniLucia LevoratoChiara BrachelenteFabrizio PassamontiFrontiers Media S.A.articleStaphylococcus pseudintermediusmethicillin resistancefluorescent antibody techniquepenicillin-binding protein 2aantimicrobial drug resistancepyodermaVeterinary medicineSF600-1100ENFrontiers in Veterinary Science, Vol 8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Staphylococcus pseudintermedius methicillin resistance fluorescent antibody technique penicillin-binding protein 2a antimicrobial drug resistance pyoderma Veterinary medicine SF600-1100
spellingShingle	Staphylococcus pseudintermedius methicillin resistance fluorescent antibody technique penicillin-binding protein 2a antimicrobial drug resistance pyoderma Veterinary medicine SF600-1100 Serenella Silvestri Elisa Rampacci Valentina Stefanetti Michele Trotta Caterina Fani Lucia Levorato Chiara Brachelente Fabrizio Passamonti Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test
description	The indiscriminate use of first-line drugs contributed to the spread of resistant bacteria, a major concern for both human and veterinary medicine. Methicillin resistance is acquired through the mecA gene, which encodes for the PBP2a protein and lends the resistance to β-lactams. Verifying the correspondence between gene harboring and protein expression and accelerating methicillin resistance diagnosis is critical to improve the management of antimicrobial administration and to reduce the spread of drug resistances. We tested the applicability of immunofluorescence targeting PBP2a protein to identify a new potential methicillin resistance screening test, ancillary to conventional culture methods. We collected 26 clinical Staphylococcus pseudintermedius (SP) isolates: 25 from canine pyoderma and 1 from dermatitis in a dog owner. SP is one of the most important etiological agents in canine pyoderma and can harbor the mecA gene. We performed PCR for mecA gene detection, broth microdilution (BMD) for phenotypic methicillin resistance, and immunofluorescence targeting PBP2a protein. Compared to the PCR as the gold standard, immunofluorescence showed an apparent prevalence of 34.6% vs. a true prevalence of 53.8%, with 100% specificity, 64.3% sensitivity, and 80.8% diagnostic accuracy. PBP2a expression showed isolate-dependent variability: in some isolates, most of the bacterial cells showed an intense and clearly membranous pattern, while in others only a few of them could be detected. Performing the assay in duplicate improved the diagnostic accuracy. Since the mecA gene is shared among the members of the Staphylococcus genus, the test can be applied to identify methicillin resistance independently from the staphylococcal species, both in human and animal samples. Being a rapid and easy method and providing the unique possibility to study the expression of PBP2a by directly visualizing the morphology, it could represent a new interesting tool for both research and diagnostics. To accelerate methicillin resistance diagnosis, it would be worth further testing of its performance on cytological samples.
format	article
author	Serenella Silvestri Elisa Rampacci Valentina Stefanetti Michele Trotta Caterina Fani Lucia Levorato Chiara Brachelente Fabrizio Passamonti
author_facet	Serenella Silvestri Elisa Rampacci Valentina Stefanetti Michele Trotta Caterina Fani Lucia Levorato Chiara Brachelente Fabrizio Passamonti
author_sort	Serenella Silvestri
title	Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test
title_short	Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test
title_full	Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test
title_fullStr	Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test
title_full_unstemmed	Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test
title_sort	immunofluorescence targeting pbp2a protein: a new potential methicillin resistance screening test
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/d8036d52548940279274a6a7738a5dd7
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Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test

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