KoNS in Rindermastitis: Schnelle Identifikation und molekulare Charakterisierung der Antibiotikaresistenzgene

• Forschungsstelle: BLV
• Projektnummer: 1.11.21
• Projekttitel Englisch: CoNS in bovine mastitis: Rapid identification and molecular characterization of antibiotic resistance genes

Schlüsselwörter (Deutsch)

Koagualse-negative Staphylokokken, Mastiden, Antibiotika-Resistenz, Mikroarray, Massenspektrometer, Maldi Tof, Identifizierung, Differenzierung

Schlüsselwörter (Englisch)

Coagulase-negative staphylococci, Mastitis, antibiotic resistance, microarray, Mass Spectrometry Maldi Tof, identification, differentiation

Kurzbeschreibung (Deutsch)

Im Rahmen dieses Projektes werden Koagulase-negative Staphylokokken (KoNS) aus Rindermastitis identifiziert und deren Antibiotikaresistenz-Profil bestimmt. Weiter wird ein schnelles Identifikations-Schema entwickelt. Ca. 1000 KoNS aus akuten, chronischen und subklinischen Mastitis werden mittels molekularen Techniken und biochemischen Reaktionen auf der Spezies-Stufe identifiziert und mit dem Maldi Tof Massenspektrometer analysiert. Die Resultate werden verwendet, um eine schnelle Identifikations-Methode mit dem Massenspektrometer zu validieren und implementieren. Die minimale Hemmstoffkonzentrationen (MHK) von verschiedenen Antibiotika werden mittels Bouillon-Verdünnungsmethode ermittelt und die Antibiotikaresistenzgene werden mit Hilfe von Mikroarrays identifiziert. Die Daten werden im Laufe des Projektes dem Institut für Veterinary Public Health der Vetsuisse Fakultät der Universität Bern für Modell-Entwicklung und Risikoanalyse zur Verfügung gestellt. Dieses Projekt wird ein Überblick geben über die Prävalenz von KoNS in Mastitis-Milch in der Schweiz und diese in Beziehung setzen zu den klinischen Fälle. Zudem wird die molekulare Verbreitung von Antibiotikaresistenzgenen in der unterschätzten Gruppe von Mastitis-Erreger bestimmt.

Kurzbeschreibung (Englisch)

The aim of the project is to identify coagulase-negative staphylococci (CoNS) causing bovine mastitis, develop and validate a rapid identification system and determine their resistance profile. A collection of ca. 1000 CoNS from acute, chronic and subclinical mastitis will be identified to the species level using, molecular techniques and biochemical reactions and analyzed by Maldi Tof mass spectrometer technology. The generated results will be used for the validation and implementation of rapid identification of bovine isolates by mass spectrometry . Antibiotic resistance will be determined by the measurement of the minimal inhibitory concentrations (MIC) using broth dilution technique and the antibiotic resistance genes will be detected using a microarray. The data will be made available to the Veterinary Public Health Institute of the Vetsuisse Faculty of the University of Bern (VPHI) for modeling and risk assessment analysis. This project will give an overview on the prevalence of CoNS isolated from mastitis milk in Switzerland in association with the clinical traits as well as determine the molecular distribution of antibiotic resistance in this underestimated group of pathogens.

Projektziele (Englisch)

Development of a rapid and cheap identification method for CoNS

A collection of staphylococci other than S. aureus is being currently collected since October 2009 at the ZOBA. Based on the number of isolates from previous years, ca. 1300 “other staphylococci” will be available for this project . The isolates are routinely only identified as Staphylococcus sp. and not at the species level. All the isolates will be identified at the species level using the specific marker hsp60 (Kwok and Chow, 2003) and 16S rDNA and the results will be compared to the phenotypic data generated by VITEK^®2 compact (bioMérieux SA, Marcy l'Etoile, France). Reactions obtained with VITEK^®2 will allow to choose species-specific metabolitic reactions for the selection of adequate commercially available chromogenic agar (CHROMagar, Paris, France) as well as for the preparation of custom made chromogenic agar (Biosynth AG, Staad, Switzerland). Isolates will be streaked onto the different chromogenic agars which will allow to differentiate bacterial species by the bias of enzymatic reactions and resulting colorations. Comparison of the different reactions will allow to generate groups of CoNS that share the same metabolitic reactions. Based on these results, a simple identification scheme will be created, and the species will be divided into groups which contains closely related CoNS (e.g. S. epidermidis group, S. sciuri group, etc). The high number of strains used for the development and validation of this identification scheme will allow to obtain a reliable identification system with high sensitivity and specificity. It should be useful for many veterinary laboratory that have to perform reliable mastitis diagnostic at low cost. The method will be made available to interested laboratories including ZOBA for implementation into routine mastitis diagnostic.

Phenotypic and genotypic determination of antibiotic resistance

The antibiotic resistance profile of CoNS isolates will be determined by the measurement of minimal inhibitory concentrations (MICs) of antibiotics and by the detection of antibiotic resistance genes using a microarray. The phenotypic and genotypic resistance profiles will allow to determine the prevalence and distribution of antibiotic resistance genes in CoNS from bovine mastitis. The comparison of MIC with the molecular detection of the antibiotic genes will also determine whether some genes remains silent in vitro. The type of antibiotic resistance genes will give a first indication whether they may be present on transferable elements. The presence of methicillin-resistant isolates will also be determined. Isolates containing the mecA gene as well as isolates displaying a multidrug resistance profile will be further characterized. SCCmec elements will be characterized by multiplex PCR (Kondo et al., 2007). This part of the project will reveal whether antibiotic resistance in more predominant in one CoNS species and whether multidrug-resistant CoNS are present in mastitis milk.

Data transfer to VPH Institute

Data (identification of the CoNS, distribution of antibiotic resistance and antibiotic resistance genes) generated during the CoNS project will be made available as soon as available to Bart van den Borne who can use them confidentially to develop a model and perform risk assessment analysis. These data should not be published prior publication of the CoNS project or without permission of the principal investigator Vincent Perreten. Model and risk assessment studies using the data and performed at the VPH Institute can then be published independently of the Institute of veterinary Bacteriology. Periodic meetings will be organized for updates and exchange of information.

Publikationen / Ergebnisse (Englisch)

Frey, Y. (2013) Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis. Dissertation, Vetsuisse Faculty, University of Bern.

Frey, Y.; Rodriguez, J.P.; Thomann, A.; Schwendener, S.; Perreten, V. (2013) Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk. Journal of Dairy Science 96 (4): 2247-57.

Frey, Y. (2012) Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis. Oral presentation, workshop “Bovine mastitis research in Switzerland”, Bern.

Frey, Y.; Rodriguez, J.P.; Thomann, A.; Schwendener, S.; Perreten, V. (2012) Antibiotic resistance in coagulase-negative staphylococci from bovine mastitis. Poster Presentation at the Joint annual meeting of the SSI, SSHH, SSM and SSTMP, St. Gallen, Switzerland.

Perreten V. Molecular diagnosis of bovine mastitis by PCR/ESI-MS (2012) Oral presentation, Joint annual meeting of the SSI, SSHH, SSM and SSTMP, St. Gallen, Switzerland.

Frey, Y.; Schwendener, S.; Perreten, V. (2012) Genetic properties of multidrug-resistant Staphylococcus  epidermidis isolates from bovine mastitis. Poster presentation, International Symposium on Staphylococci and Staphylococcal Infections, Lyon, France.

Perreten, V. et al. (2013) Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis. J. Dairy Sci. 96 (6): 3611–3620.

Perreten, V.; A. Endimiani; R. Sampath; D.J. Ecker; R. Bonomo. (2012) Molecular diagnosis and characterisation of bovine mastitis by PCR/ESI-MS. Poster presentation, 22nd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) Meeting, London, UK.

Zugehörige Dokumente

• Datenblatt [PDF] 64 kB
• 1.11.21 Monografie YFrey.pdf [PDF] 4'155 kB
• Frey et al 2013 JDS 96_2247-2257.pdf [PDF] 435 kB
• Perreten et al 2013 J Dairy Sci 96_3611-20.pdf [PDF] 161 kB

URL-Adressen (Deutsch)

1.11.21 Perreten KoNS in Rindermastitis (063-504/4/21)