An immunoassay method based on penicillin-binding protein (PBP) was developed for the quantitative determination of 10 kinds of beta-lactam antibiotics (BLAs). First, two kinds of PBPs, which are named PBP1a and PBP2x, were expressed and purified, and they were characterized by SDS-PAGE and western blotting analysis. Then, the binding activity of PBP1a and PBP2x to template BLAs, cefquinome (CEFQ) and ampicillin (AMP), was determined. The effect of the buffer solution system, e.g., pH, ion concentration, and organic solvent, on the immune interaction efficiency between PBPs and BLAs was also evaluated. In the end, the PBP-based immunoassay method was developed and validated for the detection of 10 kinds of BLAs. Under optimal conditions, PBPs exhibited high binding affinity to BLAs. In addition, this method showed a high sensitivity for the detection of 10 kinds of BLAs with the limits of detection from 0.21 to 9.12 ng/mL, which are much lower than their corresponding maximum residual limit of European Union (4-100 ng/mL). Moreover, the developed PBP-immunoassay was employed for BLA detection from milk samples, and satisfactory recoveries (68.9-101.3 %) were obtained.

2024-10-01·Journal of Dairy Science

Antimicrobial resistance of Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus canis in quarter milk samples from Bavaria, Southern Germany, between 2012 and 2022

Article

作者: Bechtold, V ; Sorge, U S ; Gangl, A ; Huber-Schlenstedt, R ; Petzl, W

The objective of this study was to analyze the in vitro antimicrobial resistance (AMR) of Streptococcusdysgalactiae, Strep. agalactiae, and Strep. canis over a 10-yr period from 2012 to 2022 against the most commonly used antimicrobial agents. For this purpose, all quarter milk samples (QMS) submitted to the milk laboratory of the Bavarian Animal Health Service were analyzed. Each QMS was tested using the California Mastitis Test and categorized as negative, subclinical, or clinical mastitis if the milk character was abnormal. Samples with Strep. dysgalactiae, Strep. agalactiae, or Strep. canis were included and a subset of isolates were further tested for in vitro AMR by breakpoint analysis with broth microdilution. Streptococcus dysgalactiae (61%, n = 65,750) was the most abundant pathogen among those 3 species, followed by Strep. agalactiae (28%, n = 30,486), and Strep. canis (11%, n = 11,336). All 3 species showed the highest resistance to the same 4 antimicrobial agents: erythromycin, marbofloxacin, pirlimycin, and cefalexin/kanamycin with varying degrees of resistance. Throughout the study period, Strep. dysgalactiae, Strep. agalactiae, and Strep. canis were largely susceptible to the remaining antimicrobial agents tested (penicillin, amoxicillin-clavulanate, oxacillin, cefazolin, cefoperazone, cefquinome). Only less than 14% of isolates of Strep. dysgalactiae and Strep. canis were resistant against any of the antimicrobials tested. Streptococcus agalactiae was the species with the highest percentage of resistant isolates. Although the percentage of resistant isolates from Strep. canis and Strep. dysgalactiae decreased, the percentage of resistant Strep. agalactiae isolates increased after 2017. In summary, most isolates were not resistant to the most commonly used antimicrobial agents for mastitis therapy, including β-lactam antibiotics and penicillin should remain the first-choice therapy against streptococcal mastitis.

2024-10-01·Journal of Dairy Science

In vitro antimicrobial resistance of Escherichia coli, Serratia marcescens, Klebsiella oxytoca, and Klebsiella pneumoniae on Bavarian dairy farms between 2014 and 2022

Article

作者: Sorge, Ulrike S ; Gangl, Armin ; Petzl, Wolfram ; Huber-Schlenstedt, Reglindis ; Pirner, Lea H

The objective of this study was to describe the prevalence of antimicrobial resistance of Escherichia coli, Klebsiella oxytoca, Klebsiellapneumoniae, and Serratiamarcescens from quarter milk samples submitted to the udder health laboratory of the Bavarian Animal Health Services (TGD) in Southern Germany between 2014 and 2022. All samples were tested with the California Mastitis Test and analyzed with a standard microbroth dilution to determine the MIC. The antimicrobials tested were amoxicillin/clavulanate, cefazoline, kanamycin/cefalexin, cefoperazone, cefquinome, and marbofloxacin. Breakpoints were chosen in accordance with the Clinical and Laboratory Standards Institute (CLSI). Over the study period, E. coli, K. oxytoca, and K. pneumoniae showed only few resistances to all antimicrobials tested. For those pathogens MIC 50 and MIC 90 were below breakpoint for all antimicrobials except cefoperazone over the 9 years. A decrease in MIC could be seen for E. coli and K. oxytoca for all of the antimicrobials. While the MIC for K. pneumoniae stayed more stagnant, the prevalence of resistance still decreased overall. Serratiamarcescens isolates were proven intrinsically resistant to amoxicillin/clavulanate and cefazolin, and while in vitro resistances were low for all other antimicrobials tested, S. marcescens tended toward higher MIC for most of the antimicrobials over the years. Over time, there was also an overall increase in the number of isolates for all 4 pathogens per year. Starting 2018 there was a steep increase in the number of isolates particularly from clinical cases. This jump in numbers coincided with a change of the regulation for veterinary drug prescriptions in Germany in 2018 that required, among other things, antimicrobial resistance testing before a change of antibiotics in the course of treatment and the use of critically important antimicrobials. Overall, although the pathogens increased in numbers, the prevalence of their antimicrobial resistance remained low.

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