|dc.description.abstract||A high use of antibiotics is usually associated with increased levels of antimicrobial resistance (AMR). Although AMR in mastitis pathogens is generally not considered as a major problem, monitoring AMR levels in these organisms remains important, since most antibiotics that are used in Dutch dairy cows are for udder-related indications.
Little information is available on the effect of the treatment and treatment route on AMR development in dairy cows. Therefore, the general goal of these three field studies was to assess the effect of intramammary (IMM) applied antibiotics on AMR levels in major mastitis pathogens and coagulase-negative staphylococci (CNS) in milk samples and Escherichia coli in feces samples. This general goal was more specified into three different studies. In the first study, the effect of antibiotics used to cure clinical mastitis (CM) during lactation on AMR development in major mastitis pathogens and CNS was assessed. In the second and third study, the focus was on dry cow treatment (DCT). The effect of DCT on AMR levels in major mastitis pathogens and CNS was evaluated in study two, whereas study three focused on the relation between DCT and β-lactam resistance in fecal E. coli. Additionally, in part four, available monitoring data is presented on AMR levels in mastitis pathogens from Dutch dairy cattle.
1. AMR development in mastitis isolates in relation to clinical mastitis treatment
Aseptic milk samples were obtained from 74 individual cows in 11 Dutch dairy herds. These 74 cows had a total of 96 quarters with CM. Both the CM quarter and the contralateral quarter were sampled before and after treatment. Farmers were asked to record additional data on the cow’s disease and treatment history.
Of the obtained isolates, minimal inhibitory concentrations (MIC) were determined. S. aureus (n=38), CNS (n=45), S. uberis (n=37) and E. coli (n=22) were the most frequently isolated organisms from all samples. Due to low numbers, statistical analysis was limited to descriptive statistics. MIC-50 and MIC-90 values were used to compare groups of organisms isolated before and after treatment. Results were very variable; both increases and decreases were found. One farm might have significantly influenced the results, as all six multiresistant S. aureus strains were obtained from there. Unfortunately, numbers were too small to conclude whether or not the application route has an effect. Also, 16 isolates were obtained from the same quarter before and after treatment. Though, no indication for AMR development due to antibiotic use during lactation was found in this study. For mastitis pathogens, monitoring AMR on herd level seems to be more reflective of the situation in practice regarding AMR development.
2. AMR development in bacteria isolated from milk samples in relation to dry cow treatment
Ten dairy farmers took quarter milk samples from 49 cows that were dried off with benzathine cloxacillin and also from 30 cows that were dried off without antibiotics for control. The quarters were sampled before drying off (n=132) and post calving (n=273), when the milk withdrawal time was over. Unfortunately, due to practical reasons, a considerable amount of samples from before drying off was missing. Major pathogens, such as S. aureus (n=7) and S. uberis (n=4) were seldom isolated. Most commonly isolated were CNS (n=50) and Corynebacterium spp. (n=71). Again, MIC-values were determined of the isolated organisms. Although numbers in this study were too low for definitive conclusions, values for the MIC-50 and MIC-90 in the DCT-group were equal or even lower compared to the control group. For more definitive conclusions, using a similar study design with a larger number of cows seems suitable.
3. AMR development in E. coli isolated from feces samples in relation to dry cow treatment
Of the same 79 cows as described above, feces samples were collected before drying off and post calving. These samples were tested for presence of ESBL/AmpC E. coli, and the proportion of ampicillin-resistant E. coli was determined.
The latter was determined by replicating ±90 colonies onto Mueller-Hinton agar plates (Central Veterinary Institute, Lelystad, the Netherlands) with and without ampicillin (16 mg/L), and after overnight incubation at 37 °C growth results were compared. Ampicillin-resistance was uncommon: in 38 of the 48 samples with ≥ 10 isolates, no ampicillin-resistance was found.
Two out of 85 tested samples were positive for ESBL-suspected isolates. There was no growth in the semi-quantitative test, which indicates presence in low numbers. Subtyping of these two isolates was performed by specific susceptibility testing, micro-array assay and sequencing techniques. This resulted in one CTX-M-1 ESBL E. coli and one AmpC-positive E. coli.
An effect of DCT was not shown in this study. Based on these results, β-lactam-resistance in fecal E. coli of dairy cattle does not seem to be a problem at the moment, nor an emerging problem.
4. Trends in AMR in mastitis pathogens from Dutch dairy cattle over the years
The aim of this study was to create an overview of occurrence and trends in antimicrobial resistance in staphylococci, streptococci and coliforms for the period 2002-2014. Data from GD Animal Health and the Central Veterinary Institute (CVI) were combined. In general, AMR levels in the major pathogens S. aureus, S. uberis, S. dysgalactiae, E. coli and Klebsiella spp. were low for the tested antibiotics, often lower than 10%. β-lactam resistance in CNS, however, is substantial (51% penicillin-resistance and 23% oxacillin-resistance in 2014), although there seems to be a decreasing trend. Trends, however, have to be interpreted with care, due to changes in methods of susceptibility testing and progressing insights. The extend of these effects will be further analyzed in the upcoming months.||