In Vitro Susceptibility of Mastitis Pathogens Isolated from Clinical Mastitis Cases on Northern German Dairy Farms.

The present research study investigated the susceptibility of common mastitis pathogens—obtained from clinical mastitis cases on 58 Northern German dairy farms—to routinely used antimicrobials. The broth microdilution method was used for detecting the Minimal Inhibitory Concentration (MIC) of Streptococcus agalactiae (n = 51), Streptococcus dysgalactiae (n = 54), Streptococcus uberis (n = 50), Staphylococcus aureus (n = 85), non-aureus staphylococci (n = 88), Escherichia coli (n = 54) and Klebsiella species (n = 52). Streptococci and staphylococci were tested against cefquinome, cefoperazone, cephapirin, penicillin, oxacillin, cloxacillin, amoxicillin/clavulanic acid and cefalexin/kanamycin. Besides cefquinome and amoxicillin/clavulanic acid, Gram-negative pathogens were examined for their susceptibility to marbofloxacin and sulfamethoxazole/trimethoprim. The examined S. dysgalactiae isolates exhibited the comparatively lowest MICs. S. uberis and S. agalactiae were inhibited at higher amoxicillin/clavulanic acid and cephapirin concentration levels, whereas S. uberis isolates additionally exhibited elevated cefquinome MICs. Most Gram-positive mastitis pathogens were inhibited at higher cloxacillin than oxacillin concentrations. The MICs of Gram-negative pathogens were higher than previously reported, whereby 7.4%, 5.6% and 11.1% of E. coli isolates had MICs above the highest concentrations tested for cefquinome, marbofloxacin and sulfamethoxazole/trimethoprim, respectively. Individual isolates showed MICs at comparatively higher concentrations, leading to the hypothesis that a certain amount of mastitis pathogens on German dairy farms might be resistant to frequently used antimicrobials.


Introduction
The pathogens predominantly associated with bovine mastitis in Germany are substantially staphylococci, streptococci and coliforms [1]. In general, clinical mastitis cases are treated using antimicrobial substances that are locally applied into the teat canal subsequent to the occurrence of clinical signs. The treatment of clinical bovine mastitis with antimicrobial agents is frequently induced without prior knowledge of causative agents and preferred use of antimicrobials covering a broad spectrum of pathogens. According to unpublished data provided by the German Consumer Research Company (GfK), udder injectors containing cephalosporins were used most frequently in the first quarter of 2017. Both cefoperazone and cefquinome, belonging to cephalosporins of the thirdand fourth-generation, achieved a total market share of 39% in Germany. Other commonly used

Isolation and Identification of Pathogens
The isolates included in the current study were randomly selected from the strain collection of the University of Applied Sciences and Arts (Hannover, Germany). Examined mastitis pathogens were isolated from quarter foremilk samples of cows suffering from clinical mastitis. Milk sampling was performed between April 2013 and January 2018 on a total of 58 different Northern German dairy farms. Altogether, 434 isolates were included in the study, subdivided as follows: 85 Staphylococcus (S.) aureus (12 farms), 88 non-aureus staphylococci (NAS) (17 farms), 51 Streptococcus (S.) agalactiae (4 farms), 54 Streptococcus (S.) dysgalactiae (30 farms), 50 Streptococcus (S.) uberis (35 farms), 54 Escherichia (E.) coli (15 farms) and 52 Klebsiella species (15 farms). The number of at least 50 isolates, as well as the long period of time, enables a comprehensive statement about the susceptibilities of mastitis pathogens to commonly used antimicrobials in Northern Germany. The herd size of participating farms ranged from 55 to 2500 cows with an annual average milk yield between 7600 and 13,000 kg. At the time of milk sampling, the bulk milk somatic cell count (SCC) ranged from 88,000 to 334,000 cells/mL. According to the German Veterinary Association (DVG), an SCC of 100,000 cells/mL being present in the bulk tank milk is aimed for. An increase in SCC is due to an increased occurrence of inflammatory cells in the milk and thus reflects an important parameter for assessing the udder health status of a dairy herd. If the arithmetic mean value of the bulk tank milk sample is >300,000 cells/mL in two consecutive samples or a single sample exhibits >400,000 cells/mL, further measures are necessary for rapidly detecting animals affected by mastitis, including clinical investigations of individual animals as well as assessing secretions and cyto-microbiological analysis [13].
The collection of milk samples as well as the microbiological identification of pathogens were performed in accordance with the guidelines of the German Veterinary Medical Association [14]. For identifying present pathogens, 10 µL of each milk sample was plated on a quadrant of esculin blood agar (Oxoid, Germany). After 24 and 48 h of aerobic incubation at 37 • C, microbiological investigations were implemented at growing colonies. Blood agar plates were examined by colony morphology, haemolysis pattern and esculin hydrolysis. Gram staining and biochemical tests were performed for further differentiation of present pathogens. This included the examination of catalase activities (3% H 2 O 2 ; Merck, Germany) as well as the performance of a clumping factor test (DiaMondiaL Staph Plus Kit, Sekisui Virotech, Germany)-for the differentiation of S. aureus and NAS. S. aureus isolates were confirmed by the detection of the specific nuc gene according to Saiful et al. (2006) [15]. Serological tests (DiaMondiaL Streptococcal Extraction Kit Sekisui Virotech, Germany) were used for identifying esculin-negative streptococci as S. dysgalactiae or S. agalactiae according to Lancefield group C and group B, respectively. A Rambach agar, modified as previously described by Watts et al. (1993) [16], served for distinguishing S. uberis from Enterococcus species. Gram-negative rods were verified by testing the activity of cytochrome oxidase C (Bactident oxidase, Merck, Germany) and the performance in an oxidative fermentative test (OF basal medium with the addition of D (+)-glucose-monohydrate, Merck, Germany). Coliform bacteria exhibiting glucose fermentation were further differentiated by Chromocult coliform agar (Merck, Germany), since E. coli appeared as blue colonies after incubation for 24 h at 37 • C. Concerning pink colonies growing on the Chromocult coliform agar, Klebsiella species could be distinguished from other coliforms by their lack of mobility during the oxidative fermentative test.

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was performed using the broth microdilution method in accordance with the DIN EN ISO 20776-1:2006 protocol [17]. The Minimal Inhibitory Concentration (MIC) is defined as the lowest antimicrobial concentration at which bacterial proliferation is no longer visually apparent and is therefore a parameter for a pathogen's susceptibility to a certain antimicrobial agent. Within the present study, MIC values of mastitis pathogens were determined for commonly used antimicrobials in mastitis treatment in Germany. Therefore, polystyrene sterile microtiter plates (Greiner Bio One, Germany) contained antimicrobial concentrations in a two-fold dilution series. For investigating Streptococcus species, 5% of defibrinated horse blood (Oxoid, Germany) was added. Gram-positive pathogens were tested against cefquinome, cefoperazone, cephapirin, penicillin, cloxacillin, oxacillin, amoxicillin/clavulanic acid (4:1) and cefalexin/kanamycin (2:1) in concentrations ranging from 0.06 µg/mL to 32 µg/mL. The used combinations referred to the ratio that is present in commercial preparations. Besides cefquinome and amoxicillin/clavulanic acid (4:1) with concentrations ranging from 0.06 µg/mL to 32 µg/mL, marbofloxacin and sulfamethoxazole/trimethoprim (19:1) were tested against coliforms with concentrations ranging from 0.015 µg/mL to 8 µg/mL and 0.03 µg/mL to 16 µg/mL, respectively. S. aureus ATCC 29213 and E. coli ATCC 25922 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Germany) were used as reference strains. Inoculated microtiter plates were incubated at 37 • C for 16 to 20 h. Bacterial growth was determined due to the appearance of a visible turbidity, whereby the well without turbidity corresponded to the MIC of the respective pathogen.

Results
The results of the MIC examination are presented in detail in the following Tables for Gram-positive  (Tables 1-5) and Gram-negative (Tables 6 and 7) mastitis pathogens, respectively. Besides the distribution of individual isolates according to determined MIC values, MIC 50 and MIC 90 values are given as well. The MIC 50 and MIC 90 values define the lowest concentration at which proliferation of at least 50% and 90% of the tested bacteria is inhibited, respectively [17].

Results of Streptococcus agalactiae (n = 51) Isolated from Northern German Dairy Farms
The distribution of 51 S. agalactiae isolates was dense for all antimicrobials investigated, extending over less than three dilution levels. MIC 50 and MIC 90 values corresponded to the same concentration level for cefoperazone (0.25 µg/mL), oxacillin (0.5 µg/mL), amoxicillin/clavulanic acid (0.5 µg/mL) and cefalexin/kanamycin (4 µg/mL). Fifty-one S. agalactiae isolates were inhibited at cefalexin/kanamycin MIC of 4 µg/mL, representing the highest MIC 50/90 values among the tested antimicrobial agents. MIC 90 of cefquinome, cephapirin, penicillin and cloxacillin were one dilution higher than their MIC 50 values. The lowest MIC 50 as well as MIC 90 were determined as ≤0.06 µg/mL and 0.125 µg/mL for cefquinome and penicillin, respectively (Table 1).

Results of Staphylococcus aureus (n = 85) Isolated from Northern German Dairy Farms
The distribution pattern of S. aureus was dense in the case of cefalexin/kanamycin, referring to three concentrations levels. In contrast, the widest distribution pattern of 85 S. aureus isolates was determined for penicillin, ranging from ≤0.06 µg/mL to 32 µg/mL with MIC 50 and MIC 90 values of ≤0.06 µg/mL and 0.5 µg/mL, respectively. Since at least 90% of S. aureus isolates were inhibited at a cephapirin concentration of 0.5 µg/mL, the lowest MIC 90 values were detected for both penicillin and cephapirin. With the exception of penicillin, MIC 90 values were either one (cefquinome, cefoperazone, cefalexin/kanamycin) or two concentration levels (cephapirin, cloxacillin, oxacillin, amoxicillin/clavulanic acid) higher than their MIC 50 values. Cefoperazone and the combination of cefalexin/kanamycin showed the highest MIC 50 and MIC 90 values at concentrations of 2 µg/mL and 4 µg/mL, respectively. Four S. aureus isolates were inhibited at cefoperazone concentrations of 16 µg/mL, whereby three bacterial isolates also had penicillin MICs of 16 µg/mL and one of them a penicillin MIC of 32 µg/mL (Table 4). Cefquinome --

Results of Klebsiella Species (n = 52) Isolated from Northern German Dairy Farms
The highest MIC 50 (4 µg/mL) and MIC 90 (16 µg/mL) values of 52 Klebsiella species were determined for amoxicillin/clavulanic acid. Sulfamethoxazole/trimethoprim MIC 90 values were three dilutions higher than MIC 50 values, whereas MIC 90 values of remaining antimicrobials were two dilutions higher than MIC 50 values. One individual isolate was not inhibited at the highest concentrations of cefquinome (>32 µg/mL) and sulfamethoxazole/trimethoprim (>16 µg/mL), respectively (Table 6).

Discussion
Besides the strongly limited amount of available research data on the susceptibility of mastitis pathogens obtained from German dairy farms, the comparison is further complicated by various methods available for susceptibility testing. The performance of susceptibility testing in accordance with internationally approved guidelines as published by the German Institute for Standardization (referred to as DIN (Deutsches Institut für Normung e. V.)) ensures the high reproducibility of the test results. While the agar disk diffusion test represents a qualitative method, not only being inexpensive to implement and easy to use in practice, the broth microdilution method is a quantitative method considered as the "gold standard" for susceptibility testing due to its complexity and accuracy [18]. Results as well as derived interpretations differ with regard to the method used for susceptibility testing, this making direct comparison of these two methods quite difficult [19,20].
Within the present study, investigated coliforms did not differ in terms of inhibition by cefquinome and amoxicillin/clavulanic acid, whereas marbofloxacin and sulfamethoxazole/trimethoprim MIC 90 of E. coli were at least two dilutions higher than those determined for Klebsiella species. More striking differences were apparent concerning cefoperazone MIC 90 values of NAS (8 µg/mL) and S. agalactiae, as well as S. dysgalactiae (0.25 µg/mL, respectively), differing by five dilution levels. While S. dysgalactiae might be considered as susceptible to most tested antimicrobials due to the comparatively low MIC values, S. uberis frequently exhibited the highest MICs among examined Gram-positive pathogens. Even though oxacillin and cloxacillin are both semisynthetic β-lactams belonging to the group of isoxazolyl penicillins [21], most Gram-positive pathogens were inhibited at lower oxacillin concentrations.
The MIC 50/90 of S. aureus agree regarding cefquinome [25,26], cefoperazone [26] and amoxicillin/clavulanic acid [25], whereas the MIC 90 values of our trial were one dilution higher concerning oxacillin [25,26]   The resistance monitoring achieved results from susceptibility examinations from various laboratories located in different German federal states. Minst et al. (2012) already detected higher resistance rates in districts with a high density of dairy farms and assumed that this might be due to a locally increased antimicrobial use [22]. The current study focused solely on mastitis pathogens obtained from dairy farms in Northern Germany, which is characterized as a particularly densely populated region of livestock [10,29]. Data on dispensed antimicrobials in veterinary medicine indicated a considerably higher amount in the North of Germany, especially in the federal state of Lower Saxony [6]. Although resistance of staphylococci to antimicrobials was frequently higher in regions with an above-average intensity of dairy cattle in Northern Germany, a previous research study concluded that the density of swine or cattle populations is not associated with the frequency of resistant bacteria in a region [29]. In contrast, Tenhagen et al. (2018) concluded that prevalence of MRSA seems to be related to the density of livestock and might thus be elevated in densely populated regions. Furthermore, Tenhagen and colleagues also found a positive correlation between prevalence of MRSA in bulk tank milk and the respective herd size. In this regard, 13.3% of milk samples obtained from herds with >80 cows were tested positive for MRSA, whereas the amount was 7.3% in herds having <80 cows [10]. The current study included mastitis pathogens from dairy herds with a herd size ranging from 55 to 2500 dairy cows. To investigate differences in susceptibilities in relation to herd size, the participating farms were divided into 30 small dairy farms (<92 cows) and 28 large ones (>92 cows). The limiting number of 92 cows was used as this number represents the current average herd size in the Northern German region [30]. Tables 8 and 9 display the MIC values for Gram-positive and Gram-negative pathogens by differentiating between small and large dairy herds, respectively. S. agalactiae was isolated from small dairy farms only, so the results of this mastitis pathogen are not presented.  50 . However, more investigations including a greater bacterial number are needed before an accurate statement can be made. The susceptibility of Gram-positive mastitis pathogens obtained from dairy farms located in Northern Germany was previously described as favorable, whereas Gram-negative isolates exhibited varying degrees of resistance regarding all tested antimicrobials. Cephalosporins were considered to be effective against staphylococci and streptococci mastitis. However, penicillin represented the drug of choice for treating clinical mastitis induced by streptococci [31]. By establishing special guidelines, various countries aim to reduce the use of antimicrobials in order to counteract the development of resistance. This especially refers to those antimicrobial agents that were declared as "critically important" due to their major relevance in human medicine [32]. In Germany, obligatory guidelines have recently been introduced for the use of certain antimicrobial substances. The microbiological analysis of milk samples as well as susceptibility testing of present pathogens are thus required whenever use of third-and fourth-generation cephalosporins or fluorochinolones is considered for antimicrobial treatment [33]. In comparison to Germany, several Nordic countries such as Sweden, Norway, Denmark and Finland already created a common strategy for treating clinical mastitis several years ago. According to the jointly created "Nordic Guidelines for Mastitis Therapy" a general restrictive use of penicillin is pursued by an overall reduction in cephalosporins and quinolones as much as possible [12]. Chehabi  Therefore, streptococci were classified as highly susceptible to penicillin, whereby results of S. uberis (MIC 50/90 : ≤0.06/0.25 µg/mL), S. dysgalactiae and S. agalactiae (MIC 50/90 : ≤0.06/≤0.06 µg/mL) were largely consistent with our results. Whereas penicillin MIC values of Danish NAS were ≤0.06 µg/mL (MIC 50 ) and 0.5 µg/mL (MIC 90 ), results of S. aureus were higher for penicillin MIC 90 (2 µg/mL), while penicillin MIC 50 was ≤0.06 µg/mL [34]. A lower penicillin MIC 90 of Danish S. aureus was previously reported at concentrations of 0.25 µg/mL, this being identical with results of S. aureus isolates from Iceland and Switzerland (MIC 50/90 : ≤0.06/0.25 µg/mL). Penicillin MIC values of S. aureus from England, Finland, Ireland, Sweden and the United States were in accordance with our results detected at concentrations of ≤0.06 (MIC 50 ) and 0.5 µg/mL (MIC 90 ) [35]. Oxacillin MIC 50/90 of S. aureus investigated in our study was determined at concentrations of 0.25 µg/mL and 1 µg/mL, respectively. In comparison to results of previous surveys from England, Finland, Iceland, Ireland, Norway, Sweden, Switzerland and the United States, the oxacillin MIC 90 of S. aureus was also 1 µg/mL, while MIC 50 was 0.5 µg/mL. S. aureus isolates from Denmark were inhibited at lower oxacillin concentrations (MIC 50/90 : 0.25/0.5 µg/mL) [35].
An interesting point is the difference in the MIC values of the isoxazolyl penicillins oxacillin and cloxacillin, which was detected for the majority of mastitis pathogens. Besides the problem of different methods used for susceptibility testing, comparison is also hampered by the use of different antimicrobial substances approved for mastitis treatment in various countries. The differences in the MIC values of oxacillin and cloxacillin indicate that a comparison of different antimicrobial agents belonging to the same antimicrobial group is inappropriate, such as comparing the third-generation cephalosporins cefoperazone (approved in Germany) and ceftiofur (approved in the United States).  [36]. Concerning S. dysgalactiae differences to our study were detected in MIC 90 values only, as MIC 90 values of ≤0.03 µg/mL (amoxicillin/clavulanic acid), 0.06 µg/mL (cephapirin) and 0.015 µg/mL (cefquinome) were previously described [36]. Compared to our result, the MICs of S. uberis were lower in the case of amoxicillin/clavulanic acid (MIC 50/90 : 0.25/0.5 µg/mL), cephapirin (MIC 90 : 0.5 µg/mL) and cefquinome (MIC 50/90 : 0.125/0.25 µg/mL) [36]. Both Klebsiella species and E. coli previously exhibited cefquinome MIC 50/90 of 0.06/0.125 µg/mL and thus differed by one (MIC 50 ) and two (MIC 90 ) concentration levels from our trial [36]. Amoxicillin/clavulanic MIC 90 published by de Jong and coworkers (2018) was 8 µg/mL regarding both Klebsiella species and E. coli. Moreover, 90% of E. coli isolates were inhibited at a marbofloxacin concentration of 0.06 µg/mL (MIC 90 ), representing a three concentration level lower value compared to our study. Marbofloxacin MIC 50/90 against Klebsiella species were formerly detected as 0.06 µg/mL and differed concerning both values from our investigations (MIC 50/90 : 0.03/0.125 µg/mL) [36]. As a conclusion of the comparison with previous German and international studies, it can be stated that staphylococci predominantly show the same susceptibility patterns to most antimicrobials. The values for S. dysgalactiae are mainly limited to low concentration levels and the S. uberis isolates in our study also deviates strongly from the former German and international results. The high MIC values compared to cefquinome could possibly be due to the increased use of these antimicrobials on German dairy farms. However, this is not supported in the case of other pathogens which did not show increased cefquinome MICs and the fact that MIC values of other antimicrobials (e.g., cephapirin) tested against S. uberis (Table 3) were also elevated, although they are not very relevant in mastitis therapy in Germany.
The favorable results of the Danish, Norwegian and Swedish mastitis pathogens could be due to a highly restrictive use of antimicrobials in these countries. Prudent use-in order to minimize the selective pressure-should therefore be a mandatory requirement whenever the use of antimicrobial agents is considered. In the context of antimicrobial reduction in mastitis treatment, the possibility of alternative treatment methods should be considered. An alternative treatment method for mastitis cases could be the use of bacteriophages as therapeutic agents (bacteriophage therapy). Bacteriophages (also called phages) are viruses that only target prokaryotic cells, mainly of one bacterial species, for inserting their DNA or RNA for propagation. After propagation, phage-coded enzymes induce the lysis of the bacterial cell (lytic propagation cycle) which leads to the release of next generation phages that are able to infect new host cells [37]. In a mouse model conducted by Capparelli et al. (2007), a bacteriophage therapy was able to achieve a complete reduction in the S. aureus in mice that were simultaneously infected with bacteria and phages [38]. In another study, S. aureus was previously isolated from dairy cows suffering from mastitis and used to induce mastitis in mice as well. Phage therapy was able to reduce the bacterial counts as well as the clinical degree of the disease. The authors considered the use of phages for an alternative therapy option for the treatment of bovine mastitis, but also mentioned the non-comparability of mastitis in mice and cows due to anatomical and physiological conditions [39]. The fact that lytic results investigated within a mouse model cannot directly be transferred to a dairy cow suffering from mastitis has already been demonstrated by Gill et al. (2006) [40]. In this former study, solely 16.7% (3/18) (p > 0.05) of the quarters infected with S. aureus achieved a bacteriological cure after a five-day treatment with an infusion of a high concentration of bacteriophage K. The authors assumed that an effective concentration of phages in the mammary gland of cattle could not be achieved in raw milk, among other things, due to an inhibition of the bacteriophages to bind to the host cell surface [40]. The investigations by Gill et al. (2006) refer to cows suffering from subclinical mastitis and, to the best of our knowledge, clinical trials are still lacking.
Moreover, a therapy option is the application of non-antimicrobial agents like products containing proteolytic enzymes such as chymotrypsin and trypsin [41] or homeopathic remedies [42]. The former was tested in cows suffering from mild to moderate clinical mastitis, while the latter was administered to chronically infected animals. In both studies, the authors concluded that the treatment could be considered as a possible alternative to antimicrobial therapy. Nevertheless, the efficacy of both products for treating severe clinical mastitis cases may be considered doubtful [41,42].
Another possibility for avoiding the extensive use of antimicrobials could be the application of vaccines. In several trials, the application of vaccines was able to cause a reduced severity of the clinical signs accompanied with mastitis cases caused by S. uberis and E. coli [43,44]. Furthermore, S. uberis-specific vaccines were beneficial concerning the reduction in somatic cell count and bacterial count [44]. While Schukken et al. (2014) achieved a reduction in both the incidence and prevalence of intramammary infections with staphylococci [45], previous observations by Tenhagen et al. (2001) were contrary. Therefore, the application of a herd-specific vaccination in order to prevent S. aureus-induced mastitis in heifers was not successful, since neither the prevalence of intramammary infections nor the incidence of clinical mastitis was significantly approved [46]. The broad range of alternative treatment options (bacteriophages, vaccination, enzymes, homeopathic agents) must be, as already recommended by the majority of authors, further researched in order to establish these methods as effective alternatives in mastitis therapy. Nevertheless, these points might be beneficial in the pursued aim of reducing the antimicrobial use on dairy farms in the future.
The aim of the present study was rather to focus on mastitis pathogens having comparatively higher MIC values, so that success of an antimicrobial therapy might be predicted as doubtful. While in the case of most mastitis pathogens, this only referred to individual isolates, an evident proportion of E. coli isolates were not inhibited within tested concentration ranges (Table 7). This leads to the assumption that individual resistant isolates are present on German dairy farms. However, the final classification of a pathogen as resistant against a certain antimicrobial agent is performed by using clinical breakpoints. Regarding the performance of susceptibility testing, it is obligatory that the used methodology and interpretive criteria are performed following the same guidelines [20]. The Clinical and Laboratory Standards Institute (CLSI) established clinical breakpoints for only three antimicrobial agents referring to the indication of bovine mastitis: ceftiofur, penicillin/novobiocin and pirlimycin [47]. While ceftiofur and penicillin/novobiocin are even not approved for the intramammary application in Germany, pirlimycin is not relevant for mastitis treatment according to current market shares (unpublished data provided by the German Consumer Research Company (GfK)). Due to the lack of specific clinical breakpoints, identifying resistant pathogens is frequently based on breakpoints established for a different animal species, different indication or even established for human medicine. A transmission of these breakpoints is not appropriate and can cause misinterpretations of the results [20,48]. Moreover, in some trials, a classification of pathogens as susceptible and resistant did not result in the enhanced success of a therapy that was derived from the test results [49][50][51]. While the β-lactam antimicrobials are excreted to a high degree in the urine and thus reach elevated concentrations there, the clinical breakpoints related to urinary tract diseases were generally set higher for these antimicrobials [48]. Undoubtedly, the conditions of the urinary tract are not directly transferable to the mammary gland of a dairy cow and presumptive pathogens present in the bovine udder. In order to avoid the pitfalls associated with the use of improper clinical breakpoints, we focused rather on distribution patterns and MIC values. Changes in MIC values can therefore be detected by a comparison with former susceptibility trials, especially by considering regional differences. An assessment and discussion based on MIC values might thus be an appropriate alternative to strictly classifying pathogens as susceptible or resistant, supported by the fact that the statement is even questionable. However, the establishment of mastitis-specific clinical breakpoints is an indispensable tool for accurately assessing resistances and therapy decisions in future.

Conclusions
In conclusion, determined MIC values of Gram-negative pathogens were frequently higher than previously reported. Among Gram-positive mastitis pathogens, differences in MIC values were striking for some pathogen-antimicrobial combinations. Compared to previous German studies, S. uberis and S. agalactiae exhibited higher amoxicillin/clavulanic acid and cephapirin MIC values, while S. uberis isolates were also inhibited at higher cefquinome concentrations. In contrast, penicillin MIC 90 of S. aureus (0.5 µg/mL) investigated in our study was considerably lower than previously described (16 µg/mL). The majority of examined Gram-positive pathogens were inhibited at lower oxacillin than cloxacillin MIC values. Except for S. agalactiae, several mastitis isolates were inhibited at comparatively higher concentrations or not inhibited at the highest tested concentration. This leads to the hypothesis that a certain number of resistant isolates are present on Northern German dairy farms. Whereas the number usually referred to individual isolates, the quantity of presumptive resistant E. coli was higher. Nonetheless, an accurate estimation of resistance is necessarily related to establishing international clinical breakpoints referring to the indication of mastitis.

Conflicts of Interest:
The authors declare no conflict of interest.