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In vitro antimicrobial resistance profiles of Streptococcus uberis, Lactococcus spp., and Enterococcus spp. from quarter milk samples of cows between 2015 and 2019 in Southern Germany

Open ArchivePublished:March 05, 2021DOI:https://doi.org/10.3168/jds.2020-19896

      ABSTRACT

      The objective was to describe and compare antimicrobial resistance patters of esculin-hydrolyzing streptococci and streptococcal-like organisms (Streptococcus uberis, Enterococcus faecium, Enterococcus faecalis, Lactococcus garvieae, Lactococcus lactis) from routine diagnostic samples of the udder health laboratory of the Bavarian Animal Health Services between 2015 and 2019. All routine diagnostic samples of the udder health laboratory of the Bavarian Animal Health Services, that were tested with a standard microbroth dilution, were eligible to be included in this retrospective case series. A California Mastitis Test result was available for all samples. Most Strep. uberis and L. lactis were susceptible to all antibiotics tested. Enterococcus faecium had consistently the highest minimum inhibitory concentration required to inhibit the growth of 90% of tested isolates. The resistance patterns of Lactococcus garvieae were positioned between enterococci and L. lactis. The minimum inhibitory concentration for various antibiotics and pathogens tended to decrease over the 5-yr period. Regardless of the pathogen, isolates of clinical cases were less likely to express in vitro resistance than isolates of healthy or subclinical cases. Streptococcus uberis or L. lactis showed hardly any in vitro resistance to tested antibiotic groups. Penicillin should remain the first-choice antimicrobial for the therapy of Strep. uberis and Lactococcus spp. However, a success of any antimicrobial treatment of enterococcal infections seems questionable.

      Key words

      INTRODUCTION

      Mastitis is one of the most common diseases on dairy farms. It is predominantly caused by bacterial infections (
      • Sorge U.S.
      Streptococcus uberis-eine wachsende Herausforderung.
      ). The pathogens are classified as either environmental or contagious based on their predominant transmission pattern. In recent years, mastitis due to environmental pathogens has increased, whereas IMI due to contagious pathogens, such as Staphylococcus aureus or Streptococcus agalactiae, have decreased in relation. Although environmental streptococci and streptococcal-like bacteria were previously often reported as esculin-hydrolyzing streptococci, new diagnostics such as MALDI-TOF (
      • Nonnemann B.
      • Lyhs U.
      • Svennesen L.
      • Kristensen K.A.
      • Klaas I.C.
      • Pedersen K.
      Bovine mastitis bacteria resolved by MALDI-TOF mass spectrometry.
      ) simplify the routine differentiation of esculin-hydrolyzing streptococci and streptococcal-like organisms. Within this group the most common pathogens associated with udder infections are Streptococcus uberis, Enterococcus faecium, Enterococcus faecalis, Lactococcus garvieae, Lactococcus lactis, and the sporadically isolated Aerococcus viridans. Streptococcus uberis is the most common pathogen associated with clinical mastitis in Germany (
      • DVG (Deutsche Veterinärmedizinische Gesellschaft e.V.)
      Zur Prävalenz von Mastitiserregern in Milchproben in Deutschland.
      ), whereas Lactococcus spp. has been recognized only fairly recently as potentially emerging mastitis pathogen (
      • Werner B.
      • Moroni P.
      • Gioia G.
      • Lavín-Alconero L.
      • Yousaf A.
      • Charter M.E.
      • Moslock Carter B.
      • Bennett J.
      • Nydam D.V.
      • Welcome F.
      • Schukken Y.H.
      Short communication: Genotypic and phenotypic identification of environmental streptococci and association of Lactococcus lactis ssp. lactis with intramammary infections among different dairy farms.
      ;
      • Rodrigues M.X.
      • Lima S.F.
      • Higgins C.H.
      • Canniattti-Brazaca S.G.
      • Bicalho R.C.
      The Lactococcus genus as a potential emerging mastitis pathogen group: A report on an outbreak investigation.
      ).
      Based on the veterinary guidelines (
      • BTK
      Leitlinien für den sorgfältigen Umgang mit antibakteriell wirksamen Tierarzneimitteln.
      ), penicillin has been the antibiotic of choice against gram-positive streptococci in Germany. Anecdotal reports of therapy-resistant Strep. uberis mastitis have persisted in the recent years, although the majority of chronic Strep. uberis infections are known to be new infections (
      • Abureema S.
      • Smooker P.
      • Malmo J.
      • Deighton M.
      Molecular epidemiology of recurrent clinical mastitis due to Streptococcus uberis: Evidence of both an environmental source and recurring infection with the same strain.
      ). Furthermore, practitioners have reported that not all cases of esculin-hydrolyzing streptococci respond equally to antibiotic therapy (
      • Kirk J.H.
      • McCowan B.
      • Atwill E.R.
      • Glenn K.S.
      • Higginbotham G.E.
      • Collar C.A.
      • Castillo A.
      • Reed B.A.
      • Peterson N.G.
      • Cullor J.S.
      Association of minimum inhibitory concentration cluster patterns with dairy management practices for environmental bacteria isolated from bulk tank milk.
      ). Therefore, it is unclear, if these pathogens may have become increasingly resistant to common antibiotics.
      Therefore, the objective of this retrospective study was to describe and compare antimicrobial resistance (AMR) patters of esculin-hydrolyzing streptococci and streptococcal-like organisms (Strep. uberis, E. faecium, E. faecalis, L. garvieae, L. lactis) from routine diagnostic samples of the udder health laboratory of the Bavarian Animal Health Services (TGD) between 2015 and 2019.

      MATERIALS AND METHODS

      Sample Population

      For this retrospective case series all quarter milk sample submissions to the milk quality laboratory of the TGD between 2015 and 2019 were eligible to be included in this analysis. Quarter milk samples of all 4 quarters of approximately 100,000 cows had been submitted to the laboratory each year (Table 1). Most samples (∼80%) were from whole or partial herd screening during routine diagnostic and consultation work of the TGD. The other samples were submitted by farmers or their veterinarians for individual cases mostly to assess the infection status of clinical mastitis cases or as control before dry off. The SCC of each quarter was assessed by the California Mastitis Test (CMT), either on farm during sample collection by TGD technicians or upon arrival of samples in the laboratory. For this analysis the CMT scores were summarized as negative (NM), subclinical (SM), and clinical (CM) mastitis.
      Table 1Submission of quarter milk samples (QMS) of cows per year
      The samples came from QMS of entire or partial herd tests (Herd) or submission from individual cows (Individual).
      YearCows (N)Quarters (N)Herds (N)Cows with QMS submissions
      Herd, n (% of all cows/yr)Individual, n
      201594,850374,7164,81880,563 (85)14,287
      201699,674393,8124,64581,997 (82)17,677
      2017103,276406,3274,72082,346 (80)20,930
      2018116,458456,1985,77393,278 (80)23,180
      2019102,089397,7954,90482,584 (81)19,505
      1 The samples came from QMS of entire or partial herd tests (Herd) or submission from individual cows (Individual).

      Laboratory Analysis

      Upon arrival in the laboratory, all samples were cultured in accordance with guidelines of the German veterinary association to diagnose infections in quarter milk samples (
      • DVG
      Leitlinien zur Labordiagnostik der Mastitis – Probenahme und mikrobiologische Untersuchung.
      ). The identification of streptococci was based on colony morphology, hemolysis, esculin hydrolysis, CAMP factor, and Gram stain. Further differentiation of esculin-positive streptococci was conducted with an in-house method that used Enterococcus selective agar plates (kanamycin-esculin-acid-agar, Merck 1.05222.0500) and a disk test against penicillin (10 µg, Oxoid CT0043B) and rifampicin (2 µg, Oxoid CT 0078B) based on a modified method by

      Krabisch, P. and A. Gangl. 2005. Untersuchungen zur Enterokokken differenzierung im Rahmen der Mastitisdiagnostik. Vortrag, 24. Arbeits- und Fortbildungstagung des AVID 14.-16.9.2005.

      and
      • Krabisch P.
      • Gangl A.
      • Wittkowski G.
      • Fehlings K.
      Prävalenz der Antibiotika-Resistenz in Milchviehherden bei Infektionserregern mit humanmedizinischer Bedeutung.
      . Strains with unclear results were identified by MALDI-TOF (microflex MALDI Biotyper, reference database V.3.3.1.0., Bruker Daltonik GmbH).
      The routine guidelines for selection of isolates to test their sensitivity to antimicrobials were as follows: For samples from herd screenings, 2 to 3 isolates per herd were included from samples with ≤2 pathogens, and if the quarter was diagnosed with at least SM, had been previously treated, or upon specific request by the herd veterinarian or farmer. A comparable scheme was applied to submissions of quarter milk samples of individual cows.
      The streptococci were tested for sensitivity to common antibiotics for intramammary therapy by using the method of MIC by broth microdilution (breakpoint method, mastitis 3 plate, Merlin Diagnostika GmbH). The commercial tray tested against the β-lactam antibiotics penicillin, aminopenicillins (ampicillin, amoxicillin-clavulanate) and penicillinase-stable isoxazolyl penicillins (oxacillin), as well as cephalosporins (first generation: cefazolin, third generation: cefoperazon, fourth generation: cefquinome), lincosamides (pirlimycin), quinolones (marbofloxacin), macrolides (erythromycin), and aminoglycosides (kanamycin-cefalexin). The program MCN 6 (version MCN 6.00–08.01.2018 Rel. 89; Demo Computer GmbH and Merlin Diagnostica GmbH) was used for the interpretation of the MIC. Breakpoints for sensitivity are based upon CLSI Vet01-A4 (
      • CLSI (Clinical and Laboratory Standards Institute)
      Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals: Approved Standards.
      ) and CLSI Vet01S fifth edition (
      • CLSI (Clinical and Laboratory Standards Institute)
      Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals.
      ). The breakpoint for penicillin for the indication mastitis cow was based on the NCCLS guidelines (
      • NCCLS
      Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard.
      ). If no breakpoint for the type of bacteria and the indication mastitis dairy cow existed, human breakpoints or typical intrinsic resistance patterns were automatically used by the program. Enterococci were considered intrinsically resistant to cephalosporins, isoxazolyl penicillins, lincosamides, macrolides, and aminoglycosides (
      • Brandis H.
      • Köhler W.
      • Eggers H.J.
      • Pulverer G.
      Lehrbuch der Medizinischen Mikrobiologie.
      ;
      • Leclercq R.
      • Cantón R.
      • Brown D.F.J.
      • Giske C.G.
      • Heisig P.
      • MacGowan A.P.
      • Mouton J.W.
      • Nordmann P.
      • Rodloff A.C.
      • Rossolini G.M.
      • Soussy C.J.
      • Steinbakk M.
      • Winstanley T.G.
      • Kahlmeter G.
      EUCAST expert rules in antimicrobial susceptibility testing.
      ) and therefore only evaluated against penicillin, ampicillin, and amoxicillin-clavulanate. Lactococcus garvieae was considered naturally resistant against oxacillin (isoxazolyl penicillins) and lincosamides (Pot et al., 1995). Not further differentiated isolates of esculin-hydrolyzing Streptococcus spp. or Enterococcus spp. were excluded from this analysis. Intermediate results were included as resistant and only acquired resistance was reported.

      Statistical Analysis

      The statistical analysis was done in SAS 9.4 (SAS Institute Inc.). The PROC FREQ procedures were used to summarize MIC observations for each pathogen by year and by mastitis status. The MIC50 and MIC90 were the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively. Fisher's exact test was used to compare proportions of resistant pathogens by mastitis status. The number of antimicrobials for that the pathogen was considered resistant to in vitro was summarized to calculate the pathogens' resistance to multiple antibiotics. Graphics were completed in Excel (Microsoft Office Professional Plus 2010). Missing data were ignored and α was set at 0.05.

      RESULTS

      Sample Population Descriptions

      Table 2 provides an overview of the isolates analyzed with broth microdilution between 2015 and 2019. The results of the broth microdilution of not further differentiated Enterococcus spp. (5,816 of 16,098) and esculin-hydrolyzing Streptococcus spp. (1,422 of 9,581) isolates were excluded from this analysis. There was a fairly high number of individual source herds for each pathogen and therefore, on average, 1.1 to 2.5 isolates/farm were included in this analysis (Table 2). Due to lower frequencies of detection, enterococcal isolates (>51%) were more than twice as likely to be assessed by MIC than Strep. uberis isolates (21%). Across all pathogens, isolates of clinical samples were, on average, more likely (90%) to be assessed for MIC than those of healthy (16%) or subclinical quarters (38%). If all available isolates were included (i.e., including the disk diffusion test results during pathogen differentiation) only 60 of 66,507 Strep. uberis isolates (0.1%) were considered resistant to penicillin, though 40/60 had an MIC just one concentration above the break point. Similarly, only very few to none of the isolates of all evaluated E. faecalis (1/1,319), L. garvieae (2/9,582), or L. lactis (0/1,497) were resistant to penicillin. The exception of the group was E. faecium with 58 of 304 isolates (19%) resistant to penicillin.
      Table 2Overview of isolates from quarter milk samples analyzed with broth microdilution between 2015 and 2019
      PathogenAll isolates (N)Analyzed with broth microdilution
      Isolates (n)Herds (n)CMT
      CMT = California Mastitis Test.
      negative (%)
      Subclinical mastitis (%)Clinical mastitis (%)
      Streptococcus uberis66,50714,1467,1371.443.355.3
      Lactococcus garvieae11,2752,8811,1656.987.85.3
      Lactococcus lactis1,5504763424.081.514.5
      Enterococcus faecalis1,3897114029.084.46.6
      Enterococcus faecium3091781631.785.412.9
      1 CMT = California Mastitis Test.

      Minimum Inhibitory Concentrations

      Tables 3 to 13 show the distribution of MIC, MIC50, and MIC90 for the different antimicrobials and pathogens. Across all pathogens, only sporadic isolates were resistant to penicillin, ampicillin, or amoxicillin-clavulanate (Tables 3, 5, 12).
      The growth of the majority of Strep. uberis isolates was consistently inhibited at the lowest concentration of antimicrobials. Streptococcus uberis showed higher MIC90 for only erythromycin, pirlimycin, and marbofloxacin, but the majority of isolates (>70%) was considered susceptible regardless.
      The MIC90 of E. faecium was commonly the highest for most of the tested antimicrobial classes compared with those of Strep. uberis, L. lactis, and L. garvieae. Overall, L. lactis isolates had generally lower MIC to most antimicrobials than L. garvieae. In addition, isolates from CM samples were generally slightly less likely to be resistant than isolates of NM or SM quarters across all pathogens and antibiotics tested (Figure 1).
      Figure thumbnail gr1
      Figure 1Percentage of resistant mastitis pathogen isolates based on MIC by mastitis severity score (Fisher's exact test: **P ≤ 0.01; *P < 0.05). (A) Streptococcus uberis (negative: n = 201, subclinical: n = 6,109, clinical: n = 7,380), (B) Enterococcus faecalis (negative: n = 64, subclinical: n = 599, clinical: n = 47), (C) Enterococcus faecium (negative: n = 3, subclinical: n = 152, clinical: n = 23), (D) Lactococcus garvieae (negative: n = 198, subclinical: n = 2,529, clinical: n = 153), and (E) Lactococcus lactis (negative: n = 19, subclinical: n = 388, clinical: n = 69). IR = intrinsic resistance, Amoxi./Clav. = amoxicillin-clavulanate.

      Changes in MIC Between 2015 and 2019

      Over the 5 years, most pathogens showed a shift toward lower MIC across various antimicrobials. For instance, only 46% of E. faecalis isolates showed an MIC of ≤1 µg/mL against penicillin in 2015 but this increased to 64% in 2019 (Table 3). Comparable trends in penicillin MIC were seen during the same time period for the Lactococcus spp. The proportion of L. garvieae and L. lactis isolates with a lower MIC for penicillin (≤0.25 µg/mL) increased from 42 to 52% and 81 to 86%, respectively (Table 3).
      Table 3Distribution of MIC of penicillin for isolates of quarter milk samples by year; vertical lines indicate breakpoints
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      PathogenYearnMIC (μg/mL)
      ≤0.1250.250.51248>8
      Streptococcus uberisAll14,14699%0%0%0%0%
      20151,8291,82081
      20162,4082,40431
      20172,6772,669413
      20183,9913,96617611
      20193,2413,2278231
      MIC50/90
      Enterococcus faecalisAll7110%1%8%44%44%2%1%1%
      201512217476241
      201622811310210813
      20172201223949064
      20189111443411
      2019501625153
      MIC50MIC90
      Enterococcus faeciumAll1785%2%3%9%24%19%5%33%
      2015382212105214
      201642125118114
      201749316158313
      201820125219
      20192923121128
      MIC50/90
      Lactococcus garvieaeAll2,8811%44%50%4%1%0%0%
      201575753134063111
      201676953093915212
      20178699416408315
      20183315158154122
      20191554747421
      MIC50/90
      Lactococcus lactisAll47613%69%8%6%3%1%1%
      20151341692101132
      201610897489233
      201713120929541
      201857939612
      201946831331
      MIC50MIC90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Furthermore, fewer L. lactis isolates (22 to 9%) were considered resistant to oxacillin (MIC ≥4 µg/mL; Table 4) and fewer E. faecium isolates were resistant at >16 µg/mL to ampicillin in 2019 (14%) compared with 2015 (29%). However, otherwise little changed at the in vitro resistance to ampicillin among the pathogens (Table 5).
      Table 4Distribution of MIC of oxacillin for isolates of quarter milk samples by year; vertical lines indicate breakpoints
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Enterococcusspp. and Lactococcus garvieae have known intrinsic resistance against isoxazolyl penicillins.
      PathogenYearnMIC (μg/mL)
      ≤124>4
      Streptococcus uberisAll14,14698%2%0%0%
      20151,8291,7913053
      20162,4082,3574146
      20172,6772,6482483
      20183,9913,885761614
      20193,2413,1488364
      MIC50/90
      Lactococcus lactisAll47665%19%8%8%
      201513474311217
      201610863221310
      2017131991949
      20185741862
      201946321031
      MIC50MIC90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      2 Enterococcusspp. and Lactococcus garvieae have known intrinsic resistance against isoxazolyl penicillins.
      Table 5Distribution of MIC of ampicillin for isolates of quarter milk samples by year; vertical lines indicate breakpoints
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      PathogenYearnMIC (μg/mL)
      ≤4816>16
      Streptococcus uberisAll14,146100%
      20151,8291,829
      20162,4082,408
      20172,6772,677
      20183,9913,991
      20193,2413,241
      MIC50/90
      Enterococcus faecalisAll711100%0%
      2015122122
      2016228228
      20172202191
      201891891
      20195050
      MIC50/90
      Enterococcus faeciumAll17874%2%2%21%
      2015382711
      20164230129
      2017493829
      2018201325
      2019292414
      MIC50MIC90
      Lactococcus garvieaeAll2,881100% — —0%
      20157577561
      2016769769
      2017869869
      2018331331
      2019155155
      MIC50/90
      Lactococcus lactisAll476100% — —
      2015134134
      2016108108
      2017131131
      20185757
      20194646
      MIC50/90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Also, for cephalosporines of different generations, a slight shift toward lower MIC was observed for Strep. uberis, L. lactis, and L. garvieae: cefazolin (Table 6), cefoperazon (Table 7), and cefquinome (Table 8).
      Table 6Distribution of MIC of cefazolin for isolates of quarter milk samples by year; vertical lines indicate breakpoints
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to cephalosporins (Leclercq et al., 2013).
      PathogenYearnMIC (μg/mL)
      ≤481632>32
      Streptococcus uberisAll14,146100%0%0%0%0%
      20151,8291,820531
      20162,4082,401421
      20172,6772,66782
      20183,9913,981721
      20193,2413,23632
      MIC50/90
      Lactococcus garvieaeAll2,88194%2%3%1%0%
      2015757709829101
      20167696991343113
      2017869828191552
      201833130811111
      2019155152111
      MIC50/90
      Lactococcus lactisAll47688%4%4%3%1%
      201513411011751
      20161088931033
      201713112434
      2018575511
      201946433
      MIC50/90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      2 Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to cephalosporins (
      • Leclercq R.
      • Cantón R.
      • Brown D.F.J.
      • Giske C.G.
      • Heisig P.
      • MacGowan A.P.
      • Mouton J.W.
      • Nordmann P.
      • Rodloff A.C.
      • Rossolini G.M.
      • Soussy C.J.
      • Steinbakk M.
      • Winstanley T.G.
      • Kahlmeter G.
      EUCAST expert rules in antimicrobial susceptibility testing.
      ).
      Table 7Distribution of MIC of cefoperazone for isolates of quarter milk samples by year; vertical lines indicate breakpoints
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to cephalosporins (Leclercq et al., 2013).
      PathogenYearnMIC (μg/mL)
      ≤24816>16
      Streptococcus uberisAll14,14699%1%0%0%0%
      20151,8291,815131
      20162,4082,394113
      20172,6772,6591412
      20183,9913,96819211
      20193,2413,22311322
      MIC50/90
      Lactococcus garvieaeAll2,88136%57%3%3%1%
      201575720149139215
      2016769158530193527
      201786931550723168
      20183312447287
      201915510942211
      MIC50/90
      Lactococcus lactisAll47683%6%3%6%3%
      20151341049894
      20161088381106
      2017131116582
      201857512220
      2019464321
      MIC50/90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      2 Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to cephalosporins (
      • Leclercq R.
      • Cantón R.
      • Brown D.F.J.
      • Giske C.G.
      • Heisig P.
      • MacGowan A.P.
      • Mouton J.W.
      • Nordmann P.
      • Rodloff A.C.
      • Rossolini G.M.
      • Soussy C.J.
      • Steinbakk M.
      • Winstanley T.G.
      • Kahlmeter G.
      EUCAST expert rules in antimicrobial susceptibility testing.
      ).
      Table 8Distribution of MIC of cefquinome for isolates of quarter milk samples by year; vertical lines indicate breakpoints
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to cephalosporins (Leclercq et al., 2013).
      PathogenYearnMIC (μg/mL)
      ≤1248>8
      Streptococcus uberisAll14,146100%0%0%0%0%
      20151,8291,8245
      20162,4082,395103
      20172,6772,666812
      20183,9913,972145
      20193,2413,224152
      MIC50/90
      Lactococcus garvieaeAll2,88197%3%0%0%0%
      20157577342111
      20167697224511
      201786985114112
      20183313208111
      201915515221
      MIC50/90
      Lactococcus lactisAll47688.9%5.5%3.4%1.3%1.1%
      201513411315321
      2016108964323
      2017131118652
      20185753121
      201946433
      MIC50/90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      2 Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to cephalosporins (
      • Leclercq R.
      • Cantón R.
      • Brown D.F.J.
      • Giske C.G.
      • Heisig P.
      • MacGowan A.P.
      • Mouton J.W.
      • Nordmann P.
      • Rodloff A.C.
      • Rossolini G.M.
      • Soussy C.J.
      • Steinbakk M.
      • Winstanley T.G.
      • Kahlmeter G.
      EUCAST expert rules in antimicrobial susceptibility testing.
      ).
      The percent of Strep. uberis isolates that were susceptible to ≤0.125 µg/mL erythromycin increased between 2015 and 2019 from 60 to 80%, respectively (Table 9). Similarly, the proportion of isolates of L. garvieae with an MIC of ≤0.125 µg/mL tripled from 19 to 63%, and the proportion of isolates considered resistant decreased from 26 to 8% over the 5-yr period. Also, L. lactis isolates showed lower MIC and the proportion of resistant isolates decreased from 14 to 2% (Table 9).
      Table 9Distribution of MIC of erythromycin for isolates of quarter milk samples by year; vertical lines indicate breakpoints
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to cephalosporins (Leclercq et al., 2013).
      PathogenYearnMIC (μg/mL)
      ≤0.1250.250.5124>4
      Streptococcus uberisAll14,14676%7%3%3%2%1%8%
      20151,8291,10127371658526208
      20162,4081,827161551033425203
      20172,6772,09214379813824219
      20183,9913,197217831513628279
      20193,2412,59021471831424245
      MIC50MIC90
      Lactococcus garvieaeAll2,88152%34%6%2%2%2%2%
      201575714541511918212514
      20167694132502423291020
      2017869601198221813512
      201833122975117351
      2019155974651114
      MIC50MIC90
      Lactococcus lactisAll47666%22%3%2%2%1%4%
      201513457584510
      2016108741535236
      201713197207421
      2018574851120
      2019463871
      MIC50MIC90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      2 Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to cephalosporins (
      • Leclercq R.
      • Cantón R.
      • Brown D.F.J.
      • Giske C.G.
      • Heisig P.
      • MacGowan A.P.
      • Mouton J.W.
      • Nordmann P.
      • Rodloff A.C.
      • Rossolini G.M.
      • Soussy C.J.
      • Steinbakk M.
      • Winstanley T.G.
      • Kahlmeter G.
      EUCAST expert rules in antimicrobial susceptibility testing.
      ).
      The prevalence of AMR against pirlimycin (Table 10) and marbofloxacin (Table 11) remained fairly consistent for Strep. uberis. However, a large drop in resistant isolates was observed for L. lactis (18 to 4%) in those 5 years (Table 10).
      Table 10Distribution of MIC of pirlimycin for isolates of quarter milk samples by year; vertical lines indicate breakpoint
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Enterococcus faecium, Enterococcus faecalis (Leclercq et al., 2013), and Lactococcus garvieae (Pot et al., 1995) are intrinsically resistant to lincosamides.
      PathogenYearnMIC (μg/mL)
      ≤124>4
      Streptococcus uberisAll14,14679%4%4%14%
      20151,8291,4378262248
      20162,4081,91594118281
      20172,6772,161105101309
      20183,9913,145142164540
      20193,2412,472114129526
      MIC50MIC90
      Lactococcus lactisAll47666%19%3%12%
      20151347832519
      20161087510221
      2017131892778
      201857371118
      2019463592
      MIC50MIC90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      2 Enterococcus faecium, Enterococcus faecalis (
      • Leclercq R.
      • Cantón R.
      • Brown D.F.J.
      • Giske C.G.
      • Heisig P.
      • MacGowan A.P.
      • Mouton J.W.
      • Nordmann P.
      • Rodloff A.C.
      • Rossolini G.M.
      • Soussy C.J.
      • Steinbakk M.
      • Winstanley T.G.
      • Kahlmeter G.
      EUCAST expert rules in antimicrobial susceptibility testing.
      ), and Lactococcus garvieae (
      • Pot B.
      • Devriese L.A.
      • Ursi D.
      • Vandamme P.
      • Haesebrouck F.
      • Kersters K.
      Phenotypic identification and differentiation of Lactococcus strains isolated from animals.
      ) are intrinsically resistant to lincosamides.
      Table 11Distribution of MIC of marbofloxacin for isolates from quarter milk samples by year; vertical lines indicate breakpoints
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to quinolones (Awosile et al., 2018).
      PathogenYearnMIC (μg/mL)
      ≤0.250.512>2
      Streptococcus uberisAll14,1462%18%72%7%1%
      20151,829373701,26813321
      20162,408704431,68418130
      20172,677694271,97518719
      20183,991958912,73324923
      20193,241564312,45825046
      MIC50/90
      Lactococcus garvieaeAll2,8810%4%85%9%1%
      2015757020642887
      20167691286576320
      2017869452750576
      201833113287310
      20191556121271
      MIC50MIC90
      Lactococcus lactisAll4760%17%76%6%2%
      201513415106112
      2016108227493
      201713112210341
      20185794422
      20194611341
      MIC50/90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      2 Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to quinolones (
      • Awosile B.B.
      • McClure J.T.
      • Saab M.E.
      • Heider L.C.
      Antimicrobial resistance in bacteria isolated from cats and dogs from the Atlantic provinces, Canada from 1994–2013.
      ).
      Only E. faecium had enough resistant isolates against amoxicillin-clavulanate to allow a comparison over the years (Table 12). Similar to changes in their resistance to other antimicrobials, their fraction decreased from 24 to 10% and the isolates with an MIC ≥4/2 µg/mL increased from 71 to 86% between 2015 and 2019 (Table 12). Comparable to pirlimycin and marbofloxacin, the distribution of MIC against kanamycin-cefalexin varied little over time across pathogens (Table 13).
      Table 12Distribution of MIC of amoxicillin-clavulanate for isolates from quarter milk samples by year; vertical lines indicate breakpoints
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      PathogenYearnMIC (μg/mL)
      ≤4/28/416/832/16>32/16
      Streptococcus uberisAll14,146100%0%0%0%
      20151,8291,82261
      20162,4082,399711
      20172,6772,6761
      20183,9913,98182
      20193,2413,2392
      MIC50/90
      Enterococcus faecalisAll711100%0%
      2015122122
      20162282253
      2017220220
      20189191
      20195050
      MIC50/90
      Enterococcus faeciumAll17876%5%11%6%3%
      20153827263
      201642313341
      201749392621
      20182014151
      20192925112
      MIC50MIC90
      Lactococcus garvieaeAll2,881100%0%0%
      201575775421
      20167697663
      20178698663
      2018331331
      2019155155
      MIC50/90
      Lactococcus lactisAll47699%1%0%
      2015134134
      20161081053
      20171311283
      201857561
      20194646
      MIC50/90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Table 13Distribution of MIC of kanamycin-cefalexin for isolates from quarter milk samples by year; vertical lines indicate breakpoints
      The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to aminoglycosides (Leclercq et al., 2013).
      PathogenYearnMIC (μg/mL)
      ≤4/0.48/0.816/1.632/3.2>32/3.2
      Streptococcus uberisAll14,14697%2%1%0%0%
      20151,8291,76051162
      20162,4082,32254311
      20172,6772,606422423
      20183,9913,852101353
      20193,2403,137573466
      MIC50/90
      Lactococcus garvieaeAll2,88142%35%19%3%1%
      2015757360238131199
      20167693812521151011
      2017869307316199389
      20183319912486157
      201915558702061
      MIC50MIC90
      Lactococcus lactisAll47676%17%5%1%1%
      201513410617542
      2016108881541
      20171319723101
      20185735184
      2019463781
      MIC50MIC90
      1 The MIC50 and MIC90 denote the MIC where 50 and 90% of isolates were susceptible to tested antibiotics, respectively.
      2 Enterococcus faecium and Enterococcus faecalis are intrinsically resistant to aminoglycosides (
      • Leclercq R.
      • Cantón R.
      • Brown D.F.J.
      • Giske C.G.
      • Heisig P.
      • MacGowan A.P.
      • Mouton J.W.
      • Nordmann P.
      • Rodloff A.C.
      • Rossolini G.M.
      • Soussy C.J.
      • Steinbakk M.
      • Winstanley T.G.
      • Kahlmeter G.
      EUCAST expert rules in antimicrobial susceptibility testing.
      ).

      In Vitro Resistance to Multiple Antimicrobials

      Figure 2 provides an overview over the distribution of number of antimicrobials that isolates of different pathogens were in vitro resistant to for the years 2015 to 2019 combined. Streptococcus uberis showed rarely in vitro resistances during testing against individual antibiotics. Consequently, 65% of isolates (all years, Figure 2A) were susceptible to all 11 tested antimicrobials (n = 9,284), and 26% of isolates were resistant to a single antibiotic tested. Here the 3 most common antimicrobials were pirlimycin (n = 1,511), erythromycin (n = 1,309), marbofloxacin (n = 750), or a combination of these antimicrobials (erythromycin and pirlimycin, n = 754; erythromycin and marbofloxacin, n = 108). Only sporadic isolates were resistant to 5 (n = 5), 6 (n = 2), 7 (n = 1), or 8 (n = 2) antibiotics. None were resistant to more than 8 of the tested antimicrobials.
      Figure thumbnail gr2
      Figure 2Number of antimicrobial substances tested that mastitis pathogen isolates tested in vitro resistant to by year. X: Enterococcus faecalis and Enterococcus faecium were only tested against 4 antimicrobial classes and Lactococcus garvieae only against 9 classes because of their known intrinsic resistance to the other antimicrobials.
      Most E. faecalis (99%; Figure 2B) and E. faecium (67%; Figure 2C) were susceptible to the antimicrobials without known intrinsic resistance. However, 19% of E. faecium isolates (n = 34) were considered resistant to all tested antimicrobials (penicillin, ampicillin, amoxicillin-clavulanate) in addition to their intrinsic resistance against the other 7 antimicrobials. The remaining E. faecium isolates were resistant to penicillin (n = 16, 9%) or penicillin and ampicillin (n = 8, 5%). This made E. faecium less susceptible to available antimicrobials than E. faecalis.
      Also, L. garvieae and L. lactis differed in their resistance patterns. Besides its intrinsic resistance to oxacillin and pirlimycin, L. garvieae had 14.9% isolates that were resistant to kanamycin-cefalexin, 5.3% to marbofloxacin, and 4.3% to erythromycin (Figure 2 D). Resistance against 2, 3, or ≥4 antimicrobials was observed in 6, 4, and 2% of L. garvieae isolates. One isolate was resistant against all antimicrobials tested with the exception of amoxicillin/cefalexin. In contrast, most L. lactis (71%) did not show any AMR during the 5-yr period. Approximately 10% of L. lactis isolates were resistant to only one antimicrobial (2.3% pirlimycin, 2.1% marbofloxacin, 2.1% kanamycin-cefalexin, 1.9% erythromycin, and 1.5% oxacillin) and the remaining isolates were resistant to 2 (5%), 3 (5%), or ≥4 (9%) antimicrobials, which included one isolate with resistance to 8 of 11 antimicrobials tested.

      DISCUSSION

      The strength of this study was the large number of samples, representing a multitude of farms and clinical scores per year and isolate. Some farms will have submitted samples on multiple occasions over the 5-yr period, which was ignored in this analysis due to the high number of farms per pathogen.
      Across all antibiotics tested, similar patterns of resistance or MIC50/90 emerged. Streptococcus uberis always showed the lowest, whereas E. faecium consistently showed the highest MIC90, even where no intrinsic resistance was present. The Lactococcus spp. were somewhere between Strep. uberis and Enterococcus spp. The resistance patterns (including its intrinsic resistance to oxacillin and pirlimycin) of L. garvieae, previously classified as Enterococcus seriolicida (
      • Teixeira L.M.
      • Merquiro V.L.
      • Carvalho M.C.
      • Fracalanzza S.E.
      • Steigerwalt A.G.
      • Brenner D.J.
      • Facklam R.R.
      Phenotypic and genotypic characterization of atypical Lactococcus garvieae strains isolated from water buffaloes with subclinical mastitis and confirmation of L. garvieae as a senior subjective synonym of Enterococcus seriolicida..
      ), were closer to the 2 enterococci, whereas L. lactis was most comparable to Strep. uberis.
      Most pathogens were sensitive to penicillin throughout all 5 years. Almost all Strep. uberis isolates had an MIC ≤0.125 µg/mL for penicillin and were considered susceptible to penicillin, which was similar to 98% observed by
      • Minst K.
      • Märtlbauer E.
      • Miller T.
      • Meyer C.
      Short communication: Streptococcus species isolated from mastitis milk samples in Germany and their resistance to antimicrobial agents.
      from a neighboring district of Germany or the observations of
      • McDougall S.
      • Clausen L.
      • Ha H.-J.
      • Gibson I.
      • Bryan M.
      • Hadjirin N.
      • Lay E.
      • Raisen C.
      • Ba X.
      • Restif O.
      • Parkhill J.
      • Holmes M.A.
      Mechanisms of β-lactam resistance of Streptococcus uberis isolated from bovine mastitis cases.
      from New Zealand. However, here the proportion of isolates susceptible to penicillin is higher than the 70% from Europe (
      • Thomas V.
      • de Jong A.
      • Moyaert H.
      • Simjee S.
      • El Garch F.
      • Morrissey I.
      • Marion H.
      • Vallé M.
      Antimicrobial susceptibility monitoring of mastitis pathogens isolated from acute cases of clinical mastitis in dairy cows across Europe: VetPath results.
      ), 72% from northern Germany (
      • Bolte J.
      • Zhang Y.
      • Wente N.
      • Krömker V.
      In vitro susceptibility of mastitis pathogens isolated from clinical mastitis cases on northern German dairy farms.
      ), or 81% from clinical isolates from China (
      • Cheng J.
      • Qu W.
      • Barkema H.W.
      • Nobrega D.B.
      • Gao J.
      • Liu G.
      • De Buck J.
      • Kastelic J.P.
      • Sun H.
      • Han B.
      Antimicrobial resistance profiles of 5 common bovine mastitis pathogens in large Chinese dairy herds.
      ). Although the percentages slightly differ, it has to be noted that in all studies the majority of Strep. uberis isolates was considered susceptible to penicillin. In particular, the persistently low MIC90 of Strep. uberis, which was identical to
      • Minst K.
      • Märtlbauer E.
      • Miller T.
      • Meyer C.
      Short communication: Streptococcus species isolated from mastitis milk samples in Germany and their resistance to antimicrobial agents.
      , shows this in vitro susceptibility. Although we cannot comment on changes to lower MIC as our commercial plate did not have concentrations below 0.125 µg/mL, the anecdotal reports of local veterinarians and farmers that penicillin has become less effective as treatment against Strep. uberis cannot be supported by the in vitro observations of this study. This is important as Strep. uberis is the most commonly isolated pathogen of clinical mastitis (e.g., 32.2% in 2018) in southern Germany (
      • Sorge U.S.
      Streptococcus uberis-eine wachsende Herausforderung.
      ). Therefore, it seems more likely that therapy failures are indeed new infections as reported by various authors (
      • McDougall S.
      • Parkinson T.J.
      • Leyland M.
      • Anniss F.M.
      • Fenwick S.G.
      Duration of infection and strain variation in Streptococcus uberis isolated from cows' milk.
      ;
      • Abureema S.
      • Smooker P.
      • Malmo J.
      • Deighton M.
      Molecular epidemiology of recurrent clinical mastitis due to Streptococcus uberis: Evidence of both an environmental source and recurring infection with the same strain.
      ). Also, the other pathogens remained susceptible to penicillin or even showed a shift toward lower in vitro MIC. Based on our breakpoint of 8 µg/mL virtually all lactococci were sensitive to penicillin (only one of 2,881 L. garvieae was considered resistant). This agrees with observations by
      • Plumed-Ferrer C.
      • Barberio A.
      • Franklin-Guild R.
      • Werner B.
      • McDonough P.
      • Bennett J.
      • Gioia G.
      • Rota N.
      • Welcome F.
      • Nydam D.V.
      • Moroni P.
      Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Lactococcus lactis ssp. lactis and Lactococcus garvieae isolated from bovine intramammary infections.
      from the United States who also used the same breakpoint.
      Although the MIC of E. faecalis isolates against penicillin decreased over time, this study had slightly more resistant enterococci than
      • Cameron M.
      • Saab M.
      • Heider L.
      • McClure J.T.
      • Rodriguez-Lecompte J.C.
      • Sanchez J.
      Antimicrobial susceptibility patterns of environmental streptococci recorded from bovine milk samples in the maritime provinces of Canada.
      . The presence of intrinsic resistance against cephalosporins, isoxazolyl penicillins, lincosamides, quinolones, macrolides, and aminoglycosides (
      • Kristich C.J.
      • Little J.L.
      • Hall C.L.
      • Hoff J.S.
      Reciprocal regulation of cephalosporin resistance in Enterococcus faecalis..
      ;
      • Leclercq R.
      • Cantón R.
      • Brown D.F.J.
      • Giske C.G.
      • Heisig P.
      • MacGowan A.P.
      • Mouton J.W.
      • Nordmann P.
      • Rodloff A.C.
      • Rossolini G.M.
      • Soussy C.J.
      • Steinbakk M.
      • Winstanley T.G.
      • Kahlmeter G.
      EUCAST expert rules in antimicrobial susceptibility testing.
      ;
      • Awosile B.B.
      • McClure J.T.
      • Saab M.E.
      • Heider L.C.
      Antimicrobial resistance in bacteria isolated from cats and dogs from the Atlantic provinces, Canada from 1994–2013.
      ) clearly shows that enterococcal species have more intrinsic factors against antimicrobials than any other pathogen from the class of esculin-hydrolyzing streptococci (

      Malinowski, E., H. Lassa, and Z. Gajewski. 2010. Antimicrobial sensitivity of streptococci and enterococci from mastitis in cows. Proc. World Association for Buiatrics, Santiago de Chile, Chile.

      ;
      • Kristich C.J.
      • Little J.L.
      • Hall C.L.
      • Hoff J.S.
      Reciprocal regulation of cephalosporin resistance in Enterococcus faecalis..
      ;
      • Garrido A.M.
      • Galvez A.
      • Pulido R.P.
      Antimicrobial resistance in Enterococci.
      ). The frequent occurrence of acquired multidrug resistance in human isolates (
      • Garrido A.M.
      • Galvez A.
      • Pulido R.P.
      Antimicrobial resistance in Enterococci.
      ) was also observed in these isolates of bovine mastitis samples. Besides the enterococci, none of the other pathogens presented such high proportions of resistant isolates to almost all antimicrobials tested (e.g., 19% of E. faecium isolates were resistant to all tested antimicrobials), which agrees with
      • Rossitto P.V.
      • Ruiz L.
      • Kikuchi Y.
      • Glenn K.
      • Luiz K.
      • Wattes J.L.
      • Cullor J.S.
      Antibiotic susceptibility patterns for environmental streptococci isolated from bovine mastitis in central California dairies.
      . This observation emphasizes the importance of a pathogen differentiation beyond environmental streptococci as already the presence of intrinsic resistance differed vastly between, for example, Strep. uberis, Enterococcus spp., and even both lactococci. It further supports the reports by
      • Kirk J.H.
      • McCowan B.
      • Atwill E.R.
      • Glenn K.S.
      • Higginbotham G.E.
      • Collar C.A.
      • Castillo A.
      • Reed B.A.
      • Peterson N.G.
      • Cullor J.S.
      Association of minimum inhibitory concentration cluster patterns with dairy management practices for environmental bacteria isolated from bulk tank milk.
      that not all environmental streptococci react to antimicrobial therapy alike. Based on these observations, it has to be questioned whether the antibiotic treatment of enterococci mastitis is sensible, with or without specific bovine mastitis breakpoints.
      A surprising finding was that isolates from CM were consistently less likely resistant to antibiotics than those of NM or SC, virtually across all pathogens and antibiotics. Other authors (
      • Saini V.
      • Mcclure J.T.
      • Léger D.
      • Keefe G.P.
      • Scholl D.T.
      • Morck D.W.
      • Barkema H.W.
      Antimicrobial resistance profiles of common mastitis pathogens on Canadian dairy farms.
      ) had not reported this for other mastitis pathogens. However, our observation aligns with observations by
      • Cameron M.
      • Saab M.
      • Heider L.
      • McClure J.T.
      • Rodriguez-Lecompte J.C.
      • Sanchez J.
      Antimicrobial susceptibility patterns of environmental streptococci recorded from bovine milk samples in the maritime provinces of Canada.
      , who found an association between higher bulk tank SCC and lower prevalence of AMR in environmental streptococci.
      • Cameron M.
      • Saab M.
      • Heider L.
      • McClure J.T.
      • Rodriguez-Lecompte J.C.
      • Sanchez J.
      Antimicrobial susceptibility patterns of environmental streptococci recorded from bovine milk samples in the maritime provinces of Canada.
      speculated that virulence and resistance might be coded on the same genetic element. Alas, low virulence might be associated with more AMR and reverse. If the AMR genes are coded on plasmids, their replication would slow growth and therefore pose a competitive disadvantage for such bacteria. A slower growth has indeed been described for methicillin-resistant Staphylococcus aureus (
      • Ender M.
      • McCallum N.
      • Adhikari R.
      • Berger-Bächi B.
      Fitness cost of SCCmec and methicillin resistance levels in Staphylococcus aureus..
      ). However, whether the lower percent of resistant bacteria actually translates to a good therapeutic effect of such antimicrobials in vivo has to be determined with future studies. There are very few break points for clinical mastitis due to environmental streptococci (
      • CLSI (Clinical and Laboratory Standards Institute)
      Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals.
      ) and in vitro susceptibility does not automatically equate therapeutic success in vivo (
      • Wallmann J.
      • Kaspar H.
      Empfindlichkeitsprüfung von antibakteriell wirkenden substanzen: Bewertung von Antibiogrammen. Dt.
      ).

      CONCLUSIONS

      Streptococcus uberis or L. lactis showed hardly any in vitro resistance to tested antibiotic groups. Penicillin should remain the first-choice antimicrobial for the therapy of Strep. uberis and Lactococcus spp. However, a success of any antimicrobial treatments of enterococcal infections seems doubtful.

      ACKNOWLEDGMENTS

      This study was made possible with the financial support of the Free State of Bavaria and the Bavarian Joint Founding Scheme for the Control and Eradication of Contagious Livestock Diseases (Bayerische Tierseuchenkasse). The authors have not stated any conflicts of interest.

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