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Invited review: Antimicrobial resistance in bovine mastitis pathogens: A review of genetic determinants and prevalence of resistance in European countries

Open AccessPublished:November 01, 2022DOI:https://doi.org/10.3168/jds.2022-22267

      ABSTRACT

      Antimicrobial resistance is an urgent and growing problem worldwide, both for human and animal health. In the animal health sector actions have been taken as concerns grow regarding the development and spread of antimicrobial resistance. Mastitis is the most common infection in dairy cattle. We aimed to summarize the genetic determinants found in staphylococci, streptococci, and Enterobacteriaceae isolated from mastitic milk samples and provide a comparison of percentage resistance to a variety of antimicrobials in European countries.

      Key words

      INTRODUCTION

      Bovine mastitis causes an economic loss to the dairy industry worldwide. In Ireland, it has been estimated that a 30% decrease in bulk milk somatic cell count levels could increase industry returns by €37.7 million per annum (
      • Geary U.
      • Begley N.
      • McCoy F.
      • O'Brien B.
      • O'Grady L.
      • Shalloo L.
      Estimating the impact of mastitis on the profitability of Irish dairy farms.
      ). Control of mastitis can be challenging, due to the high number of subclinical infections that go undetected but still lead to reduced milk production, and to the withdrawal periods that treatment in dairy cattle follow to control for antimicrobial residues in milk for human consumption. In addition, the increase of resistance worldwide has demonstrated how identification of the causing agents and their resistance patterns is key for appropriate antimicrobial therapy of infected animals (
      • Ruegg P.L.
      What is success? A narrative review of research evaluating outcomes of antibiotics used for treatment of clinical mastitis.
      ).
      Antimicrobial resistance (AMR) is a rising threat, with new and emerging mechanisms of resistance appearing and spreading globally. As antibiotics become less effective, certain infections are becoming harder, and even impossible, to treat. Public health concerns are, therefore, increasing with the growing challenge that AMR poses. The presence of pathogenic bacteria in milk is considered a Food Safety issue through the consumption of raw milk ( https://www.cdc.gov/foodsafety/rawmilk/raw-milk-questions-and-answers.html ), while living or working in close contact with dairy cattle increases infection risk (
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      • Jánosi S.
      • Somogyi P.
      • Dán Á.
      • van Bloois L.G.
      • van Duijkeren E.
      • Wagenaar J.A.
      MRSA transmission between cows and humans.
      ). Methicillin-resistant Staphylococcus aureus (MRSA) and multidrug resistant gram-negative bacteria have been detected in raw milk or raw-milk products (
      • Skočková A.
      • Bogdanovičová K.
      • Koláčková I.
      • Karpíšková R.
      Antimicrobial-resistant and extended-spectrum β-lactamase-producing Escherichia coli in raw cow's milk.
      ;
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      Short communication: Characterization of methicillin-resistant Staphylococcus aureus isolated from raw milk fresh cheese in Colombia.
      ). Therefore, surveillance of zoonotic pathogens in animals is one of the European Union's (EU) goals. However, nonzoonotic pathogens are also a threat, first to animal health and welfare, but also to public health as they are a source of transferable genetic resistance.
      The magnitude of the AMR problem has resulted in it being a high priority for health policy makers worldwide, with many implications that will affect the human health, animal health, and environmental sector (One Health) in the future. However, surveillance of veterinary clinical isolates is not systematically performed and can be challenging, as there are gaps in the knowledge and tools needed to correctly evaluate the bacteria (
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      • Szakacs L.
      • Kissiedou-Bob M.
      • Ciortea R.
      • Grilc E.
      • Klavs I.
      • Turk K.
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      • Vrdelja M.
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      • Massanes M.
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      • Periañez L.
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      • Gahigiro Merino C.
      • Portillo Bordonabe M.E.
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      • Beristáin Rementeriá X.
      • Peñalva G.
      • Cisneros J.M.
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      • Del Rio L.
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      • Alioto D.
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      • Havarria T.
      • Hulth A.
      • Carlin K.
      • Edman L.
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      • Aspevall O.
      • Haggar A.
      • Lindal E.
      • Burgos A.
      • Ottoson J.
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      • Nordenfelt A.
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      • Laine M.
      • Fagerstedt P.
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      • Soder J.
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      • Mader R.
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      • Dal Pozzo F.
      • Slowey R.
      • Urdahl A.M.
      • Vatopoulos A.
      • Zafeiridis C.
      • Madec J.Y.
      Defining the scope of the European Antimicrobial Resistance Surveillance network in veterinary medicine (EARS-Vet): A bottom-up and One Health approach.
      ).
      The aim of this review is to summarize the current situation of AMR identified in the main pathogens causing bovine mastitis. Genetic determinants detected worldwide in Staphylococcus spp., Streptococcus spp., Escherichia coli, and Klebsiella pneumoniae are discussed. Additionally, prevalence of resistance to different antimicrobials and the future of surveillance programs are exposed with a focus in Europe.

      ETIOLOGY OF MASTITIS

      No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.
      Mastitis is inflammation of the mammary gland resulting as a consequence of microbial infection (
      • Blowey R.
      • Edmondson P.
      Mastitis Control in Dairy Herds.
      ). Many bacterial species, yeasts, or fungi have been isolated from the mammary gland (
      • Watts J.L.
      Etiological agents of bovine mastitis.
      ). However, the etiological agents most commonly found to be involved in bovine mastitis are Staph. aureus, Streptococcus agalactiae, Streptococcus uberis, E. coli, CNS, and other Streptococcus spp. (such as Streptococcus dysgalactiae;
      • Keane O.M.
      • Budd K.E.
      • Flynn J.
      • McCoy F.
      Pathogen profile of clinical mastitis in Irish milk-recording herds reveals a complex aetiology.
      ;
      • Poutrel B.
      • Bareille S.
      • Lequeux G.
      • Leboeuf F.
      Prevalence of mastitis pathogens in France: Antimicrobial susceptibility of Staphylococcus aureus, Streptococcus uberis, and Escherichia coli.
      ;
      • Krishnamoorthy P.
      • Suresh K.P.
      • Jayamma K.S.
      • Shome B.R.
      • Patil S.S.
      • Amachawadi R.G.
      An understanding of the global status of major bacterial pathogens of milk concerning bovine mastitis: A systematic review and meta-analysis (scientometrics).
      ). These microorganisms can roughly be classified as contagious or environmental pathogens. The first group includes microorganisms adapted to survive within the mammary gland (such as Staph. aureus or Strep. agalactiae, for example) and their transmission is primarily from cow to cow, mostly during the milking process (
      • Blowey R.
      • Edmondson P.
      Mastitis Control in Dairy Herds.
      ). In contrast, environmental pathogens come from the contaminated environment, are opportunistic invaders, and their infections usually take place between milkings and during the dry period (for example, Strep. uberis or E. coli and other coliforms;
      • Bradley A.
      Bovine mastitis: An evolving disease.
      ).
      Intramammary infections can occur in clinical, subclinical forms, or asymptomatic forms. Clinical forms may take an acute or chronic course of infection, but pathological signs such as swelling, heat, hardness, redness or pain of the udder, changes in the milk appearance, and reduction of milk yield can always be observed (
      • Argaw A.
      Review on epidemiology of clinical and subclinical mastitis on dairy cows.
      ). However, subclinical infections are not visible externally, but cause production losses and changes in milk parameters.
      • Botrel M.A.
      • Haenni M.
      • Morignat E.
      • Sulpice P.
      • Madec J.Y.
      • Calavas D.
      Distribution and antimicrobial resistance of clinical and subclinical mastitis pathogens in Dairy Cows in Rhône-Alpes, France.
      established Strep. uberis, E. coli, and coagulase-positive staphylococci as the major causative agents of clinical mastitis in France, and CNS and Strep. dysgalactiae to be predominantly implicated in subclinical mastitis. The epidemiology of mastitis has changed in the last 70 years, due to the introduction of mastitis control strategies and regulations on milk and milk products. For these reasons, environmental pathogens are now a more common cause of mastitis than they were previously (
      • Bradley A.
      Bovine mastitis: An evolving disease.
      ).

      GENETIC DETERMINANTS OF RESISTANCE IN MASTITIS PATHOGENS: GRAM-POSITIVE MICROORGANISMS

      As indicated above, several bacterial species can cause mastitis. This section summarizes the most common acquired AMR mechanisms employed by both gram-positive and gram-negative organisms isolated from bovine milk samples. Gram-positive microorganisms responsible for mastitis infections are mainly from the Staphylococcus and Streptococcus genera.

      Staphylococcus

      Staphylococcus aureus is the main cause of clinical bovine mastitis. It is a common commensal of nares and skin, and can become an opportunistic pathogen leading to superficial and invasive infections both in humans and animals (
      • Lowy F.D.
      Staphylococcus aureus infections.
      ;
      • Foster T.J.
      • Geoghegan J.A.
      Staphylococcus aureus.
      ). It expresses a plethora of surface proteins, some of which are shared with CNS, that act as virulence factors with functions such as adhesion to surfaces, promotion of biofilm formation, invasion of epithelial cells, or immune evasion (
      • Foster T.J.
      • Geoghegan J.A.
      • Ganesh V.K.
      • Höök M.
      Adhesion, invasion and evasion: The many functions of the surface proteins of Staphylococcus aureus.
      ). In addition, CNS are frequently isolated from bovine milk, usually causing subclinical mastitis (
      • Pyörälä S.
      • Taponen S.
      Coagulase-negative staphylococci-emerging mastitis pathogens.
      ). Examples of these are Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus warneri, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus chromogenes, and Staphylococcus epidermidis, among others (
      • Li L.
      • Feng W.
      • Zhang Z.
      • Xue H.
      • Zhao X.
      Macrolide-lincosamide-streptogramin resistance phenotypes and genotypes of coagulase-positive Staphylococcus aureus and coagulase-negative staphylococcal isolates from bovine mastitis.
      ;
      • Wendlandt S.
      • Kadlec K.
      • Feßler A.T.
      • Schwarz S.
      Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.
      ;
      • Khazandi M.
      • Al-Farha A.A.B.
      • Coombs G.W.
      • O'Dea M.
      • Pang S.
      • Trott D.J.
      • Aviles R.R.
      • Hemmatzadeh F.
      • Venter H.
      • Ogunniyi A.D.
      • Hoare A.
      • Abraham S.
      • Petrovski K.R.
      Genomic characterization of coagulase-negative staphylococci including methicillin-resistant Staphylococcus sciuri causing bovine mastitis.
      ).
      With regard to intrinsic or natural resistance, Staph. aureus showed reduced fitness in the presence of ciprofloxacin, daptomycin, gentamicin, linezolid, oxacillin, or vancomycin during the activation of intrinsic factors such as mprF, ndh, fmtA, graR, or dltA (
      • Blake K.L.
      • O'Neill A.J.
      Transposon library screening for identification of genetic loci participating in intrinsic susceptibility and acquired resistance to antistaphylococcal agents.
      ;
      • Rajagopal M.
      • Martin M.J.
      • Santiago M.
      • Lee W.
      • Kos V.N.
      • Meredith T.
      • Gilmore M.S.
      • Walker S.
      Multidrug intrinsic resistance factors in Staphylococcus aureus identified by profiling fitness within high-diversity transposon libraries.
      ). Activation of some chromosomically encoded multidrug-efflux pumps such as NorA or LmrS can confer resistance or reduced susceptibility to quinolones or lincomycin, respectively (
      • Floyd J.L.
      • Smith K.P.
      • Kumar S.H.
      • Floyd J.T.
      • Varela M.F.
      LmrS is a multidrug efflux pump of the major facilitator superfamily from Staphylococcus aureus.
      ;
      • Costa S.S.
      • Viveiros M.
      • Amaral L.
      • Couto I.
      Multidrug efflux pumps in Staphylococcus aureus: An update.
      ). In addition, novobiocin is a coumarin antibiotic to which CNS, such as Staphylococcus saprophyticus, Staph. sciuri, Staphylococcus vitulinus, Staphylococcus fleuretti, Staphylococcus cohnii, Staphylococcus equorum, Staphylococcus kloosii, Staphylococcus arlettae, Staphylococcus gallinarum, Staphylococcus nepalensis, Staphylococcus succinus, or Staph. xylosus, are intrinsically resistant due to the expression of a novobiocin-resistant GyrB protein (
      • Vickers A.A.
      • Chopra I.
      • O'Neill A.J.
      Intrinsic novobiocin resistance in Staphylococcus saprophyticus.
      ;
      • Nobrega D.B.
      • Naushad S.
      • Naqvi S.A.
      • Condas L.A.Z.
      • Saini V.
      • Kastelic J.P.
      • Luby C.
      • De Buck J.
      • Barkema H.W.
      Prevalence and genetic basis of antimicrobial resistance in non-aureus staphylococci isolated from Canadian dairy herds.
      ). Finally, a β-lactamase encoding gene, blaARL, has been identified in Staphylococcus arlettae (CNS) from bovine mastitic milk in Switzerland and Canada, located in the chromosomal DNA with its regulatory genes blaIARL and blaR1ARL (
      • Andreis S.N.
      • Perreten V.
      • Schwendener S.
      Novel β-lactamase blaARL in Staphylococcus arlettae.
      ;
      • Nobrega D.B.
      • De Buck J.
      • Barkema H.W.
      Antimicrobial resistance in non-aureus staphylococci isolated from milk is associated with systemic but not intramammary administration of antimicrobials in dairy cattle.
      ).
      Intrinsic resistance factors do not always confer full resistance, and may be presented as a lower phenotypic susceptibility to the particular antimicrobial. It is also possible to find acquired resistance mechanisms in isolates where intrinsic resistance factors are present. Staphylococcus spp. isolated from bovine mastitis cases have been reported to acquire resistance to several antimicrobial classes including β-lactams, tetracyclines, aminoglycosides, amphenicols, macrolides, trimethoprim, lipopeptides, and lincosamides (
      • Lowy F.D.
      Antimicrobial resistance: The example of Staphylococcus aureus.
      ;
      • Pantosti A.
      • Sanchini A.
      • Monaco M.
      Mechanisms of antibiotic resistance in Staphylococcus aureus.
      ;
      • Wendlandt S.
      • Feßler A.T.
      • Monecke S.
      • Ehricht R.
      • Schwarz S.
      • Kadlec K.
      The diversity of antimicrobial resistance genes among staphylococci of animal origin.
      ;
      • Nobrega D.B.
      • Naushad S.
      • Naqvi S.A.
      • Condas L.A.Z.
      • Saini V.
      • Kastelic J.P.
      • Luby C.
      • De Buck J.
      • Barkema H.W.
      Prevalence and genetic basis of antimicrobial resistance in non-aureus staphylococci isolated from Canadian dairy herds.
      ; Table 1).
      Table 1Percent of resistance of Staphylococcus from bovine mastitis to various antimicrobials in EU countries
      Pen: penicillin; Amp: ampicillin, Amc: amoxicillin + clavulanic acid; Oxa: oxacillin; Fox: cefoxitin; Fur: ceftiofur; Ery: Erythromycin; Pir: pirlimycin; Lin: lincomycin; Cli: clindamycin; Spc: spectinomycin; Gen: gentamicin; Kan: kanamycin; Neo: neomycin; Str: streptomycin; Cip: ciprofloxacin; Enr: enrofloxacin; Tet: tetracycline; Smx: sulfamethoxazole; Tmp: trimethoprim; Sxt: sulfamethoxazole + trimethoprim. In the case of Staph. aureus from Portugal, cloxacillin was tested instead of Oxa/Fox.
      CountryT
      T = type: D, diagnostic; S, survey. CPS: coagulase-positive staphylococcus.
      Disc
      Disc = disc diffusion.
      or MIC
      Standard
      CLSI: Clinical and Laboratory Standards Institute; EUCAST: European Committee on Antimicrobial Susceptibility Testing; CA-SFM: Comité de l'Antibiogramme–Société Française de Microbiologie; BSAC: British Society for Antimicrobial Chemotherapy; AHVLA: Animal Health and Veterinary Laboratories Agency.
      PenAmpAmcOxa/FoxFurEryPirLinCliSpcGenKanNeoStrCipEnrTetSmxTmpSxtReference
      Staphylococcus aureus
       Austria (n = 100)DMICCLSI/publications10241
      Percent resistance was calculated using the % susceptibility published.
      41
      • Wald R.
      • Hess C.
      • Urbantke V.
      • Wittek T.
      • Baumgartner M.
      Characterization of Staphylococcus species isolated from bovine quarter milk samples.
       Belgium (n = 768)DDiscCLSI12.72.12.63.18.31.8
      • Supré K.
      • Lommelen K.
      • De Meulemeester L.
      Antimicrobial susceptibility and distribution of inhibition zone diameters of bovine mastitis pathogens in Flanders, Belgium.
       Czech Republic (n = 46)SDiscCLSI65
      • Baumgartner M.
      • Štromerová N.H.
      • Bzdil J.
      • Siegwalt G.
      Susceptibility and Resistance of Selected Pathogens of the Mammary Gland of Cattle from Austria and Czech Republic in 2017.
       Croatia* (n = 140)DDiscCLSI51.48.660
      Oxacillin was tested.
      31.411.462.2
      Oxytetracycline was tested.
      45.7
      • Sukalić T.
      • Samardžija M.
      • Jurmanović J.
      • Bačanek B.
      • Grbavac J.
      • Cvetnić Ž.
      • Končurat A.
      • Pavljak I.
      • Đuričić D.
      Antimikrobna osjetljivost uzročnika mastitisa krava s područja sjeverozapadne Hrvatske u razdoblju od 2014. do 2018 (Antimicrobial susceptibility of bovine mastitis pathogens from northwestern Croatia in the period 2014 to 2018).
       Denmark (n = 63)DMICCLSI/EUCAST17.51.64.852.40001.628.600
      • Chehabi C.N.
      • Nonnemann B.
      • Astrup L.B.
      • Farre M.
      • Pedersen K.
      In vitro antimicrobial resistance of causative agents to clinical mastitis in Danish Dairy cows.
       England and Wales (n = 28)DDiscBSAC/AHVLA17.97.107.1
      • UK-VARSS
      UK Veterinary Antibiotic Resistance and Sales Surveillance Report UK-VARSS 2018 99.
       Finland (n = 196)SMICNCCLS/SVARM52.14.11.50.5004.105.11.5
      • Pitkälä A.
      • Haveri M.
      • Pyörälä S.
      • Myllys V.
      • Honkanen-Buzalski T.
      Bovine mastitis in Finland 2001—Prevalence, distribution of bacteria, and antimicrobial resistance.
       France * (CPS) (n = 401)DDiscCA-SFM1785211213042
      • Anses
      Resapath: French surveillance network for antimicrobial resistance in bacteria from diseased animals. 2019 annual report.
       Germany (n = 56)SDiscEUCAST85.70010.77.1
      • El Behiry A.
      • Schlenker G.
      • Szabo I.
      • Roesler U.
      In vitro susceptibility of Staphylococcus aureus strains isolated from cows with subclinical mastitis to different antimicrobial agents.
       Italy (n = 120)DDiscCLSI/CASFM64.59.413.142.986.517.525.13.4
      • Intorre L.
      • Vanni M.
      • Meucci V.
      • Tognetti R.
      • Cerri D.
      • Turchi B.
      • Cammi G.
      • Arrigoni N.
      • Garbarino C.
      Antimicrobial resistance of Staphylococcus aureus isolated from bovine milk in Italy from 2005 to 2011.
       Ireland (n = 154)DDiscCLSI44.8003.21.91.90DAFM et al., 2021
       Norway (n = 20)DDiscEUCAST500050050
      • Fergestad M.E.
      • De Visscher A.
      • L'Abee-Lund T.
      • Tchamba C.N.
      • Mainil J.G.
      • Thiry D.
      • De Vliegher S.
      • Wasteson Y.
      Antimicrobial resistance and virulence characteristics in 3 collections of staphylococci from bovine milk samples.
       Lithuania (n = 176)SDiscCLSI76.778.438.1Klimiene et al., 2011
       Portugal (n = 28,126)DDiscCLSI44.712.32.92.200.7
      • Rocha B.
      • Mendonca D.
      • Niza-Ribeiro J.
      Trends in antibacterial resistance of major bovine mastitis pathogens in Portugal.
       Slovakia (n = 65)SDiscCLSI/EUCAST10.86.1
      Cloxacillin was tested.
      10.821.544.610.8
      • Holko I.
      • Tančin V.
      • Vršková M.
      • Tvarožková K.
      Prevalence and antimicrobial susceptibility of udder pathogens isolated from dairy cows in Slovakia.
       Sweden (n = 227)SMICEUCAST2.60.600.900.800.98.80
      • Duse A.
      • Persson-Waller K.
      • Pedersen K.
      Microbial aetiology, antibiotic susceptibility and pathogen-specific risk factors for udder pathogens from clinical mastitis in dairy cows.
       Switzerland (n = 58)SMICCLSI14202
      • Käppeli N.
      • Morach M.
      • Corti S.
      • Eicher C.
      • Stephan R.
      • Johler S.
      Staphylococcus aureus related to bovine mastitis in Switzerland: Clonal diversity, virulence gene profiles, and antimicrobial resistance of isolates collected throughout 2017.
       Ukraine (n = 59)DDiscCLSI41.5116.9521.43
      • Elias L.
      • Balasubramanyam A.S.
      • Ayshpur O.Y.
      • Mushtuk I.U.
      • Sheremet N.O.
      • Gumeniuk V.V.
      • Musser J.M.B.
      • Rogovskyy A.S.
      Antimicrobial susceptibility of Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli isolated from mastitic dairy cattle in Ukraine.
      CNS
       Austria (n = 100)DMICCLSI/publications1742
      • Wald R.
      • Hess C.
      • Urbantke V.
      • Wittek T.
      • Baumgartner M.
      Characterization of Staphylococcus species isolated from bovine quarter milk samples.
       Czech Republic (n = 68)SDiscCLSI43
      • Baumgartner M.
      • Štromerová N.H.
      • Bzdil J.
      • Siegwalt G.
      Susceptibility and Resistance of Selected Pathogens of the Mammary Gland of Cattle from Austria and Czech Republic in 2017.
       Croatia* (n = 189)DDiscCLSI52.116.164.1
      Oxacillin was tested.
      50.81879.9
      Oxytetracycline was tested.
      53.4
      • Sukalić T.
      • Samardžija M.
      • Jurmanović J.
      • Bačanek B.
      • Grbavac J.
      • Cvetnić Ž.
      • Končurat A.
      • Pavljak I.
      • Đuričić D.
      Antimikrobna osjetljivost uzročnika mastitisa krava s područja sjeverozapadne Hrvatske u razdoblju od 2014. do 2018 (Antimicrobial susceptibility of bovine mastitis pathogens from northwestern Croatia in the period 2014 to 2018).
       Denmark (n = 49)DMICCLSI/EUCAST22.42010.220010.220.48.20
      • Chehabi C.N.
      • Nonnemann B.
      • Astrup L.B.
      • Farre M.
      • Pedersen K.
      In vitro antimicrobial resistance of causative agents to clinical mastitis in Danish Dairy cows.
       Finland (n = 400)S/DMICEUCAST41.8346.30.82.37.50016.5505.3
      • Taponen S.
      • Nykäsenoja S.
      • Pohjanvirta T.
      • Pitkälä A.
      • Pyörälä S.
      Species distribution and in vitro antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine mastitic milk.
       France* (n = 488)DDiscCASFM26312821123191183

      Anses. 2019. Résapath: Réseau d'épidémiosurveillance de l'antibiorésistance des bactéries pathogènes animales, bilan 2018. (French surveillance network for antimicrobial resistance in bacteria from diseased animals, report 2018.) Lyon et Ploufragan-Plouzané-Niort.

       Germany (n = 14)SDiscCLSI/EUCAST74.2800107.14Behiry et al., 2012
       Lithuania (n = 95)SMICEUCAST67.44.213.79.54.218.910.5
      • Klimiene I.
      • Virgailis M.
      • Pavilonis A.
      • Siugzdiniene R.
      • Mockeliunas R.
      • Ruzauskas M.
      Phenotypical and genotypical antimicrobial resistance of coagulase-negative staphylococci isolated from cow mastitis.
       Portugal (n = 204)SDiscCLSI9.30016.7
      • Seixas R.
      • Santos J.P.
      • Bexiga R.
      • Vilela C.L.
      • Oliveira M.
      Short communication: Antimicrobial resistance and virulence characterization of methicillin-resistant staphylococci isolates from bovine mastitis cases in Portugal.
       Slovakia (n = 187)SDiscCLSI/EUCAST5.914.4
      Cloxacillin was tested.
      4.820.936.4
      • Holko I.
      • Tančin V.
      • Vršková M.
      • Tvarožková K.
      Prevalence and antimicrobial susceptibility of udder pathogens isolated from dairy cows in Slovakia.
       Sweden (n = 21)SMICEUCAST30.46.29.514.3000
      • Duse A.
      • Persson-Waller K.
      • Pedersen K.
      Microbial aetiology, antibiotic susceptibility and pathogen-specific risk factors for udder pathogens from clinical mastitis in dairy cows.
      1 Pen: penicillin; Amp: ampicillin, Amc: amoxicillin + clavulanic acid; Oxa: oxacillin; Fox: cefoxitin; Fur: ceftiofur; Ery: Erythromycin; Pir: pirlimycin; Lin: lincomycin; Cli: clindamycin; Spc: spectinomycin; Gen: gentamicin; Kan: kanamycin; Neo: neomycin; Str: streptomycin; Cip: ciprofloxacin; Enr: enrofloxacin; Tet: tetracycline; Smx: sulfamethoxazole; Tmp: trimethoprim; Sxt: sulfamethoxazole + trimethoprim. In the case of Staph. aureus from Portugal, cloxacillin was tested instead of Oxa/Fox.
      2 T = type: D, diagnostic; S, survey. CPS: coagulase-positive staphylococcus.
      3 Disc = disc diffusion.
      4 CLSI: Clinical and Laboratory Standards Institute; EUCAST: European Committee on Antimicrobial Susceptibility Testing; CA-SFM: Comité de l'Antibiogramme–Société Française de Microbiologie; BSAC: British Society for Antimicrobial Chemotherapy; AHVLA: Animal Health and Veterinary Laboratories Agency.
      * Percent resistance was calculated using the % susceptibility published.
      ** Oxytetracycline was tested.
      *** Oxacillin was tested.
      Cloxacillin was tested.

      Resistance to β-Lactams

      The increase in penicillin-resistant isolates encountered during the 1940s and 1950s led to the introduction of another β-lactam antimicrobial, methicillin, as a therapeutic solution. Methicillin is a semisynthetic penicillinase-resistant penicillin. However, shortly after methicillin-resistant MRSA was detected. Even though it was first reported in a British hospital in the early 1960s, MRSA is now a worldwide cause of healthcare, community, and livestock infections (
      • Lyon B.R.
      • Skurray R.
      Antimicrobial resistance of Staphylococcus aureus: Genetic basis.
      ;
      • Lowy F.D.
      Antimicrobial resistance: The example of Staphylococcus aureus.
      ).
      The genetic basis for resistance against both β-lactam antimicrobials, penicillin, and methicillin differs in their genetic elements and location within the cell, but the regulation mechanisms have some similarities. The blaZ gene encodes the PC1 β-lactamase responsible for penicillin resistance both in Staph. aureus and CNS species, including isolates from mastitis such as Staph. epidermidis, Staph. haemolyticus, or Staph. chromogenes (
      • Olsen J.E.
      • Christensen H.
      • Aarestrup F.M.
      Diversity and evolution of blaZ from Staphylococcus aureus and coagulase-negative staphylococci.
      ;
      • Nobrega D.B.
      • De Buck J.
      • Barkema H.W.
      Antimicrobial resistance in non-aureus staphylococci isolated from milk is associated with systemic but not intramammary administration of antimicrobials in dairy cattle.
      ;
      • Bolte J.
      • Zhang Y.
      • Wente N.
      • Mahmmod Y.S.
      • Svennesen L.
      • Krömker V.
      Comparison of phenotypic and genotypic antimicrobial resistance patterns associated with Staphylococcus aureus mastitis in German and Danish dairy cows.
      ). The blaZ gene is located in the mobile genetic element transposon Tn552, either in the bacterial chromosome or a plasmid, and is under the control of 2 regulatory genes, blaR1 and blaI, organized in the blaZ-blaR1-blaI operon (
      • Olsen J.E.
      • Christensen H.
      • Aarestrup F.M.
      Diversity and evolution of blaZ from Staphylococcus aureus and coagulase-negative staphylococci.
      ;
      • Llarrull L.I.
      • Mobashery S.
      Dissection of events in the resistance to β-lactam antibiotics mediated by the protein BlaR1 from Staphylococcus aureus.
      ).
      Resistance to methicillin and oxacillin is mediated by the mecA gene, which encodes PBP2a (with low β-lactam affinity), and its expression is regulated by the inducer-repressor genes mecR1 and mecI (
      • Blázquez B.
      • Llarrull L.I.
      • Luque-Ortega J.R.
      • Alfonso C.
      • Boggess B.
      • Mobashery S.
      Regulation of the expression of the β-lactam antibiotic-resistance determinants in methicillin-resistant Staphylococcus aureus (MRSA).
      ). The mecA gene is located with its regulators (mec gene complex) and a cassette chromosome recombinase (ccr gene complex) responsible for the mobility of the system, in a mobile genetic element named staphylococcal cassette chromosome mec (SCCmec) which integrates in the bacterial chromosome always at the same site (att site within host chromosomal gene orfX;
      • Katayama Y.
      • Ito T.
      • Hiramatsu K.
      A new mobile genetic element, staphylococcal cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus.
      ;
      • Ito T.
      • Hiramatsu K.
      • Oliveira D.C.
      • De Lencastre H.
      • Zhang K.
      • Westh H.
      • O'Brien F.
      • Giffard P.M.
      • Coleman D.
      • Tenover F.C.
      • Boyle-Vavra S.
      • Skov R.L.
      • Enright M.C.
      • Kreiswirth B.
      • Kwan S.K.
      • Grundmann H.
      • Laurent F.
      • Sollid J.E.
      • Kearns A.M.
      • Goering R.
      • John J.F.
      • Daum R.
      • Soderquist B.
      Classification of staphylococcal cassette chromosome mec (SCCmec): Guidelines for reporting novel SCCmec elements.
      ). The transfer mechanism is still not fully understood, but conjugation, transduction, and transformation have been reported (
      • Cafini F.
      • Romero V.M.
      • Morikawa K.
      Mechanisms of horizontal gene transfer.
      ). The bla regulatory system can also regulate mecA, particularly when mecR1 and mecI are not present (
      • Hackbarth C.J.
      • Chambers H.F.
      blaI and blaR1 regulate β-lactamase and PBP 2a production in methicillin-resistant Staphylococcus aureus.
      ;
      • Liu P.
      • Xue H.
      • Wu Z.
      • Ma J.
      • Zhao X.
      Effect of bla regulators on the susceptible phenotype and phenotypic conversion for oxacillin-susceptible mecA-positive staphylococcal isolates.
      ). Thirteen different types of SCCmec have been discovered to date and a mecA homolog, mecC, has also been found to confer resistance to penicillinase-resistant penicillins (
      • Ballhausen B.
      • Kriegeskorte A.
      • Schleimer N.
      • Peters G.
      • Becker K.
      The mecA homolog mecC confers resistance against β-lactams in Staphylococcus aureus irrespective of the genetic strain background.
      ;
      • Baig S.
      • Johannesen T.B.
      • Overballe-Petersen S.
      • Larsen J.
      • Larsen A.R.
      • Stegger M.
      Novel SCCmec type XIII (9A) identified in an ST152 methicillin-resistant Staphylococcus aureus.
      ). This was first isolated in England and Denmark from human and bovine isolates, but has also been detected elsewhere (
      • García-Álvarez L.
      • Holden M.T.G.
      • Lindsay H.
      • Webb C.R.
      • Brown D.F.J.
      • Curran M.D.
      • Walpole E.
      • Brooks K.
      • Pickard D.J.
      • Teale C.
      • Parkhill J.
      • Bentley S.D.
      • Edwards G.F.
      • Girvan E.K.
      • Kearns A.M.
      • Pichon B.
      • Hill R.L.R.
      • Larsen A.R.
      • Skov R.L.
      • Peacock S.J.
      • Maskell D.J.
      • Holmes M.A.
      Methicillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: A descriptive study.
      ). In fact, herd-level prevalence of mecC isolates in English and Welsh dairy farms was 2.15% (
      • Paterson G.K.
      • Morgan F.J.E.
      • Harrison E.M.
      • Peacock S.J.
      • Parkhill J.
      • Zadoks R.N.
      • Holmes M.A.
      Prevalence and properties of mecC methicillin-resistant Staphylococcus aureus (MRSA) in bovine bulk tank milk in Great Britain.
      ). It has been shown that Africa, Latin America, and Asia have higher levels of oxacillin or cefoxitin resistance than Europe or North America (
      • Molineri A.I.
      • Camussone C.
      • Zbrun M.V.
      • Suárez Archilla G.
      • Cristiani M.
      • Neder V.
      • Calvinho L.
      • Signorini M.
      Antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis: Systematic review and meta-analysis.
      ).
      The CNS are thought to have a role in the spread of resistance within staphylococcal species isolated from clinical and other public and environmental settings, or animals. Indeed, CNS can carry mecA genes in SCCmec elements, show resistance to oxacillin, and ancestral forms of SSCmec have been identified in some species (
      • Hussain Z.
      • Stoakes L.
      • Massey V.
      • Diagre D.
      • Fitzgerald V.
      • El Sayed S.
      • Lannigan R.
      Correlation of oxacillin MIC with mecA gene carriage in coagulase-negative staphylococci.
      ;
      • Cafini F.
      • Romero V.M.
      • Morikawa K.
      Mechanisms of horizontal gene transfer.
      ;
      • Saber H.
      • Jasni A.S.
      • Tengku Jamaluddin T.Z.M.
      • Ibrahim R.
      A review of staphylococcal cassette chromosome mec (SCCmec) types in coagulase-negative staphylococci (CoNS) species.
      ;
      • Xu Z.
      • Shah H.N.
      • Misra R.
      • Chen J.
      • Zhang W.
      • Liu Y.
      • Cutler R.R.
      • Mkrtchyan H.V.
      The prevalence, antibiotic resistance and mecA characterization of coagulase negative staphylococci recovered from non-healthcare settings in London, UK.
      ). The mecA gene homologs such as mecA1 or mecA2 have been identified in Staph. sciuri or Staph. vitulinus, respectively; however, these do not confer β-lactam resistance (
      • Miragaia M.
      Factors contributing to the evolution of mecA-mediated β-lactam resistance in staphylococci: Update and new insights from whole genome sequencing (WGS).
      ). Methicillin resistance due to mecA is commonly observed in Staphylococcus spp. causing bovine mastitis infections (
      • Turutoglu H.
      • Hasoksuz M.
      • Ozturk D.
      • Yildirim M.
      • Sagnak S.
      Methicillin and aminoglycoside resistance in Staphylococcus aureus isolates from bovine mastitis and sequence analysis of their mecA genes.
      ;
      • Soares L.C.
      • Pereira I.A.
      • Pribul B.R.
      • Oliva M.S.
      • Coelho S.M.O.
      • Souza M.M.S.
      Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis.
      ;
      • Pu W.
      • Su Y.
      • Li J.X.
      • Li C.H.
      • Yang Z.Q.
      • Deng H.P.
      • Ni C.X.
      High incidence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with bovine mastitis in China.
      ;
      • Khazandi M.
      • Al-Farha A.A.B.
      • Coombs G.W.
      • O'Dea M.
      • Pang S.
      • Trott D.J.
      • Aviles R.R.
      • Hemmatzadeh F.
      • Venter H.
      • Ogunniyi A.D.
      • Hoare A.
      • Abraham S.
      • Petrovski K.R.
      Genomic characterization of coagulase-negative staphylococci including methicillin-resistant Staphylococcus sciuri causing bovine mastitis.
      ). However, methicillin-susceptible isolates of Staph. aureus from bovine mastitis carrying the mecA gene have been detected; it is therefore important to combine genotypic and phenotypic tests to obtain certain results (
      • Pu W.
      • Su Y.
      • Li J.X.
      • Li C.H.
      • Yang Z.Q.
      • Deng H.P.
      • Ni C.X.
      High incidence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with bovine mastitis in China.
      ).

      Resistance to Tetracyclines

      Resistance to tetracyclines in staphylococcal species from mastitis samples is mainly related to the genes tet(K) and tet(L), which code for membrane-associated efflux proteins of the Major Facilitator Superfamily, and are transferred by plasmids (
      • Enany S.
      • Alexander L.E.C.
      The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus.
      ;
      • Schwarz S.
      • Feßler A.T.
      • Loncaric I.
      • Wu C.
      • Kadlec K.
      • Wang Y.
      • Shen J.
      Antimicrobial resistance among staphylococci of animal origin.
      ). Gene tet(38) is a chromosomally encoded efflux pump that can be overexpressed from a plasmid (
      • Truong-Bolduc Q.C.
      • Villet R.A.
      • Estabrooks Z.A.
      • Hooper D.C.
      Native efflux pumps contribute resistance to antimicrobials of skin and the ability of Staphylococcus aureus to colonize skin.
      ;
      • Chen C.
      • Hooper D.C.
      Effect of Staphylococcus aureus Tet38 native efflux pump on in vivo response to tetracycline in a murine subcutaneous abscess model.
      ). Additionally, the gene tet(M) is also frequently found and codes for a ribosome-protective protein. The tet(M) gene is usually located in conjugative transposons Tn916-Tn1545 (
      • Liu H.
      • Li S.
      • Meng L.
      • Dong L.
      • Zhao S.
      • Lan X.
      • Wang J.
      • Zheng N.
      Prevalence, antimicrobial susceptibility, and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China.
      ;
      • Schwarz S.
      • Feßler A.T.
      • Loncaric I.
      • Wu C.
      • Kadlec K.
      • Wang Y.
      • Shen J.
      Antimicrobial resistance among staphylococci of animal origin.
      ).
      These genes were detected in Staph. aureus and CNS from dairy farms in Switzerland, Canada, China, Australia, or Germany, for instance (
      • Feßler A.
      • Scott C.
      • Kadlec K.
      • Ehricht R.
      • Monecke S.
      • Schwarz S.
      Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis.
      ;
      • Frey Y.
      • Rodriguez J.P.
      • Thomann A.
      • Schwendener S.
      • Perreten V.
      Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk.
      ;
      • Ali A.
      • Shrief L.
      Molecular characterization of tetracycline-resistant genes in Staphylococcus aureus isolated from dairy cows and she-camels suffering from subclinical mastitis.
      ;
      • Khazandi M.
      • Al-Farha A.A.B.
      • Coombs G.W.
      • O'Dea M.
      • Pang S.
      • Trott D.J.
      • Aviles R.R.
      • Hemmatzadeh F.
      • Venter H.
      • Ogunniyi A.D.
      • Hoare A.
      • Abraham S.
      • Petrovski K.R.
      Genomic characterization of coagulase-negative staphylococci including methicillin-resistant Staphylococcus sciuri causing bovine mastitis.
      ;
      • Nobrega D.B.
      • Naushad S.
      • Naqvi S.A.
      • Condas L.A.Z.
      • Saini V.
      • Kastelic J.P.
      • Luby C.
      • De Buck J.
      • Barkema H.W.
      Prevalence and genetic basis of antimicrobial resistance in non-aureus staphylococci isolated from Canadian dairy herds.
      ;
      • Qu Y.
      • Zhao H.
      • Nobrega D.B.
      • Cobo E.R.
      • Han B.
      • Zhao Z.
      • Li S.
      • Li M.
      • Barkema H.W.
      • Gao J.
      Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis.
      ;
      • Lima M.C.
      • De Barros M.
      • Scatamburlo T.M.
      • Polveiro R.C.
      • De Castro L.K.
      • Guimarães S.H.S.
      • Da Costa S.L.
      • Da Costa M.M.
      • Moreira M.A.S.
      Profiles of Staphylococcus aureus isolated from goat persistent mastitis before and after treatment with enrofloxacin.
      ).
      • Qu Y.
      • Zhao H.
      • Nobrega D.B.
      • Cobo E.R.
      • Han B.
      • Zhao Z.
      • Li S.
      • Li M.
      • Barkema H.W.
      • Gao J.
      Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis.
      reported that the genes tet(K), tet(L), tet(M) were more common in non-aureus isolates than in Staph. aureus. It is possible to find combinations of 2 or more genes in the same isolate, particularly tet(K) and tet(M) (
      • Wendlandt S.
      • Feßler A.T.
      • Monecke S.
      • Ehricht R.
      • Schwarz S.
      • Kadlec K.
      The diversity of antimicrobial resistance genes among staphylococci of animal origin.
      ).

      Resistance to Aminoglycosides and Aminocyclitols

      Resistance to aminoglycosides can be mediated through several genes that code for inactivating enzymes in staphylococcal species. The gene aphA3 codes for a phosphotransferase and mediates resistance to kanamycin, neomycin, and amikacin. In contrast, aacA-aphD codes for an acetyltransferase and phosphotransferase conferring resistance to gentamicin, kanamycin, tobramycin, and amikacin if overexpressed (
      • Schwarz S.
      • Feßler A.T.
      • Loncaric I.
      • Wu C.
      • Kadlec K.
      • Wang Y.
      • Shen J.
      Antimicrobial resistance among staphylococci of animal origin.
      ). Both can be localized in a transposon, on a plasmid, or in the bacterial chromosome. In a recent study about staphylococci from bovine mastitis in China, aphA3 was detected at a higher proportion in non-aureus than Staph. aureus isolates, whereas aacA-aphD was more commonly found in Staph. aureus (
      • Qu Y.
      • Zhao H.
      • Nobrega D.B.
      • Cobo E.R.
      • Han B.
      • Zhao Z.
      • Li S.
      • Li M.
      • Barkema H.W.
      • Gao J.
      Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis.
      ). Both genes have also been identified in MRSA from Germany or Turkey, increasing the resistance spectrum of these isolates (
      • Turutoglu H.
      • Hasoksuz M.
      • Ozturk D.
      • Yildirim M.
      • Sagnak S.
      Methicillin and aminoglycoside resistance in Staphylococcus aureus isolates from bovine mastitis and sequence analysis of their mecA genes.
      ;
      • Feßler A.
      • Scott C.
      • Kadlec K.
      • Ehricht R.
      • Monecke S.
      • Schwarz S.
      Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis.
      ). The gene aadD (kanamycin, neomycin, and tobramycin resistance) has been identified both in Staph. aureus and non-aureus isolates from cases of bovine mastitis (
      • Feßler A.
      • Scott C.
      • Kadlec K.
      • Ehricht R.
      • Monecke S.
      • Schwarz S.
      Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis.
      ;
      • Qu Y.
      • Zhao H.
      • Nobrega D.B.
      • Cobo E.R.
      • Han B.
      • Zhao Z.
      • Li S.
      • Li M.
      • Barkema H.W.
      • Gao J.
      Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis.
      ). In addition, the genes aadE, ant(6)-Ia, and str mediate streptomycin resistance, and together with the genes lsa(E) and lnu(B), aadE is part of a multiresistant gene cluster. They have been found in mastitis Staph. aureus and CNS isolates (
      • Frey Y.
      • Rodriguez J.P.
      • Thomann A.
      • Schwendener S.
      • Perreten V.
      Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk.
      ;
      • Silva N.C.C.
      • Guimarães F.F.
      • Manzi M.P.
      • Júnior A.F.
      • Gómez-Sanz E.
      • Gómez P.
      • Langoni H.
      • Rall V.L.M.
      • Torres C.
      Methicillin-resistant Staphylococcus aureus of lineage ST398 as cause of mastitis in cows.
      ;
      • Wendlandt S.
      • Kadlec K.
      • Feßler A.T.
      • Schwarz S.
      Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.
      ;
      • Antók F.I.
      • Mayrhofer R.
      • Marbach H.
      • Masengesho J.C.
      • Keinprecht H.
      • Nyirimbuga V.
      • Fischer O.
      • Lepuschitz S.
      • Ruppitsch W.
      • Ehling-Schulz M.
      • Feßler A.T.
      • Schwarz S.
      • Monecke S.
      • Ehricht R.
      • Grunert T.
      • Spergser J.
      • Loncaric I.
      Characterization of antibiotic and biocide resistance genes and virulence factors of Staphylococcus species associated with bovine mastitis in Rwanda.
      ). Staphylococcus aureus mastitis isolates from Colombia showed the presence of aminoglycoside resistance genes aph(3′)IIIa (kanamycin, neomycin, amikacin, gentamicin B, paromomycin), ant(4′)Ia3 (tobramycin, amikacin), or aac(6′)/aph(2ʺ)-3 (gentamicin, tobramycin, amikacin), whereas in China the latest was found along with aph(3′)-III (kanamycin, neomycin, amikacin, gentamicin B, paromomycin;
      • Wang D.
      • Wang Z.
      • Yan Z.
      • Wu J.
      • Ali T.
      • Li J.
      • Lv Y.
      • Han B.
      Bovine mastitis Staphylococcus aureus: Antibiotic susceptibility profile, resistance genes and molecular typing of methicillin-resistant and methicillin-sensitive strains in China.
      ;
      • Jiménez Velásquez S.C.
      • Torres Higuera L.D.
      • Parra Arango J.L.
      • Rodríguez Bautista J.L.
      • García Castro F.E.
      • Patiño Burbano R.E.
      Perfil de resistencia antimicrobiana en aislamientos de Staphylococcus spp. obtenidos de leche bovina en Colombia (Profile of antimicrobial resistance in isolates of Staphylococcus spp. obtained from bovine milk in Colombia).
      ). In addition,
      • Frey Y.
      • Rodriguez J.P.
      • Thomann A.
      • Schwendener S.
      • Perreten V.
      Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk.
      showed that CNS from bovine mastitis milk from Switzerland also carry the aminoglycoside resistance genes ant(6)-Ia (streptomycin), aac(6′)-Ie–aph(2′)-Ia (gentamicin, tobramycin, amikacin), or aph(3′)-III (kanamycin, neomycin, amikacin, gentamicin B, paromomycin).
      In the case of resistance to aminocyclitols, genes spc and spw (spectinomycin resistance genes) have been detected in methicillin-resistant Staph. aureus and CNS from bovine mastitis (
      • Feßler A.
      • Scott C.
      • Kadlec K.
      • Ehricht R.
      • Monecke S.
      • Schwarz S.
      Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis.
      ;
      • Frey Y.
      • Rodriguez J.P.
      • Thomann A.
      • Schwendener S.
      • Perreten V.
      Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk.
      ;
      • Silva N.C.C.
      • Guimarães F.F.
      • Manzi M.P.
      • Júnior A.F.
      • Gómez-Sanz E.
      • Gómez P.
      • Langoni H.
      • Rall V.L.M.
      • Torres C.
      Methicillin-resistant Staphylococcus aureus of lineage ST398 as cause of mastitis in cows.
      ;
      • Wendlandt S.
      • Kadlec K.
      • Feßler A.T.
      • Schwarz S.
      Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.
      ;
      • Kadlec K.
      • Entorf M.
      • Peters T.
      Occurrence and characteristics of livestock-associated methicillin-resistant Staphylococcus aureus in quarter milk samples from dairy cows in Germany.
      ).
      Recent studies from China have detected class 1 integrons and gene cassettes in Staph. aureus from bovine mastitis (
      • Li L.
      • Zhao X.
      Characterization of the resistance class 1 integrons in Staphylococcus aureus isolates from milk of lactating dairy cattle in Northwestern China.
      ). Gene cassettes dfrA1-aadA1, aadA2, dfrA12-orfX2-aadA2, and aadA1 were most prevalent in China, and all isolates were phenotypically resistant to aminoglycosides, with some also showing trimethoprim-sulfamethoxazole resistance (
      • Li L.
      • Zhao X.
      Characterization of the resistance class 1 integrons in Staphylococcus aureus isolates from milk of lactating dairy cattle in Northwestern China.
      ).
      • Abd El-Rahman A.M.
      • Torky H.A.
      • Ebied S.K.H.M.
      Molecular detection of integron in Staphylococcus aureus isolated from ruminants.
      identified a class 1 integron in Staph. aureus of ruminant origin containing a dfrA15 gene cassette. These are considered a new finding, as integrons in gram-positive bacteria are not well characterized. However, the number of studies showing similar findings from human isolates as well is increasing, resulting in multidrug resistance in Staph. aureus associated with the presence of class 1 or class 2 integrons (
      • Mostafa M.
      • Siadat S.D.
      • Shahcheraghi F.
      • Vaziri F.
      • Japoni-Nejad A.
      • Vand Yousefi J.
      • Rajaei B.
      • Harifi Mood E.
      • Ebrahim Zadeh N.
      • Moshiri A.
      • Seyed Siamdoust S.A.
      • Rahbar M.
      Variability in gene cassette patterns of class 1 and 2 integrons associated with multi drug resistance patterns in Staphylococcus aureus clinical isolates in Tehran, Iran.
      ).

      Resistance to Macrolides or Lincosamides or Streptogramins

      Macrolide, lincosamide, and streptogramin B resistance may be associated. This may be caused by certain genes that confer resistance to antimicrobial classes that share the same mode of action. In this case, expression of one or more erm class A, B, C, T, 43, 44, or 48 genes can induce a modification of the target site at the rRNA and inhibit the binding of these compounds to the ribosome (
      • Wipf J.R.K.
      • Schwendener S.
      • Perreten V.
      The novel macrolide-lincosamide-streptogramin B resistance gene erm(44) is associated with a prophage in Staphylococcus xylosus.
      ;
      • Schwarz S.
      • Feßler A.T.
      • Loncaric I.
      • Wu C.
      • Kadlec K.
      • Wang Y.
      • Shen J.
      Antimicrobial resistance among staphylococci of animal origin.
      ). Common erm genes reported from bovine mastitis MRSA in German studies include erm(A), erm(B), erm(C), and erm(T) (
      • Feßler A.
      • Scott C.
      • Kadlec K.
      • Ehricht R.
      • Monecke S.
      • Schwarz S.
      Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis.
      ;
      • Kadlec K.
      • Entorf M.
      • Peters T.
      Occurrence and characteristics of livestock-associated methicillin-resistant Staphylococcus aureus in quarter milk samples from dairy cows in Germany.
      ). The erm(A) and erm(B) genes are associated with transposons Tn554 and Tn917/Tn551. The erm(A) gene is usually integrated into SCCmec elements and erm(B) is related to multiresistant plasmids (
      • Wendlandt S.
      • Shen J.
      • Kadlec K.
      • Wang Y.
      • Li B.
      • Zhang W.J.
      • Feßler A.T.
      • Wu C.
      • Schwarz S.
      Multidrug resistance genes in staphylococci from animals that confer resistance to critically and highly important antimicrobial agents in human medicine.
      ;
      • Schwarz S.
      • Feßler A.T.
      • Loncaric I.
      • Wu C.
      • Kadlec K.
      • Wang Y.
      • Shen J.
      Antimicrobial resistance among staphylococci of animal origin.
      ). The erm(C) gene is usually located in small plasmid that does not carry further resistant genes (
      • Lodder G.
      • Werckenthin C.
      • Schwarz S.
      • Dyke K.
      Molecular analysis of naturally occurring ermC-encoding plasmids in staphylococci isolated from animals with and without previous contact with macrolide/lincosamide antibiotics.
      ). The erm(B) and erm(C) genes were found in CNS and Staph. aureus from mastitis cases in China (
      • Li L.
      • Feng W.
      • Zhang Z.
      • Xue H.
      • Zhao X.
      Macrolide-lincosamide-streptogramin resistance phenotypes and genotypes of coagulase-positive Staphylococcus aureus and coagulase-negative staphylococcal isolates from bovine mastitis.
      ). The ermT gene can be harbored in a multiresistant plasmid and has been found to be more common in Staph. aureus than non-aureus staphylococci of bovine mastitis origin (
      • Qu Y.
      • Zhao H.
      • Nobrega D.B.
      • Cobo E.R.
      • Han B.
      • Zhao Z.
      • Li S.
      • Li M.
      • Barkema H.W.
      • Gao J.
      Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis.
      ).
      Other genes that employ different mechanisms are msr, mph, ere, Inu, vga, lsa, or sal genes. msr(A) and msr(B), which code for an efflux pump from the ABC-F subfamily protein, confer resistance to macrolide and streptogramin B. mph(C) codes for a macrolide phosphotransferase that inactivates some macrolide antibiotics, and ere(A) codes for an esterase that hydrolyzes the macrocyclic nucleus. lnu(A) and lnu(B) encode nucleotidyltrasferases and confer resistance to lincosamides (
      • Schwarz S.
      • Feßler A.T.
      • Loncaric I.
      • Wu C.
      • Kadlec K.
      • Wang Y.
      • Shen J.
      Antimicrobial resistance among staphylococci of animal origin.
      ). Resistance to lincosamides can also be achieved by the expression of plasmid genes vga(A), vga(C), lsa(E) and sal(A), which code for ABC transporters and mediate resistance to pleuromutilins, streptogramin A and lincosamides (
      • Gentry D.R.
      • McCloskey L.
      • Gwynn M.N.
      • Rittenhouse S.F.
      • Scangarella N.
      • Shawar R.
      • Holmes D.J.
      Genetic characterization of Vga ABC proteins conferring reduced susceptibility to pleuromutilins in Staphylococcus aureus.
      ;
      • Kadlec K.
      • Pomba C.F.
      • Couto N.
      • Schwarz S.
      Small plasmids carrying vga(A) or vga(C) genes mediate resistance to lincosamides, pleuromutilins, and streptogramin A antibiotics in methicillin-resistant Staphylococcus aureus ST398 from swine.
      ;
      • Hot C.
      • Berthet N.
      • Chesneau O.
      Characterization of sal(A), a novel gene responsible for lincosamide and streptogramin A resistance in Staphylococcus sciuri.
      ;
      • Wendlandt S.
      • Kadlec K.
      • Feßler A.T.
      • Schwarz S.
      Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.
      ,
      • Wendlandt S.
      • Lozano C.
      • Kadlec K.
      • Gómez-Sanz E.
      • Zarazaga M.
      • Torres C.
      • Schwarz S.
      The enterococcal ABC transporter gene lsa(E) confers combined resistance to lincosamides, pleuromutilins, and streptogramin A antibiotics in methicillin-susceptible and methicillinresistant Staphylococcus aureus.
      ). Most of these genes have been detected both in Staph. aureus and CNS from mastitis origin (
      • Lüthje P.
      • von Köckritz-Blickwede M.
      • Schwarz S.
      Identification and characterization of nine novel types of small staphylococcal plasmids carrying the lincosamide nucleotidyltransferase gene lnu(A).
      ;
      • Feßler A.
      • Scott C.
      • Kadlec K.
      • Ehricht R.
      • Monecke S.
      • Schwarz S.
      Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis.
      ;
      • Silva N.C.C.
      • Guimarães F.F.
      • Manzi M.P.
      • Júnior A.F.
      • Gómez-Sanz E.
      • Gómez P.
      • Langoni H.
      • Rall V.L.M.
      • Torres C.
      Methicillin-resistant Staphylococcus aureus of lineage ST398 as cause of mastitis in cows.
      ;
      • Li L.
      • Feng W.
      • Zhang Z.
      • Xue H.
      • Zhao X.
      Macrolide-lincosamide-streptogramin resistance phenotypes and genotypes of coagulase-positive Staphylococcus aureus and coagulase-negative staphylococcal isolates from bovine mastitis.
      ;
      • Liu H.
      • Li S.
      • Meng L.
      • Dong L.
      • Zhao S.
      • Lan X.
      • Wang J.
      • Zheng N.
      Prevalence, antimicrobial susceptibility, and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China.
      ;
      • Nobrega D.B.
      • Naushad S.
      • Naqvi S.A.
      • Condas L.A.Z.
      • Saini V.
      • Kastelic J.P.
      • Luby C.
      • De Buck J.
      • Barkema H.W.
      Prevalence and genetic basis of antimicrobial resistance in non-aureus staphylococci isolated from Canadian dairy herds.
      ;
      • Qu Y.
      • Zhao H.
      • Nobrega D.B.
      • Cobo E.R.
      • Han B.
      • Zhao Z.
      • Li S.
      • Li M.
      • Barkema H.W.
      • Gao J.
      Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis.
      ;
      • Antók F.I.
      • Mayrhofer R.
      • Marbach H.
      • Masengesho J.C.
      • Keinprecht H.
      • Nyirimbuga V.
      • Fischer O.
      • Lepuschitz S.
      • Ruppitsch W.
      • Ehling-Schulz M.
      • Feßler A.T.
      • Schwarz S.
      • Monecke S.
      • Ehricht R.
      • Grunert T.
      • Spergser J.
      • Loncaric I.
      Characterization of antibiotic and biocide resistance genes and virulence factors of Staphylococcus species associated with bovine mastitis in Rwanda.
      ). However, sal(A) has only been detected in Staph. sciuri (CNS) from bovine mastitis to date (
      • Wendlandt S.
      • Kadlec K.
      • Feßler A.T.
      • Schwarz S.
      Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.
      ).

      Other Resistance Genes

      Other resistance genes that have been detected in staphylococci from bovine mastitis include those conferring resistance to trimethoprim [dfr(A), dfr(D), dfr(G), dfr(K)], fluoroquinolones (gyrA mutation, grlA mutation, mepA), phenicols (fexA), vancomycin (vanA), or sulfonamides (AMR-associated residues in the folP gene;
      • Feßler A.
      • Scott C.
      • Kadlec K.
      • Ehricht R.
      • Monecke S.
      • Schwarz S.
      Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis.
      ;
      • Frey Y.
      • Rodriguez J.P.
      • Thomann A.
      • Schwendener S.
      • Perreten V.
      Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk.
      ; Silva et al., 2014;
      • Wendlandt S.
      • Kadlec K.
      • Feßler A.T.
      • Schwarz S.
      Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.
      ;
      • Nobrega D.B.
      • Naushad S.
      • Naqvi S.A.
      • Condas L.A.Z.
      • Saini V.
      • Kastelic J.P.
      • Luby C.
      • De Buck J.
      • Barkema H.W.
      Prevalence and genetic basis of antimicrobial resistance in non-aureus staphylococci isolated from Canadian dairy herds.
      ;
      • Qu Y.
      • Zhao H.
      • Nobrega D.B.
      • Cobo E.R.
      • Han B.
      • Zhao Z.
      • Li S.
      • Li M.
      • Barkema H.W.
      • Gao J.
      Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis.
      ;
      • Antók F.I.
      • Mayrhofer R.
      • Marbach H.
      • Masengesho J.C.
      • Keinprecht H.
      • Nyirimbuga V.
      • Fischer O.
      • Lepuschitz S.
      • Ruppitsch W.
      • Ehling-Schulz M.
      • Feßler A.T.
      • Schwarz S.
      • Monecke S.
      • Ehricht R.
      • Grunert T.
      • Spergser J.
      • Loncaric I.
      Characterization of antibiotic and biocide resistance genes and virulence factors of Staphylococcus species associated with bovine mastitis in Rwanda.
      ;
      • Naushad S.
      • Nobrega D.B.
      • Naqvi S.A.
      • Barkema H.W.
      • De Buck J.
      Genomic analysis of bovine Staphylococcus aureus isolates from milk to elucidate diversity and determine the distributions of antimicrobial and virulence genes and their association with mastitis.
      ;
      • Pérez V.K.C.
      • Custódio D.A.C.
      • Silva E.M.M.
      • de Oliveira J.
      • Guimarães A.S.
      • Brito M.A.V.P.
      • Souza-Filho A.F.
      • Heinemann M.B.
      • Lage A.P.
      • Dorneles E.M.S.
      Virulence factors and antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis in Brazil.
      ;
      • Ndahetuye J.B.
      • Leijon M.
      • Båge R.
      • Artursson K.
      • Persson Y.
      Genetic characterization of Staphylococcus aureus from subclinical mastitis cases in dairy cows in Rwanda.
      ;
      • Patel K.
      • Godden S.M.
      • Royster E.E.
      • Crooker B.A.
      • Johnson T.J.
      • Smith E.A.
      • Sreevatsan S.
      Prevalence, antibiotic resistance, virulence and genetic diversity of Staphylococcus aureus isolated from bulk tank milk samples of U.S. dairy herds.
      ). Some staphylococci not intrinsically resistant to novobiocin including Staph. aureus can acquire this resistance by accumulation of point mutations in the genes parE and gyrB (
      • Fujimoto-Nakamura M.
      • Ito H.
      • Oyamada Y.
      • Nishino T.
      • Yamagishi J.I.
      Accumulation of mutations in both gyrB and parE genes is associated with high-level resistance to novobiocin in Staphylococcus aureus.
      ).

      Streptococcus

      The Streptococcus spp. most commonly isolated from bovine mastitis samples in Ireland are Strep. uberis and Strep. dysgalactiae (
      • Department of Agriculture, Food and the Marine of Ireland, Agri-Food and Bioscience Institute, and Animal Health Ireland (DAFM, AFBI, and AHI)
      All-Island Animal Disease Surveillance Report 2020.
      ). This was also true in studies from France, Sweden and Finland, but not in Portugal or Germany where Strep. agalactiae had a similar or higher prevalence than Strep. uberis and Strep. dysgalactiae (
      • Rato M.G.
      • Bexiga R.
      • Florindo C.
      • Cavaco L.M.
      • Vilela C.L.
      • Santos-Sanches I.
      Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis.
      ;
      • Vakkamäki J.
      • Taponen S.
      • Heikkilä A.M.
      • Pyörälä S.
      Bacteriological etiology and treatment of mastitis in Finnish dairy herds.
      ;
      • Poutrel B.
      • Bareille S.
      • Lequeux G.
      • Leboeuf F.
      Prevalence of mastitis pathogens in France: Antimicrobial susceptibility of Staphylococcus aureus, Streptococcus uberis, and Escherichia coli.
      ;
      • Bolte J.
      • Zhang Y.
      • Wente N.
      • Krömker V.
      In vitro susceptibility of mastitis pathogens isolated from clinical mastitis cases on northern German dairy farms.
      ;
      • Duse A.
      • Persson-Waller K.
      • Pedersen K.
      Microbial aetiology, antibiotic susceptibility and pathogen-specific risk factors for udder pathogens from clinical mastitis in dairy cows.
      ).
      Streptococci typically have low-level intrinsically resistant to quinolones due to the overexpression of ABC efflux pumps PmrA, PatA, or PatB, for instance (
      • Garvey M.I.
      • Piddock L.J.V.
      The efflux pump inhibitor reserpine selects multidrug-resistant Streptococcus pneumoniae strains that overexpress the ABC transporters patA and patB.
      ). Low-level intrinsic resistance to aminoglycosides can also be present in streptococci as they are facultative anaerobic bacterium, whereas they are fully resistant to fusidic acid (
      • 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.
      ;
      • El Moujaber G.
      • Osman M.
      • Rafei R.
      • Dabboussi F.
      • Hamze M.
      Molecular mechanisms and epidemiology of resistance in Streptococcus pneumoniae in the Middle East region.
      ). Acquired resistance from mastitis streptococci isolates is described below (Table 2).
      Table 2Percent of resistance of Streptococcus from bovine mastitis to various antimicrobials in EU countries
      Pen: penicillin; Amp: ampicillin; Amc: amoxicillin + clavulanic acid; Fur: ceftiofur; Chl: chloramphenicol; Ery: Erythromycin; Tyl: tylosin; Pir: pirlimycin; Lin: lincomycin; Cli: clindamycin; Spc: spectinomycin; Gen: gentamicin; Kan: kanamycin; Neo: neomycin; Str: streptomycin; Cip: ciprofloxacin; Enr: enrofloxacin; Tet: tetracycline; Smx: sulfamethoxazole; Tmp: trimethoprim; Sxt: sulfamethoxazole + trimethoprim.
      CountryT
      T = type: D, diagnostic; S, survey.
      Disc
      Disc = disc diffusion.
      or MIC
      Standard
      CLSI: Clinical and Laboratory Standards Institute; EUCAST: European Committee on Antimicrobial Susceptibility Testing; CA-SFM: Comité de l'Antibiogramme–Société Française de Microbiologie; BSAC: British Society for Antimicrobial Chemotherapy; AHVLA: Animal Health and Veterinary Laboratories Agency.
      PenAmpAmcFurChlEryTylPirLinCliSpcGenkanNeoStrCipEnrTetSmxTmpSxtReference
      Streptococcus uberis
       Austria (n = 124)SDiscCLSI2
      • Baumgartner M.
      • Štromerová N.H.
      • Bzdil J.
      • Siegwalt G.
      Susceptibility and Resistance of Selected Pathogens of the Mammary Gland of Cattle from Austria and Czech Republic in 2017.
       Belgium (n = 939)DDiscCLSI1.60.130.440.82.5
      • Supré K.
      • Lommelen K.
      • De Meulemeester L.
      Antimicrobial susceptibility and distribution of inhibition zone diameters of bovine mastitis pathogens in Flanders, Belgium.
       Czech Republic (n = 163)SMICCLSI/EUCASTT/CA-SFM01.21.830.1052.12.563.20
      • Šlosárková S.
      • Nedbalcová K.
      • Bzdil J.
      • Fleischer P.
      • Zouharová M.
      • Staněk S.
      • Kašná E.
      • Pechová A.
      Antimicrobial susceptibility of streptococci most frequently isolated from Czech dairy cows with mastitis.
       Denmark (n = 61)DMICCLSI/EUCAST006.63.398.4021.31.60
      • Chehabi C.N.
      • Nonnemann B.
      • Astrup L.B.
      • Farre M.
      • Pedersen K.
      In vitro antimicrobial resistance of causative agents to clinical mastitis in Danish Dairy cows.
       England and Wales (n = 44)DDiscBSAC/AHVLA0002.339.534.1
      • UK-VARSS
      UK Veterinary Antibiotic Resistance and Sales Surveillance Report UK-VARSS 2018 99.
       Finland (n = 89)SMICNCCLS/SVARM0001.10
      • Pitkälä A.
      • Haveri M.
      • Pyörälä S.
      • Myllys V.
      • Honkanen-Buzalski T.
      Bovine mastitis in Finland 2001—Prevalence, distribution of bacteria, and antimicrobial resistance.
       France
      Percent resistance was calculated using the % susceptibility published.
      (n = 969)
      DDiscCA-SFM1418152612321610
      • Anses
      Resapath: French surveillance network for antimicrobial resistance in bacteria from diseased animals. 2019 annual report.
       Ireland (n = 165)DDiscCLSI00015.222.211.5DAFM et al., 2021
       Lithuania (n = 25)SDiscCLSI12128Klimiene et al., 2011
       Poland (n = 53)SMICCLSI069683034
      • Kaczorek E.
      • Małaczewska J.
      • Wójcik R.
      • Rękawek W.
      • Siwicki A.K.
      Phenotypic and genotypic antimicrobial susceptibility pattern of Streptococcus spp. isolated from cases of clinical mastitis in dairy cattle in Poland.
       Portugal (n = 30)SDiscCLSI00026.753.38010060
      • Rato M.G.
      • Bexiga R.
      • Florindo C.
      • Cavaco L.M.
      • Vilela C.L.
      • Santos-Sanches I.
      Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis.
       Slovakia (n = 57)SDiscCLSI/EUCAST10.53.55.37478.98.85.3
      • Holko I.
      • Tančin V.
      • Vršková M.
      • Tvarožková K.
      Prevalence and antimicrobial susceptibility of udder pathogens isolated from dairy cows in Slovakia.
       Sweden (n = 89)SMICEUCAST000022.2
      • Duse A.
      • Persson-Waller K.
      • Pedersen K.
      Microbial aetiology, antibiotic susceptibility and pathogen-specific risk factors for udder pathogens from clinical mastitis in dairy cows.
       Switzerland (n = 221)DDiscCLSI7.87.70.449.736.299.5
      • Rüegsegger F.
      • Ruf J.
      • Tschuor A.
      • Sigrist Y.
      • Rosskopf M.
      • Hässig M.
      Antimicrobial susceptibility of mastitis pathogens of dairy cows in Switzerland.
      Streptococcus dysgalactiae
       Austria (n = 35)SDiscCLSI0
      • Baumgartner M.
      • Štromerová N.H.
      • Bzdil J.
      • Siegwalt G.
      Susceptibility and Resistance of Selected Pathogens of the Mammary Gland of Cattle from Austria and Czech Republic in 2017.
       Belgium* (n = 444)DDiscCLSI0.70.219.593.21.4
      • Supré K.
      • Lommelen K.
      • De Meulemeester L.
      Antimicrobial susceptibility and distribution of inhibition zone diameters of bovine mastitis pathogens in Flanders, Belgium.
       Czech Republic (n = 25)SMICCLSI/EUCASTT/CA-SFM000120280600
      • Šlosárková S.
      • Nedbalcová K.
      • Bzdil J.
      • Fleischer P.
      • Zouharová M.
      • Staněk S.
      • Kašná E.
      • Pechová A.
      Antimicrobial susceptibility of streptococci most frequently isolated from Czech dairy cows with mastitis.
       Denmark (n = 33)DMICCLSI/EUCAST006.6012.19.19.100
      • Chehabi C.N.
      • Nonnemann B.
      • Astrup L.B.
      • Farre M.
      • Pedersen K.
      In vitro antimicrobial resistance of causative agents to clinical mastitis in Danish Dairy cows.
       France* (n = 167)DDiscCA-SFM1114110754888
      • Anses
      Resapath: French surveillance network for antimicrobial resistance in bacteria from diseased animals. 2019 annual report.
       Ireland (n = 52)DDiscCLSI0001.93.857.7DAFM et al., 2021
       Lithuania (n = 30)SDiscCLSI23.326.6-6.7Klimiene et al., 2011
       Poland (n = 41)SMICCLSI0226851561
      • Kaczorek E.
      • Małaczewska J.
      • Wójcik R.
      • Rękawek W.
      • Siwicki A.K.
      Phenotypic and genotypic antimicrobial susceptibility pattern of Streptococcus spp. isolated from cases of clinical mastitis in dairy cattle in Poland.
       Portugal (n = 18)SDiscCLSI00022.238.938.977.8100
      • Rato M.G.
      • Bexiga R.
      • Florindo C.
      • Cavaco L.M.
      • Vilela C.L.
      • Santos-Sanches I.
      Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis.
       England and Wales (n = 21)DDiscBSAC/AHVLA0004.85.390.5
      • UK-VARSS
      UK Veterinary Antibiotic Resistance and Sales Surveillance Report UK-VARSS 2018 99.
       Sweden (n = 120)SMICEUCAST0
      • Duse A.
      • Persson-Waller K.
      • Pedersen K.
      Microbial aetiology, antibiotic susceptibility and pathogen-specific risk factors for udder pathogens from clinical mastitis in dairy cows.
       Switzerland (n = 221)DDiscCLSI75.2037.639.997.5
      • Rüegsegger F.
      • Ruf J.
      • Tschuor A.
      • Sigrist Y.
      • Rosskopf M.
      • Hässig M.
      Antimicrobial susceptibility of mastitis pathogens of dairy cows in Switzerland.
      Streptococcus agalactiae
       Croatia* (n = 33)DDiscCLSI36.457.833.336.478.8
      Oxacillin was tested.
      63.6
      • Sukalić T.
      • Samardžija M.
      • Jurmanović J.
      • Bačanek B.
      • Grbavac J.
      • Cvetnić Ž.
      • Končurat A.
      • Pavljak I.
      • Đuričić D.
      Antimikrobna osjetljivost uzročnika mastitisa krava s područja sjeverozapadne Hrvatske u razdoblju od 2014. do 2018 (Antimicrobial susceptibility of bovine mastitis pathogens from northwestern Croatia in the period 2014 to 2018).
       Denmark (n = 13)DMICCLSI/EUCAST000100076.900
      • Chehabi C.N.
      • Nonnemann B.
      • Astrup L.B.
      • Farre M.
      • Pedersen K.
      In vitro antimicrobial resistance of causative agents to clinical mastitis in Danish Dairy cows.
       Lithuania (n = 134)SDiscCLSI28.346.316.4
      • Klimienė I.
      • Ružauskas M.
      • Špakauskas V.
      • Matusevičius A.
      • Mockeliunas R.
      • Pereckiene A.
      • Butrimaite-Ambrozevičiene C.
      • Virgailis M.
      Antimicrobial resistance patterns to β-lactams of gram-positive cocci isolated from bovine mastitis in Lithuania.
       Poland (n = 27)SMICCLSI0710096044
      • Kaczorek E.
      • Małaczewska J.
      • Wójcik R.
      • Rękawek W.
      • Siwicki A.K.
      Phenotypic and genotypic antimicrobial susceptibility pattern of Streptococcus spp. isolated from cases of clinical mastitis in dairy cattle in Poland.
       Portugal (n = 60)SDiscCLSI00018.318.393.3−96.756.7
      • Rato M.G.
      • Bexiga R.
      • Florindo C.
      • Cavaco L.M.
      • Vilela C.L.
      • Santos-Sanches I.
      Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis.
       Slovakia (n = 30)SDiscCLSI/EUCAST23.36.7807023.313.3
      • Holko I.
      • Tančin V.
      • Vršková M.
      • Tvarožková K.
      Prevalence and antimicrobial susceptibility of udder pathogens isolated from dairy cows in Slovakia.
       Ukraine (n = 43)DDiscCLSI25.5880
      • Elias L.
      • Balasubramanyam A.S.
      • Ayshpur O.Y.
      • Mushtuk I.U.
      • Sheremet N.O.
      • Gumeniuk V.V.
      • Musser J.M.B.
      • Rogovskyy A.S.
      Antimicrobial susceptibility of Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli isolated from mastitic dairy cattle in Ukraine.
      1 Pen: penicillin; Amp: ampicillin; Amc: amoxicillin + clavulanic acid; Fur: ceftiofur; Chl: chloramphenicol; Ery: Erythromycin; Tyl: tylosin; Pir: pirlimycin; Lin: lincomycin; Cli: clindamycin; Spc: spectinomycin; Gen: gentamicin; Kan: kanamycin; Neo: neomycin; Str: streptomycin; Cip: ciprofloxacin; Enr: enrofloxacin; Tet: tetracycline; Smx: sulfamethoxazole; Tmp: trimethoprim; Sxt: sulfamethoxazole + trimethoprim.
      2 T = type: D, diagnostic; S, survey.
      3 Disc = disc diffusion.
      4 CLSI: Clinical and Laboratory Standards Institute; EUCAST: European Committee on Antimicrobial Susceptibility Testing; CA-SFM: Comité de l'Antibiogramme–Société Française de Microbiologie; BSAC: British Society for Antimicrobial Chemotherapy; AHVLA: Animal Health and Veterinary Laboratories Agency.
      * Percent resistance was calculated using the % susceptibility published.
      *** Oxacillin was tested.

      Resistance to Macrolides or Lincosamides or Streptogramins

      Macrolide, lincosamide, and streptogramin B resistance phenotypes are observed in streptococci from bovine mastitis.
      • Rato M.G.
      • Bexiga R.
      • Florindo C.
      • Cavaco L.M.
      • Vilela C.L.
      • Santos-Sanches I.
      Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis.
      identified the gene erm(B) in Strep. uberis, the genes erm(A) or erm(B) in Strep. agalactiae, and both erm(A) and erm(B) in Strep. dysgalactiae isolates. The mechanism of resistance employed by the expression of these genes is target protection, following methylation of the ribosome at position 2058 (
      • Haenni M.
      • Lupo A.
      • Madec J.-Y.
      Antimicrobial resistance in Streptococcus spp.
      ). They are usually found in mobile genetic elements and can be associated with tetracycline resistance.
      The ermB are among the most commonly detected genes that result in a macrolide, lincosamide, and streptogramin B resistant phenotype, but others such as mef, msr, or mre families, which code for efflux pumps, are also significant (
      • Haenni M.
      • Lupo A.
      • Madec J.-Y.
      Antimicrobial resistance in Streptococcus spp.
      ,
      • Haenni M.
      • Saras E.
      • Chaussière S.
      • Treilles M.
      • Madec J.Y.
      ermB-mediated erythromycin resistance in Streptococcus uberis from bovine mastitis.
      ). In a study from the United States, only erm(B) was present in Strep. uberis (
      • Loch I.M.
      • Glenn K.
      • Zadoks R.N.
      Macrolide and lincosamide resistance genes of environmental streptococci from bovine milk.
      ). In Germany erm(B) was only detected in Strep. uberis, but erm(B) and erm(C) were detected in Strep. agalactiae and Strep. dysgalactiae. Additionally, mef(A) and msr(D) were present in all 3 species (
      • Entorf M.
      • Feßler A.T.
      • Kaspar H.
      • Kadlec K.
      • Peters T.
      • Schwarz S.
      Comparative erythromycin and tylosin susceptibility testing of streptococci from bovine mastitis.
      ). Combination of these genes in the same isolate is very common.
      • Duarte R.S.
      • Bellei B.C.
      • Miranda O.P.
      • Brito M.A.V.P.
      • Teixeira L.M.
      Distribution of antimicrobial resistance and virulence-related genes among Brazilian group B streptococci recovered from bovine and human sources.
      found the mreA gene in 9/38 Strep. agalactiae isolates from bovine mastitis in Brazil (
      • Duarte R.S.
      • Bellei B.C.
      • Miranda O.P.
      • Brito M.A.V.P.
      • Teixeira L.M.
      Distribution of antimicrobial resistance and virulence-related genes among Brazilian group B streptococci recovered from bovine and human sources.
      ). All isolates also carried erm(B) and 6 of them erm(A). A recent study from Australia on Strep. uberis from dairy herds found a new gene, mel or mef(A), as well as mrsE (
      • Vezina B.
      • Al-harbi H.
      • Ramay H.R.
      • Soust M.
      • Moore R.J.
      • Olchowy T.W.J.
      • Alawneh J.I.
      Sequence characterisation and novel insights into bovine mastitis-associated Streptococcus uberis in dairy herds.
      ).
      Acquisition of an mph gene confers resistance to macrolides only through the production of inactivating enzymes (
      • Chesneau O.
      • Tsvetkova K.
      • Courvalin P.
      Resistance phenotypes conferred by macrolide phosphotransferases.
      ). Emergence of mph(B) has been documented in French Strep. uberis isolates from clinical mastitis cows (
      • Achard A.
      • Guérin-Faublée V.
      • Pichereau V.
      • Villers C.
      • Leclercq R.
      Emergence of macrolide resistance gene mph(B) in Streptococcus uberis and cooperative effects with rdmC-like gene.
      ). Finally, resistance to lincosamide only is achieved through genes that code for inactivating enzymes that adenylate these compounds on position 3 or 4. These are mainly from the lnu family. lnuA has been detected in Strep. dysgalactiae and Strep. agalactiae from Egypt, or Strep. uberis and Strep. dysgalactiae from Poland for example (
      • Kaczorek E.
      • Małaczewska J.
      • Wójcik R.
      • Rękawek W.
      • Siwicki A.K.
      Phenotypic and genotypic antimicrobial susceptibility pattern of Streptococcus spp. isolated from cases of clinical mastitis in dairy cattle in Poland.
      ;
      • Ahmed W.
      • Neubauer H.
      • Tomaso H.
      • El Hofy F.I.
      • Monecke S.
      • Abd El-Tawab A.A.
      • Hotzel H.
      Characterization of enterococci- and ESBL-producing Escherichia coli isolated from milk of bovides with mastitis in Egypt.
      ). However, lnuB (also known as linB) is by far the most common gene (
      • Haenni M.
      • Lupo A.
      • Madec J.-Y.
      Antimicrobial resistance in Streptococcus spp.
      ). It has been found in Strep. uberis and Strep. dysgalactiae subsp. dysgalactiae and Strep. agalactiae (
      • Schmitt-van de Leemput E.
      • Zadoks R.N.
      Genotypic and phenotypic detection of macrolide and lincosamide resistance in Streptococcus uberis.
      ;
      • Rato M.G.
      • Bexiga R.
      • Florindo C.
      • Cavaco L.M.
      • Vilela C.L.
      • Santos-Sanches I.
      Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis.
      ;
      • Vélez J.R.
      • Cameron M.
      • Rodríguez-Lecompte J.C.
      • Xia F.
      • Heider L.C.
      • Saab M.
      • McClure J.T.
      • Sánchez J.
      Whole-genome sequence analysis of antimicrobial resistance genes in Streptococcus uberis and Streptococcus dysgalactiae isolates from Canadian dairy herds.
      ;
      • Reyes J.
      • Rodriguez-Lecompte J.C.
      • Blanchard A.
      • McClure J.T.
      • Sánchez J.
      Molecular variability of Streptococcus uberis isolates from intramammary infections in Canadian dairy farms from the maritime region.
      ;
      • Hernandez L.
      • Bottini E.
      • Cadona J.
      • Cacciato C.
      • Monteavaro C.
      • Bustamante A.
      • Sanso A.M.
      Multidrug resistance and molecular characterization of Streptococcus agalactiae isolates from dairy cattle with mastitis.
      ). lnu(D) has been detected in Strep. uberis from France and lnu(C) in Strep. uberis from Australia (
      • Petinaki E.
      • Guérin-Faublée V.
      • Pichereau V.
      • Villers C.
      • Achard A.
      • Malbruny B.
      • Leclercq R.
      Lincomycin resistance gene lnu(D) in Streptococcus uberis.
      ;
      • Haenni M.
      • Saras E.
      • Chaussière S.
      • Treilles M.
      • Madec J.Y.
      ermB-mediated erythromycin resistance in Streptococcus uberis from bovine mastitis.
      ;
      • Vezina B.
      • Al-harbi H.
      • Ramay H.R.
      • Soust M.
      • Moore R.J.
      • Olchowy T.W.J.
      • Alawneh J.I.
      Sequence characterisation and novel insights into bovine mastitis-associated Streptococcus uberis in dairy herds.
      ).

      Resistance to Tetracyclines

      A slow but continuous increase of resistance to tetracycline in Strep. uberis from French dairy farms was reported between 2006 and 2016 (
      • Boireau C.
      • Cazeau G.
      • Jarrige N.
      • Calavas D.
      • Madec J.Y.
      • Leblond A.
      • Haenni M.
      • Gay É.
      Antimicrobial resistance in bacteria isolated from mastitis in dairy cattle in France, 2006–2016.
      ). Levels of resistance vary between streptococcal species, with Strep. dysgalactiae commonly showing a higher resistance prevalence (
      • Mevius D.J.
      • Koene M.G.J.
      • Wit B.
      • van Pelt W.
      • Bondt N.
      Monitoring of Antimicrobial Resistance and Antibiotic Usage in Animals in the Netherlands in 2008.
      ;
      • Persson Y.
      • Nyman A.K.J.
      • Grönlund-Andersson U.
      Etiology and antimicrobial susceptibility of udder pathogens from cases of subclinical mastitis in dairy cows in Sweden.
      ;
      • Cameron M.
      • Saab M.
      • Heider L.
      • McClure J.T.
      • Rodriguez-Lecompte J.C.
      • Sanchez J.
      Antimicrobial susceptibility patterns of environmental streptococci recovered from bovine milk samples in the Maritime Provinces of Canada.
      ;
      • de Jong A.
      • Garch F.E.
      • Simjee S.
      • Moyaert H.
      • Rose M.
      • Youala M.
      • Siegwart E.
      Monitoring of antimicrobial susceptibility of udder pathogens recovered from cases of clinical mastitis in dairy cows across Europe: VetPath results.
      ). Genes involved include membrane efflux systems [tet(K) tet(L)] and ribosomal protection enzymes [tet(M), tet(O), tet(S) (
      • Haenni M.
      • Lupo A.
      • Madec J.-Y.
      Antimicrobial resistance in Streptococcus spp.
      ]. Overall, tet(M) is the most commonly found although all 5 genes have been detected in Strep. uberis, Strep. agalactiae, or Strep. dysgalactiae from France, Brazil, Canada, Portugal, Poland, Egypt, Argentina, or China, for example (
      • Duarte R.S.
      • Bellei B.C.
      • Miranda O.P.
      • Brito M.A.V.P.
      • Teixeira L.M.
      Distribution of antimicrobial resistance and virulence-related genes among Brazilian group B streptococci recovered from bovine and human sources.
      ;
      • Haenni M.
      • Saras E.
      • Bertin S.
      • Leblond P.
      • Madec J.Y.
      • Payot S.
      Diversity and mobility of integrative and conjugative elements in bovine isolates of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis.
      ;
      • Rato M.G.
      • Bexiga R.
      • Florindo C.
      • Cavaco L.M.
      • Vilela C.L.
      • Santos-Sanches I.
      Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis.
      ;
      • Kaczorek E.
      • Małaczewska J.
      • Wójcik R.
      • Rękawek W.
      • Siwicki A.K.
      Phenotypic and genotypic antimicrobial susceptibility pattern of Streptococcus spp. isolated from cases of clinical mastitis in dairy cattle in Poland.
      ; Vélez et al., 2017;
      • Reyes J.
      • Rodriguez-Lecompte J.C.
      • Blanchard A.
      • McClure J.T.
      • Sánchez J.
      Molecular variability of Streptococcus uberis isolates from intramammary infections in Canadian dairy farms from the maritime region.
      ;
      • Tian X.Y.
      • Zheng N.
      • Han R.W.
      • Ho H.
      • Wang J.
      • Wang Y.T.
      • Wang S.Q.
      • Li H.G.
      • Liu H.W.
      • Yu Z.N.
      Antimicrobial resistance and virulence genes of Streptococcus isolated from dairy cows with mastitis in China.
      ;
      • Ahmed W.
      • Neubauer H.
      • Tomaso H.
      • El Hofy F.I.
      • Monecke S.
      • Abd El-Tawab A.A.
      • Hotzel H.
      Characterization of enterococci- and ESBL-producing Escherichia coli isolated from milk of bovides with mastitis in Egypt.
      ;
      • Hernandez L.
      • Bottini E.
      • Cadona J.
      • Cacciato C.
      • Monteavaro C.
      • Bustamante A.
      • Sanso A.M.
      Multidrug resistance and molecular characterization of Streptococcus agalactiae isolates from dairy cattle with mastitis.
      ).

      Resistance to β-Lactams

      Resistance to β-lactam antimicrobials in streptococci from bovine mastitis is usually low mainly because they cannot successfully acquire exogenous β-lactam resistance genes. However, decreased susceptibility, and in some cases resistance, has been detected in certain studies in low numbers (
      • Guérin-Faublée V.
      • Tardy F.
      • Bouveron C.
      • Carret G.
      Antimicrobial susceptibility of Streptococcus species isolated from clinical mastitis in dairy cows.
      ;
      • Tenhagen B.A.
      • Köster G.
      • Wallmann J.
      • Heuwieser W.
      Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg, Germany.
      ;
      • Haenni M.
      • Lupo A.
      • Madec J.-Y.
      Antimicrobial resistance in Streptococcus spp.
      ). Resistance in Strep. uberis can be acquired by mutation (substitutions) in the penicillin-binding proteins (
      • 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.
      ). This is how traditionally Streptococcus were thought to acquire β-lactam resistance. However, recent studies from Canada showed a higher prevalence of penicillin and ampicillin resistance in Strep. uberis and revealed the presence of the gene bl2b in Strep. uberis and Strep. dysgalactiae as well as TEM genes in the 2 species (TEM-1, TEM-127, TEM-136, TEM-157, TEM-163, TEM-47, TEM-89, and TEM-95 in Strep. uberis and TEM-71, TEM-1, TEM-136, TEM-157, and TEM-47 in Strep. dysgalactiae;
      • Vélez J.R.
      • Cameron M.
      • Rodríguez-Lecompte J.C.
      • Xia F.
      • Heider L.C.
      • Saab M.
      • McClure J.T.
      • Sánchez J.
      Whole-genome sequence analysis of antimicrobial resistance genes in Streptococcus uberis and Streptococcus dysgalactiae isolates from Canadian dairy herds.
      ). However, phenotypic resistance was not always observed when they were present (
      • Reyes J.
      • Rodriguez-Lecompte J.C.
      • Blanchard A.
      • McClure J.T.
      • Sánchez J.
      Molecular variability of Streptococcus uberis isolates from intramammary infections in Canadian dairy farms from the maritime region.
      ). The gene blaZ has been detected in Strep. uberis and Strep. dysgalactiae from Poland, but most isolates showed phenotypic susceptibility to penicillin (
      • Kaczorek E.
      • Małaczewska J.
      • Wójcik R.
      • Rękawek W.
      • Siwicki A.K.
      Phenotypic and genotypic antimicrobial susceptibility pattern of Streptococcus spp. isolated from cases of clinical mastitis in dairy cattle in Poland.
      ). In China, correlation between phenotypic resistance and the presence of a β-lactam resistance gene was low (3.13%), with higher numbers of phenotypic resistance (
      • Tian X.Y.
      • Zheng N.
      • Han R.W.
      • Ho H.
      • Wang J.
      • Wang Y.T.
      • Wang S.Q.
      • Li H.G.
      • Liu H.W.
      • Yu Z.N.
      Antimicrobial resistance and virulence genes of Streptococcus isolated from dairy cows with mastitis in China.
      ). The relevance and potential of these β-lactamases have therefore yet to be elucidated in streptococcal species.

      Resistance to Other Antimicrobials

      With regard to fluoroquinolones, resistance is mediated by point mutations in the quinolone resistance determinant regions of the gyrA and parC genes (
      • Drlica K.
      • Zhao X.
      DNA gyrase, topoisomerase IV, and the 4-quinolones.
      ). These have been found in Streptococcus spp. from bovine mastitis in China (
      • Zhang S.
      • Piepers S.
      • Shan R.
      • Cai L.
      • Mao S.
      • Zou J.
      • Ali T.
      • De Vliegher S.
      • Han B.
      Phenotypic and genotypic characterization of antimicrobial resistance profiles in Streptococcus dysgalactiae isolated from bovine clinical mastitis in 5 provinces of China.
      ;
      • Tian X.Y.
      • Zheng N.
      • Han R.W.
      • Ho H.
      • Wang J.
      • Wang Y.T.
      • Wang S.Q.
      • Li H.G.
      • Liu H.W.
      • Yu Z.N.
      Antimicrobial resistance and virulence genes of Streptococcus isolated from dairy cows with mastitis in China.
      ). Fifteen isolates presented quinolone resistance while only 9 carried resistance genes.
      • Tian X.Y.
      • Zheng N.
      • Han R.W.
      • Ho H.
      • Wang J.
      • Wang Y.T.
      • Wang S.Q.
      • Li H.G.
      • Liu H.W.
      • Yu Z.N.
      Antimicrobial resistance and virulence genes of Streptococcus isolated from dairy cows with mastitis in China.
      also found higher phenotypic than genetic prevalence of resistance for chloramphenicol (cat1, cat2), or sulfonamides (sul1, sul2, sul3), with the latest showing the highest correlation between both.
      Aminoglycoside resistance genes such as aphA-3 and aad-6 have been detected in Strep. uberis and Strep. dysgalactiae although in low numbers (
      • Kaczorek E.
      • Małaczewska J.
      • Wójcik R.
      • Rękawek W.
      • Siwicki A.K.
      Phenotypic and genotypic antimicrobial susceptibility pattern of Streptococcus spp. isolated from cases of clinical mastitis in dairy cattle in Poland.
      ;
      • Ahmed W.
      • Neubauer H.
      • Tomaso H.
      • El Hofy F.I.
      • Monecke S.
      • Abd El-Tawab A.A.
      • Hotzel H.
      Characterization of enterococci- and ESBL-producing Escherichia coli isolated from milk of bovides with mastitis in Egypt.
      ). Again, phenotypic resistance levels were present in a higher number of isolates due to these species showing a naturally lower susceptibility to this group of antibiotics. Finally, resistance to streptogramin A has been identified in Strep. uberis via the vatD gene (
      • Vezina B.
      • Al-harbi H.
      • Ramay H.R.
      • Soust M.
      • Moore R.J.
      • Olchowy T.W.J.
      • Alawneh J.I.
      Sequence characterisation and novel insights into bovine mastitis-associated Streptococcus uberis in dairy herds.
      ).

      GENETIC DETERMINANTS OF RESISTANCE IN MASTITIS PATHOGENS: GRAM-NEGATIVE MICROORGANISMS

      Enterobacteriaceae: E. coli and K. pneumoniae

      Escherichia coli is an opportunistic pathogen and the most frequent gram-negative bacterium responsible for bovine mastitis. It can cause IMI in cattle particularly during parturition or early lactation due to the immunosuppression taking place at these stages. Infections can result in severe clinical mastitis and sometimes become recurrent, although they are usually of shorter duration than those produced by other pathogens and a high number of them do not need treatment (
      • Blowey R.
      • Edmondson P.
      Mastitis Control in Dairy Herds.
      ). Although less commonly isolated, K. pneumoniae can also cause severe clinical mastitis inducing massive inflammation and necrosis of the mammary gland (
      • Schukken Y.
      • Chuff M.
      • Moroni P.
      • Gurjar A.
      • Santisteban C.
      • Welcome F.
      • Zadoks R.
      The “Other” gram-negative bacteria in mastitis.
      ).
      Enterobacteriaceae are considered intrinsically resistant to macrolides, aminocoumarins or glycopeptides due to the poor membrane permeability of these antimicrobial classes, unable to penetrate the outer membrane of gram-negative bacteria (
      • Klobucar K.
      • Brown E.D.
      New potentiators of ineffective antibiotics: Targeting the gram-negative outer membrane to overcome intrinsic resistance.
      ). However, the macrolide azithromycin is associated with successful treatment (
      • Gomes C.
      • Martínez-Puchol S.
      • Palma N.
      • Horna G.
      • Ruiz-Roldán L.
      • Pons M.J.
      • Ruiz J.
      Macrolide resistance mechanisms in Enterobacteriaceae: Focus on azithromycin.
      ). AmpC enzymes can be chromosomal or plasmid-determined (
      • Thomson K.S.
      Extended-spectrum-β-lactamase, AmpC, and carbapenemase issues.
      ). Chromosomically encoded AmpC genes in E. coli from milk have been detected although resistance is not always present due to low expression. For example,
      • Fazel F.
      • Jamshidi A.
      • Khoramian B.
      Phenotypic and genotypic study on antimicrobial resistance patterns of E. coli isolates from bovine mastitis.
      detected blaampC in more than 95% of E. coli isolates from clinical mastitis cases in Iran, although only 66% showed phenotypic resistance to ampicillin (
      • Fazel F.
      • Jamshidi A.
      • Khoramian B.
      Phenotypic and genotypic study on antimicrobial resistance patterns of E. coli isolates from bovine mastitis.
      ). Similarly,
      • Koovapra S.
      • Bandyopadhyay S.
      • Das G.
      • Bhattacharyya D.
      • Banerjee J.
      • Mahanti A.
      • Samanta I.
      • Nanda P.K.
      • Kumar A.
      • Mukherjee R.
      • Dimri U.
      • Singh R.K.
      Molecular signature of extended spectrum β-lactamase producing Klebsiella pneumoniae isolated from bovine milk in eastern and north-eastern India.
      , detected blaampC in 20 K. pneumoniae from bovine mastitis milk in India, but only 7 were positive phenotypically (
      • Koovapra S.
      • Bandyopadhyay S.
      • Das G.
      • Bhattacharyya D.
      • Banerjee J.
      • Mahanti A.
      • Samanta I.
      • Nanda P.K.
      • Kumar A.
      • Mukherjee R.
      • Dimri U.
      • Singh R.K.
      Molecular signature of extended spectrum β-lactamase producing Klebsiella pneumoniae isolated from bovine milk in eastern and north-eastern India.
      ). In addition, K. pneumoniae produces small amounts of SHV β-lactamases, which can generate an intrinsic resistance to ampicillin and other β-lactams such as carbencillin or ticarcillin (
      • Fu Y.
      • Zhang F.
      • Zhang W.
      • Chen X.
      • Zhao Y.
      • Ma J.
      • Bao L.
      • Song W.
      • Ohsugi T.
      • Urano T.
      • Liu S.
      Differential expression of blaSHV related to susceptibility to ampicillin in Klebsiella pneumoniae.
      ).
      Escherichia coli and K. pneumoniae isolates are important from a public health and surveillance perspective as they can act as reservoirs for antimicrobial resistance genes. Acquired resistance in Enterobacteriaceae from mastitis is summarized below (Table 3).
      Table 3Percent of resistance of Escherichia coli and Klebsiella pneumoniae from bovine mastitis to various antimicrobials in EU countries
      Amp: ampicillin; Amc: amoxicillin + clavulanic acid; Fur: ceftiofur; Ctx: cefotaxime; Chl: chloramphenicol; Ery: Erythromycin; Col: colistin; Nal: nalidixic acid; Spc: spectinomycin; Gen: gentamicin; Kan: kanamycin; Neo: neomycin; Str: streptomycin; Cip: ciprofloxacin; Enr: enrofloxacin; Tet: tetracycline; Smx: sulfamethoxazole; Tmp: trimethoprim; Sxt: sulfamethoxazole + trimethoprim.
      CountryType
      T = type: D, diagnostic; S, survey.
      Disc
      Disc = disc diffusion.
      or MIC
      Standard
      CLSI: Clinical and Laboratory Standards Institute; EUCAST: European Committee on Antimicrobial Susceptibility Testing; CA-SFM: Comité de l'Antibiogramme–Société Française de Microbiologie; BSAC: British Society for Antimicrobial Chemotherapy; AHVLA: Animal Health and Veterinary Laboratories Agency.
      AmpAmcFurCtxChlEryColNalSpcGenkanNeoStrCipEnrTetSmxTmpSxtReference
      E. coli
       Belgium (n = 563)DDiscCLSI28.87.314.79.6
      • Supré K.
      • Lommelen K.
      • De Meulemeester L.
      Antimicrobial susceptibility and distribution of inhibition zone diameters of bovine mastitis pathogens in Flanders, Belgium.
       Croatia
      Percent resistance was calculated using the % susceptibility published.
      (n = 93)
      DDiscCLSI45.582.819.478.5
      Oxacillin was tested.
      69.9
      • Sukalić T.
      • Samardžija M.
      • Jurmanović J.
      • Bačanek B.
      • Grbavac J.
      • Cvetnić Ž.
      • Končurat A.
      • Pavljak I.
      • Đuričić D.
      Antimikrobna osjetljivost uzročnika mastitisa krava s područja sjeverozapadne Hrvatske u razdoblju od 2014. do 2018 (Antimicrobial susceptibility of bovine mastitis pathogens from northwestern Croatia in the period 2014 to 2018).
       Czech Republic (n = 243)SDiscCLSI30.40.40.701.50.41.65.90.7133.3
      • Skočková A.
      • Bogdanovičová K.
      • Koláčková I.
      • Karpíšková R.
      Antimicrobial-resistant and extended-spectrum β-lactamase-producing Escherichia coli in raw cow's milk.
       Denmark (n = 62)DMICCLSI/EUCAST11.30001.601.61.60012.9011.317.716.1
      • Chehabi C.N.
      • Nonnemann B.
      • Astrup L.B.
      • Farre M.
      • Pedersen K.
      In vitro antimicrobial resistance of causative agents to clinical mastitis in Danish Dairy cows.
       Finland (n = 144)SMICEUCAST18.701.46.90.706.318.10.716.714.610.4
      • Suojala L.
      • Pohjanvirta T.
      • Simojoki H.
      • Myllyniemi A.L.
      • Pitkälä A.
      • Pelkonen S.
      • Pyörälä S.
      Phylogeny, virulence factors, and antimicrobial susceptibility of Escherichia coli isolated in clinical bovine mastitis.
       France (n = 1,114)DDiscCA-SFM22030521114220181112
      • Anses
      Resapath: French surveillance network for antimicrobial resistance in bacteria from diseased animals. 2019 annual report.
       Germany (n = 224)DMICCLSI12.10.44.50.92.28.58.5

      BVL. 2018. Resistenzsituation bei klinisch wichtigen tierpathogenen Bakterien. German Resistance Monitoring, Vet. ORCA Affairs.

       Ireland (n = 148)DDiscCLSI18.24.106.116.95.412.211.5DAFM et al., 2021
       Italy (n = 105)SMICCLSI7016237146042
      • Locatelli C.
      • Barberio A.
      • Bonamico S.
      • Casula A.
      • Moroni P.
      • Bronzo V.
      Identification of multidrug-resistant Escherichia coli from bovine clinical mastitis using a ceftiofur-supplemented medium.
       Portugal (n = 5,916)DDiscCLSI99.430.33.613.1
      • Rocha B.
      • Mendonca D.
      • Niza-Ribeiro J.
      Trends in antibacterial resistance of major bovine mastitis pathogens in Portugal.
       Slovakia (n = 65)SDiscCLSI/EUCAST22.124.735.12.6
      • Holko I.
      • Tančin V.
      • Vršková M.
      • Tvarožková K.
      Prevalence and antimicrobial susceptibility of udder pathogens isolated from dairy cows in Slovakia.
       Sweden (n = 116)DMICEUCAST8.64.3060.92.37.81.704.36.93.5
      • Duse A.
      • Persson-Waller K.
      • Pedersen K.
      Microbial aetiology, antibiotic susceptibility and pathogen-specific risk factors for udder pathogens from clinical mastitis in dairy cows.
       Switzerland (n = 82)DDiscCLSI14.71.60.82.56.6
      • Nüesch-Inderbinen M.
      • Käppeli N.
      • Morach M.
      • Eicher C.
      • Corti S.
      • Stephan R.
      Molecular types, virulence profiles, and antimicrobial resistance of Escherichia coli causing bovine mastitis.
       UK–England and Wales (n = 55)DDiscBSAC/AHVLA45.57.35.512.71.814.510.9
      • UK-VARSS
      UK Veterinary Antibiotic Resistance and Sales Surveillance Report UK-VARSS 2018 99.
       UK–Scotland (n = 118)DDiscBSAC/AHVLA22.99.31.715.32.516.911.9
      • UK-VARSS
      UK Veterinary Antibiotic Resistance and Sales Surveillance Report UK-VARSS 2018 99.
       Ukraine (n = 59)DDiscCLSI43.2656.2526.2718.75
      • Elias L.
      • Balasubramanyam A.S.
      • Ayshpur O.Y.
      • Mushtuk I.U.
      • Sheremet N.O.
      • Gumeniuk V.V.
      • Musser J.M.B.
      • Rogovskyy A.S.
      Antimicrobial susceptibility of Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli isolated from mastitic dairy cattle in Ukraine.
      K. pneumoniae
       Belgium (n = 59)DDiscCLSI1.716.93.4
      • Supré K.
      • Lommelen K.
      • De Meulemeester L.
      Antimicrobial susceptibility and distribution of inhibition zone diameters of bovine mastitis pathogens in Flanders, Belgium.
       France (n = 73)DDiscCA-SFM1205102170124
      • Anses
      Resapath: French surveillance network for antimicrobial resistance in bacteria from diseased animals. 2019 annual report.
       Denmark (n = 18)DMICCLSI/EUCAST83.30000000005.60000
      • Chehabi C.N.
      • Nonnemann B.
      • Astrup L.B.
      • Farre M.
      • Pedersen K.
      In vitro antimicrobial resistance of causative agents to clinical mastitis in Danish Dairy cows.
       England and Wales (n = 12)DDiscBSAC/AHVLA10000008.30
      • UK-VARSS
      UK Veterinary Antibiotic Resistance and Sales Surveillance Report UK-VARSS 2018 99.
       Portugal (n = 773)DDiscCLSI10037.41.36.1
      • Rocha B.
      • Mendonca D.
      • Niza-Ribeiro J.
      Trends in antibacterial resistance of major bovine mastitis pathogens in Portugal.
       Sweden (n = 45)SMICEUCAST95.404.64.69.10
      • Duse A.
      • Persson-Waller K.
      • Pedersen K.
      Microbial aetiology, antibiotic susceptibility and pathogen-specific risk factors for udder pathogens from clinical mastitis in dairy cows.
      1 Amp: ampicillin; Amc: amoxicillin + clavulanic acid; Fur: ceftiofur; Ctx: cefotaxime; Chl: chloramphenicol; Ery: Erythromycin; Col: colistin; Nal: nalidixic acid; Spc: spectinomycin; Gen: gentamicin; Kan: kanamycin; Neo: neomycin; Str: streptomycin; Cip: ciprofloxacin; Enr: enrofloxacin; Tet: tetracycline; Smx: sulfamethoxazole; Tmp: trimethoprim; Sxt: sulfamethoxazole + trimethoprim.
      2 T = type: D, diagnostic; S, survey.
      3 Disc = disc diffusion.
      4 CLSI: Clinical and Laboratory Standards Institute; EUCAST: European Committee on Antimicrobial Susceptibility Testing; CA-SFM: Comité de l'Antibiogramme–Société Française de Microbiologie; BSAC: British Society for Antimicrobial Chemotherapy; AHVLA: Animal Health and Veterinary Laboratories Agency.
      * Percent resistance was calculated using the % susceptibility published.
      *** Oxacillin was tested.

      Resistance to β-Lactams

      Resistance to β-lactams is possibly the most found in E. coli and K. pneumoniae isolates from bovine mastitis. β-Lactamases are enzymes that can hydrolyze chemical compounds with a β-lactam ring. However, a wide range of enzymes employ different hydrolyzing mechanisms and have distinct functional capabilities (
      • Bush K.
      Past and present perspectives on β-lactamases.
      ). TEM-1 and SHV-1 and SHV-2 β-lactamases inactivate penicillins and narrow-spectrum cephalosporins (
      • EFSA
      Scientific opinion on the public health risks of bacterial strains producing extended-spectrum β-lactamases and/or AmpC β-lactamases in food and food-producing animals.
      ). Their success is probably due to their easy dissemination through plasmids and other mobile genetic elements (
      • Tooke C.L.
      • Hinchliffe P.
      • Bragginton E.C.
      • Colenso C.K.
      • Hirvonen V.H.A.
      • Takebayashi Y.
      • Spencer J.
      β-Lactamases and β-lactamase inhibitors in the 21st century.
      ). The gene blaTEM-1 has been shown to be present in mastitis E. coli isolates from China, Greece, or Canada for instance, and in K. pneumoniae from Egypt or the United Kingdom (
      • Ahmed A.M.
      • Shimamoto T.
      Molecular characterization of antimicrobial resistance in gram-negative bacteria isolated from bovine mastitis in Egypt.
      ;
      • Timofte D.
      • Maciuca I.E.
      • Evans N.J.
      • Williams H.
      • Wattret A.
      • Fick J.C.
      • Williams N.J.
      Detection and molecular characterization of Escherichia coli CTX-M-15 and Klebsiella pneumoniae SHV-12 β-lactamases from bovine mastitis isolates in the United Kingdom.
      ;
      • Filioussis G.
      • Kachrimanidou M.
      • Christodoulopoulos G.
      • Kyritsi M.
      • Hadjichristodoulou C.
      • Adamopoulou M.
      • Tzivara A.
      • Kritas S.K.
      • Grinberg A.
      Short communication: Bovine mastitis caused by a multidrug-resistant, mcr-1-positive (colistin-resistant), extended-spectrum β-lactamase–producing Escherichia coli clone on a Greek dairy farm.
      ;
      • Yu Z.N.
      • Wang J.
      • Ho H.
      • Wang Y.T.
      • Huang S.N.
      • Han R.W.
      Prevalence and antimicrobial-resistance phenotypes and genotypes of Escherichia coli isolated from raw milk samples from mastitis cases in four regions of China.
      ;
      • Majumder S.
      • Jung D.
      • Ronholm J.
      • George S.
      Prevalence and mechanisms of antibiotic resistance in Escherichia coli isolated from mastitic dairy cattle in Canada.
      ). SHV-1 and 2 enzymes carried by plasmids are less common, although blaSHV-1 was detected in K. pneumoniae mastitis strains from the United States or E. coli from China, and blaSHV-1 and blaSHV-2a in K. pneumoniae from Indonesia (
      • Sudarwanto M.
      • Akineden Ö.
      • Odenthal S.
      • Gross M.
      • Usleber E.
      Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae in bulk tank milk from dairy farms in Indonesia.
      ;
      • Ali T.
      • Ur Rahman S.
      • Zhang L.
      • Shahid M.
      • Zhang S.
      • Liu G.
      • Gao J.
      • Han B.
      ESBL-producing Escherichia coli from cows suffering mastitis in China contain clinical class 1 integrons with CTX-M linked to ISCR1.
      ; Zheng et al., 2021).
      The emergence of these enzymes up until the 1960s increased the need for further development of new antimicrobials and led to the introduction of β-lactamase inhibitors in the 1970s, and third generation cephalosporins and carbapenems in the 1980s and 1990s (
      • Padmini N.
      • Ajilda A.A.K.
      • Sivakumar N.
      • Selvakumar G.
      Extended spectrum β-lactamase producing Escherichia coli and Klebsiella pneumoniae: Critical tools for antibiotic resistance pattern.
      ). However, extended spectrum β-lactamases (ESBL) and AmpC cephalosporinases started to emerge then mainly in E. coli and K. pneumoniae isolates increasing the incidence of nosocomial infections (
      • Padmini N.
      • Ajilda A.A.K.
      • Sivakumar N.
      • Selvakumar G.
      Extended spectrum β-lactamase producing Escherichia coli and Klebsiella pneumoniae: Critical tools for antibiotic resistance pattern.
      ). Extended spectrum β-lactamases can hydrolyze penicillins, first-, second-, third- and fourth-generation cephalosporins as well as monobactams such as aztreonam, but not cephamycins or carbapenems (
      • Jacoby G.A.
      • Munoz-price L.S.
      The new β-lactamases.
      ). Additionally, they are susceptible to β-lactam inhibitors such as clavulanic acid, sulbactam, or tazobactam. The genes coding for these enzymes rarely integrate in the bacterial chromosome, and are associated with insertion sequences such as ISEcp1, ISCR1, or IS26, Transposons such as Tn2 or integrons (
      • Poirel L.
      • Madec J.-Y.
      • Lupo A.
      • Schink A.-K.
      • Kieffer N.
      • Nordmann P.
      • Schwarz S.
      Antimicrobial resistance in Escherichia coli.
      ). Nowadays about 150 different enzymes have been described from each of the CTX-M, SHV, or TEM families (
      • ur Rahman S.
      • Ali T.
      • Ali I.
      • Khan N.A.
      • Han B.
      • Gao J.
      The growing genetic and functional diversity of extended spectrum β-lactamases.
      ). The blaCTX-M-15 gene has been found in numerous mastitis E. coli and K. pneumoniae isolates from around the globe. Other examples for ESBL detected in E. coli include CTX-M-1, CTX-M-2, CTX-M-3, CTX-M-14, CTX-M-55, CTX-M-96, SHV-12 from Germany, Switzerland, Japan, France, or Colombia (