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Rapid Communication| Volume 97, ISSUE 11, P6964-6969, November 2014

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Short communication: Genotypic and phenotypic identification of environmental streptococci and association of Lactococcus lactis ssp. lactis with intramammary infections among different dairy farms

Open ArchivePublished:September 18, 2014DOI:https://doi.org/10.3168/jds.2014-8314
Short communication: Genotypic and phenotypic identification of environmental streptococci and association of Lactococcus lactis ssp. lactis with intramammary infections among different dairy farms
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      Abstract

      Lactococcus species are counted among a large and closely related group of environmental streptococci and streptococci-like bacteria that include bovine mastitis pathogenic Streptococcus, Enterococcus, and Aerococcus species. Phenotypic and biochemical identification methods can be inaccurate and unreliable for species within this group, particularly for Lactococcus spp. As a result, the incidence of Lactococcus spp. on the farm may have been historically underreported and consequently little is known about the clinical importance of this genus as a mastitis pathogen. We used molecular genetic identification methods to accurately differentiate 60 environmental streptococci and streptococci-like bacteria isolated from cows with high somatic cell count and chronic intramammary infection (IMI; >2 somatic cell scores above 4) among 5 geographically distinct farms in New York and Minnesota that exhibited an observed increase in IMI. These isolates were phenotypically identified as Streptococcus uberis and Streptococcus spp. Genetic methods identified 42 isolates (70%) as Lactococcus lactis ssp. lactis, including all 10 isolates originally phenotypically identified as Streptococcus uberis. Antibiotic inhibition testing of all Lc. lactis ssp. lactis showed that 7 isolates were resistant to tetracycline. In the present study, a predominance of Lc. lactis ssp. lactis was identified in association with chronic, clinical bovine IMI among all 5 farms and characterized antimicrobial resistance for treatment therapies. Routine use by mastitis testing labs of molecular identification methods for environmental streptococci and streptococci-like bacteria can further define the role and prevalence of Lc. lactis ssp. lactis in association with bovine IMI and may lead to more targeted therapies.

      Key words

      Short Communication

      Environmental streptococci and streptococci-like bacteria are significant contributors to the incidence of clinical mastitis in the United States (
      • Gröhn Y.T.
      • Wilson D.J.
      • Gonzalez R.N.
      • Hertl J.A.
      • Schulte H.
      • Bennett G.
      • Schukken Y.H.
      Effect of pathogen-specific clinical mastitis on milk yield in dairy cows.
      J. Dairy Sci. 2004; 87: 3358-3374
      ), accounting for up to 15% of cows diagnosed annually (
      NMC (National Mastitis Council)
      Procedures for the identification of specific groups or species of microorganisms that cause mastitis.
      Microbiological procedures for the diagnosis of bovine udder infection. National Mastitis Council Inc., Washington, DC1990
      ). Only a small percentage of these infections become chronic (18% of infections exceeding 100 d), and although the cure rate for these cases is low, targeted therapy can sometimes reduce disease severity (

      Jones, G. M., and J. M. Swisher. 2009. Environmental streptococcal and coliform mastitis. Virginia Cooperative Extension Publication No. 404–234. http://pubs.ext.vt.edu/category/dairy-cattle.html.

      ).
      Streptococcus uberis has been implicated in most bovine IMI, with less than 18% of reported cases attributed to other environmental streptococci and streptococci-like bacteria (

      Jones, G. M., and J. M. Swisher. 2009. Environmental streptococcal and coliform mastitis. Virginia Cooperative Extension Publication No. 404–234. http://pubs.ext.vt.edu/category/dairy-cattle.html.

      ). Among this 18%, species of Enterococcus and Aerococcus have been well characterized in terms of incidence and role in bovine IMI; however, similar data for Lactococcus spp. are few (
      • Malinowski E.
      • Klossowska A.
      • Kaczmarowski M.
      • Kuzma K.
      Prevalence of intramammary infections in pregnant heifers.
      Bull. Vet. Inst. Pulawy. 2003; 47: 165-170
      ;
      • Kuang Y.
      • Tani K.
      • Synnott A.J.
      • Ohshima K.
      • Higuchi H.
      • Nagahata H.
      • Tanji Y.
      Characterization of bacterial population of raw milk from bovine mastitis by culture-independent PCR-DGGE method.
      Biochem. Eng. J. 2009; 45: 76-81
      ;
      • Plumed-Ferrer C.
      • Uusikylä K.
      • Korhonen J.
      • von Wright A.
      Characterization of Lactococcus lactis isolates from bovine mastitis.
      Vet. Microbiol. 2013; 167: 592-599
      ). This lack of information could be attributed to limitations in routine procedures used to speciate environmental streptococci and streptococci-like bacteria. Phenotypic and biochemical tests recommended by the National Mastitis Council for identification of environmental streptococci and streptococci-like bovine milk isolates (
      NMC (National Mastitis Council)
      Laboratory Handbook on Bovine Mastitis. Rev ed. National Mastitis Council, Madison, WI1999
      ), as well as commercially available biochemical test kits for these microorganisms, are not inclusive of Lactococcus spp. and can produce variable or erroneous results that cause misidentifications (
      • Fortin M.
      • Messier S.
      • Pare J.
      • Higgins R.
      Identification of catalase-negative, non-beta-hemolytic gram-positive cocci isolated from milk samples.
      J. Clin. Microbiol. 2003; 41: 106-109
      ;
      • Odierno L.
      • Calvinho L.
      • Traverssa P.
      • Lasagno M.
      • Bogni C.
      • Reinoso E.
      Conventional identification of Streptococcus uberis isolated from bovine mastitis in Argentinean dairy herds.
      J. Dairy Sci. 2006; 89: 3886-3890
      ;
      • Svec P.
      • Sedlácek I.
      Characterization of Lactococcus lactis ssp. lactis isolated from surface waters.
      Folia Microbiol. (Praha). 2008; 53: 53-56
      ;
      • Gordoncillo M.J.N.
      • Bautista J.A.N.
      • Hikiba M.
      • Sarmago I.G.
      • Haguingan J.M.B.
      Comparison of conventionally identified mastitis bacterial organisms with commercially available microbial identification kit (BBL Crystal ID).
      Phil. J. Vet. Med. 2010; 47: 54-57
      ). Thus, it is possible that the incidence of Lactococcus spp. associated with bovine IMI has been severely underreported.
      In recent years, molecular genetic methods such as PCR and sequencing-based methods have proven a more reliable means of accurately differentiating environmental streptococci and streptococci-like bacteria isolated from bulk tank and composite milk samples (
      • Holm C.
      • Jepsen L.
      • Larsen M.
      • Jespersen L.
      Predominant microflora of downgraded Danish bulk tank milk.
      J. Dairy Sci. 2004; 87: 1151-1157
      ;
      • Reinoso E.
      • Dieser S.
      • Calvinho L.
      • Bogni C.
      • Odierno L.
      Phenotyping and genotyping of streptococci in bovine milk in Argentinean dairy herds.
      Acta Vet. Hung. 2010; 58: 287-295
      ;
      • de Garnica M.L.
      • Saez-Nieto J.A.
      • Gonzalez R.
      • Santos J.A.
      • Gonzalo C.
      Diversity of gram-positive catalase-negative cocci in sheep bulk tank milk by comparative 16S rDNA sequence analysis.
      Int. Dairy J. 2014; 34: 142-145
      ;
      • Kanyó I.
      • Nagy D.
      Molecular identification key of the family Streptococcaceae..
      Acta Microbiol. Immunol. Hung. 2014; 61: 27-48
      ) and have, in some cases, enabled the unequivocal identification of pure cultures of Lactococcus lactis ssp. lactis and Lactococcus garvieae originating from bovine IMI cases (
      • Devriese L.A.
      • Hommez J.
      • Laevens H.
      • Pot B.
      • Vandamme P.
      • Haesebrouck F.
      Identification of aesculin-hydrolyzing streptococci, lactococci, aerococci, and enterococci from subclinical intramammary infections in dairy cows.
      Vet. Microbiol. 1999; 70: 87-94
      ;
      • Kuang Y.
      • Tani K.
      • Synnott A.J.
      • Ohshima K.
      • Higuchi H.
      • Nagahata H.
      • Tanji Y.
      Characterization of bacterial population of raw milk from bovine mastitis by culture-independent PCR-DGGE method.
      Biochem. Eng. J. 2009; 45: 76-81
      ).
      The objective of this report was to use molecular genetic identification methods to accurately differentiate environmental streptococci and streptococci-like bacteria isolated from cows with high SCC and chronic IMI among 5 geographically distinct farms in New York and Minnesota that exhibited an observed increase of IMI due to these bacteria. Where Lc. lactis ssp. lactis isolates were identified, antimicrobial resistance profiles were determined to contribute to the understanding of possible therapies and cure rates.
      Isolates were included from 3 New York State dairy farms (herds A, B, and C), where each herd exhibited a high prevalence of chronic infections as determined by 2 or more consecutive monthly test-day linear scores greater than 4.0 (SCC ≥200,000 cells/mL; 18, 21, and 23% of the total of lactating cows, respectively). Herd A, a 160-lactating cow Holstein Friesian herd in freestall facilities bedded with sand, had an average daily milk production of 40.1 kg and a rolling bulk milk SCC of 286,000 cells/mL. Quarter samples (n = 42) from 12 cows with high SCC were submitted to the Quality Milk Production Services (QMPS; Ithaca, NY) for bacteriological culture. Ten of the 12 culture-positive samples were identified as Streptococcus spp. Isolates from herds B and C were submitted through a private veterinary clinic located in New York State and were initially identified as Streptococcus spp. or Streptococcus uberis. Herd B, an 895-lactating cow Holstein-Friesian herd in freestall facilities bedded with sand, had an average daily milk production of 40.2 kg and a bulk milk SCC of 397,000 cells/mL. Eighteen isolates from 11 cows with high SCC milk were submitted to QMPS for sequence analysis. Herd C, a 230-lactating cow Holstein-Friesian herd in a freestall facility bedded with sand, had an average daily milk production of 37 kg and bulk milk SCC of 286,000 cells/mL. Eleven isolates from 11 cows with high SCC milk were submitted to QMPS for sequence analysis.
      Milk samples from 2 Minnesota dairy farms (herds D and E) with chronic infection rates of 14 and 21%, respectively, were also submitted for speciation of the pathogens. Herd D, a 1,693-lactating cow Holstein-Friesian herd in a freestall facility bedded with recycled sand, had an average daily milk production of 43 kg and a bulk tank SCC of 270,000 cells/mL. Eleven isolates from 11 cows with high SCC milk were submitted to QMPS for sequence analysis. Herd E, a 914-lactating cow Holstein-Friesian herd in a freestall facility bedded with recycled sand and sand lane, had an average daily milk production of 39 kg and a bulk tank SCC of 365,000 cells/mL. Ten isolates from 10 cows with high SCC milk were submitted to QMPS for sequence analysis.
      The initial phenotypic speciation by all laboratories was performed using National Mastitis Council laboratory identification procedure recommendations (
      NMC (National Mastitis Council)
      Laboratory Handbook on Bovine Mastitis. Rev ed. National Mastitis Council, Madison, WI1999
      ). These biochemical and serological tests, including Gram stain, β-hemolysis, catalase test, esculin hydrolysis, Christie, Atkins, Munch-Petersen (CAMP) test, growth in enterococcal medium, and growth in 6.5% NaCl, were used to identify isolates as Streptococcus spp. or Streptococcus uberis. For 2 isolates, the BBL Crystal Gram-Positive ID Kit (Becton Dickinson, Franklin Lakes, NJ) was used for further identification. Polymerase chain reaction and sequence analysis of 16S rDNA and rpoB were used for molecular identification of all isolates. For these analyses, a crude cell DNA lysate was first prepared for each isolate (
      • Furrer B.
      • Candrian U.
      • Hoefelein C.
      • Luethy J.
      Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of hemolysin gene fragments.
      J. Appl. Bacteriol. 1991; 70: 372-379
      ). Briefly, pure culture streaks were made on a tryptic soy agar with 5% sheep blood plate (Becton Dickinson) and incubated for 18 to 24 h at 37°C. One isolated colony was transferred to a 1.5-mL tube of Todd Hewitt Broth (Becton Dickinson) and incubated 18 to 24 h at 37°C. A NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) was used to determine DNA quantity. Two hundred fifty microliters of each broth culture was transferred to 1.5-mL microcentrifuge tubes and centrifuged to pellet for 10 min at 6,000 × g at room temperature (20°C ± 2°C). The supernatant was removed and discarded, and the pellet was resuspended in 95 µL of 1× PCR buffer (Roche Applied Science, Indianapolis, IN). A 4-µL aliquot of freshly prepared 50 mg/mL lysozyme (Promega, Madison, WI) solution was added to this suspension and mixed well by pipetting to result in a final concentration of 2 mg/mL lysozyme. After a 15-min incubation at room temperature, 1 µL of a 20 mg/mL proteinase K solution (Promega) was added and vortexed to mix, resulting in a final concentration of 200 µg/mL proteinase K. The tubes were incubated for up to 2 h in a heat block set at 58°C until the lysates cleared. The enzymes were inactivated for 8 min at 95°C. After equilibrating to room temperature, tubes were centrifuged for 3 s at 6,000 × g to remove condensate from the sides and lids. Crude cell lysates were stored at −20°C until further analysis.
      Two different PCR amplification steps were performed to amplify 2 different gene targets, rpoB (
      • Drancourt M.
      • Roux V.
      • Fournier P.E.
      • Raoult D.
      rpoB gene sequence-based identification of aerobic gram-positive cocci of the genera Streptococcus, Enterococcus, Gemella, Abiotrophia, and Granulicatella..
      J. Clin. Microbiol. 2004; 42: 497-504
      ) and 16S rDNA (
      • Greisen K.
      • Loeffelholz M.
      • Purohit A.
      • Leong D.
      PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid.
      J. Clin. Microbiol. 1994; 32: 335-351
      ). Primers rpoBF (5′-AARYTIGGMCCTGAAGAAAT-3′), rpoBR (5′-GCCTTTAACTTCAGACTTATCA-3′), DG74 (5′-AGGAGGTGATCCAACCGCA-3′), and P5SH (5′-TGAAGAGTTTGATCMTGGCTCAG-3′) were purchased from Integrated DNA Technologies (IDT, Coralville, IA). A PCR amplification mix was prepared consisting of 0.25 µL of a 100 µM solution of each forward and reverse primer, 25.00 µL of GoTaqGreen (Promega), 27.50 µL of H2O, and 2.0 µL of DNA. The mix was amplified on a MyCycler Thermal Cycler (Bio-Rad, Hercules, CA) with running conditions of an initial denaturation step of 5 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 50°C, and 1 min at 72°C; and a final 7-min extension time at 72°C. The PCR amplicons were analyzed by gel electrophoresis to confirm a 740-bp rpoB amplicon and a 1,040-bp 16S rDNA amplicon.
      In preparation for sequencing, excess primers and nucleotides were removed from each sample by treatment with ExoSAP-IT (USB Corporation, Cleveland, OH). Sequencing of each sample was performed in 2 directions using Big Dye Terminator chemistry on an ABI Prism 3700 DNA analyzer (Applied BioSystems, Foster City, CA). Sequences were proofread in SeqMan (version 5.08, Lasergene; DNAStar Inc., Madison, WI), compared with publicly available sequence data using the National Center for Biotechnology Information (NCBI) BLAST programs (
      • Altschul S.F.
      • Madden T.L.
      • Schäffer A.A.
      • Zhang J.
      • Zhang Z.
      • Miller W.
      • Lipman D.J.
      Gapped BLAST and PSI-BLAST: A new generation of protein database search programs.
      Nucleic Acids Res. 1997; 25: 3389-3402
      ), and identified using interpretive criteria provided by the Clinical and Laboratory Standards Institute (

      CLSI (Clinical and Laboratory Standards Institute). 2008a. Interpretive criteria for identification of bacteria and fungi by DNA target sequencing; approved guideline. CLSI document MM18-A. CLSI, Wayne, PA.

      ). Briefly, the criteria for BLAST were as follows: maximum identity ≥99.0% to species level with greater than 0.8% separation between different species and an expected value (E-value) = 0.0.
      Antimicrobial MIC for Lc. lactis ssp. lactis isolates were determined using the Sensititer Mastitis Plate Format veterinary panel and Sensititer ARIS system (Trek Diagnostics Systems, Cleveland, OH). This panel includes antibiotics and the respective MIC dilution ranges listed in Table 1. Results were interpreted according to the European Food Safety Authority (EFSA) technical guides (
      European Commission
      Opinion of the Scientific Panel on Additives and Products of Substances used in Animal Feed on the updating of the criteria used in the assessment of bacteria for resistance to antibiotics of human or veterinary importance.
      EFSA J. 2005; 223: 1-12
      ,
      European Commission
      Technical guidance prepared by the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on the update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance.
      EFSA J. 2008; 732: 1-15
      ).
      Table 1Minimum inhibitory concentrations of the 10 antimicrobial agents tested for the 42 Lactococcus lactis ssp. lactis isolates
      Shaded areas indicate the concentration ranges of each of the antimicrobials tested. Resistance breakpoints are indicated by vertical lines when available and as NA when not available. 2Resistance breakpoints proposed by the EFSA (European Commission, 2008). 3Resistance breakpoints proposed by the EFSA (European Commission, 2005).
      1 Shaded areas indicate the concentration ranges of each of the antimicrobials tested. Resistance breakpoints are indicated by vertical lines when available and as NA when not available.2Resistance breakpoints proposed by the EFSA (
      European Commission
      Technical guidance prepared by the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on the update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance.
      EFSA J. 2008; 732: 1-15
      ).      3Resistance breakpoints proposed by the EFSA (
      European Commission
      Opinion of the Scientific Panel on Additives and Products of Substances used in Animal Feed on the updating of the criteria used in the assessment of bacteria for resistance to antibiotics of human or veterinary importance.
      EFSA J. 2005; 223: 1-12
      ).
      Genotypic and phenotypic identification results for all isolates analyzed are shown in Table 2. Sequence analysis of all 60 isolates among the 5 herds identified overall 42 (70%) as Lc. lactis ssp. lactis, 10 (17%) as Enterococcus saccharolyticus, 1 (1.5%) as Enterococcus faecium, 1 (1.5%) as Enterococcus spp., 4 (7%) as Streptococcus uberis, 1 (1.5%) as Lactococcus lactis, and 1 (1.5%) as Streptococcus dysgalactiae ssp. dysgalactiae.
      Table 2Phenotypic identities and sequencing results for bacterial isolates from 5 farms (herds A, B, C, D, and E)
      Herd

      identity      		No. of

      isolates      		IsolatePhenotypic

      identification      		Sequencing result
      A10B4-261Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-262Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-263Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-264Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-265Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-266Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-267Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-268Streptococcus spp.Enterococcus faecium
      B4-269Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-270Streptococcus spp.Lactococcus lactis ssp. lactis
      B18B4-203Streptococcus uberisLactococcus lactis ssp. lactis
      B4-204Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-205Streptococcus spp.Enterococcus saccharolyticus
      B4-206Streptococcus spp.Enterococcus saccharolyticus
      B4-207Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-208Streptococcus uberisLactococcus lactis ssp. lactis
      B4-209Streptococcus uberisLactococcus lactis ssp. lactis
      B4-210Streptococcus uberisLactococcus lactis ssp. lactis
      B4-211Streptococcus uberisLactococcus lactis ssp. lactis
      B4-212Streptococcus uberisLactococcus lactis ssp. lactis
      B4-213Streptococcus uberisLactococcus lactis ssp. lactis
      B4-214Streptococcus uberisLactococcus lactis ssp. lactis
      B4-215Streptococcus uberisLactococcus lactis ssp. lactis
      B4-216Streptococcus spp.Enterococcus spp.
      B4-217Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-218Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-219Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-220Streptococcus uberisLactococcus lactis ssp. lactis
      C11B4-279Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-280Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-281Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-282Leuconostoc spp.Lactococcus lactis ssp. lactis
      B4-283Streptococcus spp.Enterococcus saccharolyticus
      B4-284Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-285Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-286Aerococcus viridansEnterococcus saccharolyticus
      B4-287Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-288Streptococcus spp.Lactococcus lactis ssp. lactis
      B4-289Streptococcus spp.Lactococcus lactis ssp. lactis
      D11W1–849Streptococcus spp.Streptococcus uberis
      W1–850Streptococcus spp.Lactococcus lactis ssp. lactis
      W1–851Streptococcus spp.Enterococcus saccharolyticus
      W1–852Streptococcus spp.Streptococcus dysgalactiae ssp. dysgalactiae
      W1–853Streptococcus spp.Lactococcus lactis ssp. lactis
      W1–854Streptococcus spp.Enterococcus saccharolyticus
      W1–855Streptococcus spp.Enterococcus saccharolyticus
      W1–856Streptococcus spp.Lactococcus lactis ssp. lactis
      W1–857Streptococcus spp.Lactococcus lactis ssp. lactis
      W1–858Streptococcus spp.Enterococcus saccharolyticus
      W1–859Streptococcus spp.Lactococcus lactis ssp. lactis
      E10W1–896Streptococcus spp.Streptococcus uberis
      W1–897Streptococcus spp.Lactococcus lactis
      W1–898Streptococcus spp.Lactococcus lactis ssp. lactis
      W1–899Streptococcus spp.Enterococcus saccharolyticus
      W1–900Streptococcus spp.Enterococcus saccharolyticus
      W1–901Streptococcus spp.Streptococcus uberis
      W1–902Streptococcus spp.Lactococcus lactis ssp. lactis
      W1–903Streptococcus spp.Lactococcus lactis ssp. lactis
      W1–904Streptococcus spp.Lactococcus lactis ssp. lactis
      W1–905Streptococcus spp.Streptococcus uberis
      For herd A, 9 of the 10 isolates phenotypically reported as Streptococcus spp. were identified by sequencing as Lc. lactis ssp. lactis, whereas 1 was identified as Enterococcus faecium. For herd B, where 8 of the 18 isolates were initially phenotypically identified as Streptococcus spp. and 10 of the isolates as Streptococcus uberis, 15 of these were identified by molecular methods as Lc. lactis ssp. lactis, 2 as Ent. saccharolyticus, and 1 as Enterococcus spp. All 10 of the isolates phenotypically identified as Streptococcus uberis were genetically identified as Lc. lactis ssp. lactis.
      For herd C, 2 isolates were biochemically identified by the BBL Crystal Gram-Positive ID kit as Leuconostoc spp. and Aerococcus viridans. These isolates were identified by sequencing as Lc. lactis ssp. lactis and Enterococcus saccharolyticus, respectively. Among the remaining 9 isolates that were phenotypically identified as Streptococcus spp., 2 were identified by molecular methods as Enterococcus saccharolyticus and 7 as Lc. lactis ssp. lactis.
      All isolates from herds D and E were initially phenotypically identified as Streptococcus spp. For herd D, DNA sequence analysis identified 5 as Lc. lactis ssp. lactis, 4 as Enterococcus saccharolyticus, 1 as Streptococcus uberis, and 1 as Streptococcus dysgalactiae ssp. dysgalactiae. For herd E, 4 of these isolates were identified by sequence analysis as Lc. lactis ssp. lactis, 1 as Lc. lactis, 3 as Streptococcus uberis, and 2 as Enterococcus saccharolyticus.
      Antibiotic inhibition testing of all Lc. lactis ssp. lactis showed that all isolates were susceptible to ampicillin, erythromycin, and penicillin, and 7 isolates were resistant to tetracycline. These data are shown in Table 1. Results showed that, among these 5 farms, Lc. lactis ssp. lactis was the predominant streptococci-like bacterium associated with bovine IMI and identified potential antimicrobial agents for therapy. Overall, use of phenotypic identification methods resulted in overreporting the incidence of Streptococcus uberis and did not provide discriminatory information about the incidence of other genera and species. In this case, phenotypic and biochemical identification produced erroneous results and failed to identify Lc. lactis ssp. lactis. From a clinical perspective, it is important to understand the role played by Lc. lactis ssp. lactis within the farm and, furthermore, to study the epidemiology in order to define the behavior of this potential mastitis pathogen and management of therapy decisions. Routine use of molecular methods for speciation of environmental streptococci and streptococci-like bacteria in association with chronic, clinical IMI will contribute to this understanding. Antimicrobial susceptibility profiling along with accurate identification will enable targeted and early intervention for reduction of chronic IMI.

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      Article info

      Publication history

      Published online: September 18, 2014
      Accepted: August 12, 2014
      Received: May 2, 2014

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      DOI: https://doi.org/10.3168/jds.2014-8314

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      © 2014 American Dairy Science Association. Published by Elsevier Inc.

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