Media and methods for Identification of Enterococci

Transcription

1 APPENDIX.1 Media and methods for Identification of Enterococci Blood agar Meat extract g Peptone g Sodium chloride - 5.0g Agar g Dist.water ml Autoclaved blood agar base is cooled and at 50 o C, 10% (v/v) defibrinated blood is added. Glucose azide broth Bromocresol purple g Dextrose - 5g Dipotassium phosphate - 5g Monopotassiu phosphate - 2g Peptic digest of animal tissue - 10g Bile Esculin Azide Agar Pepton from casein Peptone Yeast extract Sodium chloride - 5.0

2 214 Esculin Ammonium iron(iii) citrate Ox bile Sodium azide Dist. Water - 1L Agar - 13g Streptococcus Faecalis broth Casein Peptone - 20g Dextrose - 5g Potassium Phosphate, dibasic - 4g Potassium Phosphate, monobasic - 1.5g Sodium Chloride - 5g Sodium Azide - 0.5g Bromcresol Purple Dist. Water - 1L Gram staining Procedure Prepared smears using aseptic technique. After air drying and heat fixing the Gram staining procedure was followed. Poured the crystal violet for one minute. Poured off excess dye and washed gently in tap water and drained the slide against a paper towel. Exposed the smears to Gram's iodine for one minute and then added more iodine and left it on the smear for 1minute. Washed with tap water and drained carefully. Washed with 95% alcohol for

3 Appendices seconds. Washed with tap water at the end of the 30 seconds to stop the decolorization. Drained and counter stained with carbol fuschsin for 30 seconds. Washed, drained, and examined under oil. Catalase test Using a sterile inoculating loop or wooden applicator stick, a small amount of organism from a well-isolated 18- to 24-hour colony grown on nutrient agar plate were collected and placed it onto the microscope slide. Using a dropper or Pasteur pipette, placed 1 drop of 3% H 2 O 2 onto the organism on the microscope slide. Immediately covered the petri dish with a lid to limit aerosols and observed for immediate bubble formation was indicative of catalase activity. Sugar Media Phenol red broth base Peptone - 1% Beef extract - 0.1% NaCl - 0.5% Phenol Red - 20 µg/ml Desired filter sterilized carbohydrates like glucose, lactose, sucrose, adonitol, raffinose, arabinose, mannitol etc. of 1% concentration were added.

4 Appendices 217 APPENDIX-2 Media for the Detection of Virulence factors and list of antibiotics and biocides Nutrient gelatin Gelatine - 120g Meat extract - 3g Peptone - 5g Dist water - 1L Tween 80-agar Peptone g NaCl - 5.0g CaCl2 2H2O - 0.1g Tween-80 (Sigma) - 10.mL Agar g Dist.water - 1L ph 6.0 Congo red agar Congo red dye g Sucrose - 36g BHIA - 52g Water ml Neutralizer for biocide testing Polysorbate % Lecithin % Peptamin - 1%

5 218 Beef extract - 0.5% Sodium chloride - 0.5% Hucker s crystal violet Solution A Crystal violet stain - 2.0gm Ethyl alcohol - 20ml Solution B Ammonium oxalate - 0.8gm Distilled water - 80ml Mix solutions A and B. Store for 24 hours before use. The resulting stain is stable. List of disinfectants used with stated concentration Glutaraldehyde (BioShield,Tulip group India: 24.5%w/v), Benzalkonium chloride (BioShield,Tulip group India 5%w/v), Povidone 1odine (Sigma- Aldrich:10%w/v), Sodium hypochlorite (Sigma-Aldrich:10%w/v), cresylic acid (Sigma-Aldrich:90%w/v) List of antibiotics used with stated concentration (HiMedia) Penicillin P (10 units); Oxacillin OX (1 µg); Gentamycin G (30 µg); Streptomycin S (30 µg); Tetracycline T(30 µg); Clindamicin CD (30µg); Chloramphenicol C (30 µg); Ciprofloxacin CF (5 µg);nitrofurantion (30 µg); Erythromycin E (30 µg); Ampicillin A (30 µg); Amoxyclav AC (30 µg); Gentamycin G120 (120);Vancomycin VA (30 µg); Teicoplanin Te (30 µg)

6 Appendices 219 APPENDIX 3 Reagents and media for molecular identification Buffers for Plasmid Isolation Solution I 25mM Tris-HCI ph 8.O 50mM glucose 10mM EDTA Autoclave the solution and store at 4 C. Solution II 0.2N NaOH (freshly diluted from a 10N stock) 1% SDS Prepare solution II fresh and use at room temperature Solution III 5.0M Potassium acetate - 60ml Glacial acetic acid ml Water ml Prepare and store at 4 C. Transfer to an ice bucket just before use. DNA Isolation from Bacteria: Stock solutions: 0.85% (w/v) NaCl solution: NaCl g Distilled water - 90 ml

7 220 Add 0.85g NaCl to 90 ml of distilled water and make up the final volume to 100ml, autoclave and store at 40C. 0.5 M EDTA: Na2EDTA.2H g Distilled water - 20 ml Adjust the ph to 8.0 using 1M NaOH and make up the final volume to 25ml, autoclave and store at 40C. 1M Tris (ph 8.0): Tris -Cl 3.93 g Distilled water 20 ml Adjust the ph to 8.0 using 1M HCl and make up the volume to 25ml, autoclave and store at 40C. 20% SDS: SDS g Distilled water - 20 ml Make up the final volume to 25ml and store at 40C. O.1M Tris HCl (ph 8.0): 1M Tris HCl - 15 ml Distilled water ml

8 Appendices 221 Chloroform: Isoamyl alcohol (24:1): Chloroform - 48 ml Isoamyl alcohol - 2 ml Both of the components are mixed properly by inversion. 3M Sodium Acetate (ph 5.2) Sodium acetate g Distilled water - 20 ml Adjust the ph to 5.2 using glacial acetic acid and make up the final volume to 25ml, autoclave and store at 40C. TE buffer (DNA dissolving solution) 10mM Tris - 20µl from 1M Tris Stock 1mM EDTA - 4 µl from 0.5M EDTA Stock. Make up to 2 ml and store at 4 C Proteinase-K: Proteinase-K - 20 mg Sterile Distillled water - 1ml Dissolve the proteinase-k in 1 ml of autoclaved distilled water and store at 20oC. Reagents Required LB (Luria Bertani ) medium (ph 7.0) Yeast Extract g Tryptone g

9 222 Sodium chloride( NACl) g Distilled water Litre Suspension Buffer (GTE solution) Glucose - 50mM Tris-HCl (ph 8.0) - 25mM EDTA (ph 8.0) - 10mM (Autoclave and can be stored at 4oC) Lysis Buffer Sodium Hydroxide (NaOH) N Sodium Dodecyl Sulphate - 1% (SDS should not be autoclaved and stored at room temperature) Potassium acetate solution Potassium acetate 5M (ph 4.8) ml Glacial Acetic Acid ml Water ml

10 Appendices 223 APPENDIX.4 Sequence data and Phylogenetic analysis Phylogenetic analysis of van B gene sequence of vnacomycin resistant E.faecium (VREF 1) FJ X56895 GQ AB VREF1 JN JN JN JN JN JN The analysis was carried out using MEGA4.The scale bar indicates 0.1 substitutions per nucleotide positions for van A gene VERF1: Isolated Van Gene 1 JN207928: Enterococcus faecium strain VRE12 VanA ligase (vana) JN207932: Enterococcus faecalis strain VRE28 VanA ligase (vana) JN207933: Enterococcus faecalis strain VRE44 VanA ligase (vana)

11 224 JN207929: Enterococcus faecium strain VRE49 VanA ligase (vana) JN207930: Enterococcus faecium strain VRE56 VanA ligase (vana) JN029954: Enterococcus faecium strain CNM232_09 ST17 vancomycin resistance protein A (vana) X56895: E.faecium plasmid pip816 vana gene for VANA ligase FJ545640: Staphylococcus haemolyticus VanA (vana) GQ273978: Staphylococcus aureus strain ST1RCGLD-IPI vana gene cluster, partial sequence AB663321: Enterococcus cecorum orf1, orf2, vanr, vans, vanh, vana, vanx, vany, vanz genes, complete cds Sequence analysis of vanb1 (gene bank submission ID ) TTGATGACGCTCTATCTGTGCCGGATTACTTGTTAAAAAGAACCATGAATATGAAATCAACCATGTTG ATGTAGCATTTTCAGCTTTGCATGGCAAGTCAGGTGAAGATGGATCCATACAAGGTCTGTTTGAATTG TCCGGTATCCCTTTTGTAGGCTGCGATATTCAAAGCTCAGCAATTTGTATGGACAAATCGTTGACATA CATCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCTTTTGGGTTATTAATAAAGATGATAGGCCGG TGGCAGCTACGTTTACCTATCCTGTTTTTGTTAAGCCGGCGCGTTCAGGCTCATCCTTCGGTGTGAAAA AAGTCAATAGCGCGGACGAATTGGACTACGCAATTGAATCGGCAAGACAATATGACAGCAAAATCTT AATTGAGCAGGCTGTTTCGGGCTGTGAGGTCGGTTGTGCGGTATTGGGAAACAGTGCCGCGTTAGTTG TTGGCGAGGTGGACCAAATCAGGCTGCAGTACGGAATCTTTCGTATTCATCAGGAAGTCGAGCCGGA AAAAGGCTCTGAAAACGCAGTTATAACCGTTCCCGCAGACCTTTCAGCAGAGGAGCGAGGACGGATA CAGGAAACGGCAAAAAAAATATATAAAGCGCTCGGCTGTAGAGGTCTAGCCCGTGTGGATATGTTTT TACAAGATAACGGCGCCATTGTACAGCAC

12 Appendices 225 Phylogenetic analysis of vana gene sequence of vnacomycin resistant E.faecium (VREF2) VREF2 AB GQ FJ EF EF EF AF X56895 JN JN VERF2: Isolated Van Gene 2 JN207932: Enterococcus faecalis strain VRE28 VanA ligase (vana) JN029954: Enterococcus faecium strain CNM232_09 ST17 vancomycin resistance protein A (vana) X56895: E.faecium plasmid pip816 vana gene for VANA ligase AF516335: Enterococcus faecium plasmid puw786 multiple antibiotic resistance gene cluster, complete sequence EF206284: Enterococcus faecium strain F135/41 vancomycin resistance protein A (vana)

13 226 EF206285: Enterococcus faecium strain F4/13(54) vancomycin resistance protein A (vana) EF206286: Enterococcus faecalis strain A21(35) vancomycin resistance protein A (vana) FJ545640: Staphylococcus haemolyticus VanA (vana) gene, partial cds GQ273978: Staphylococcus aureus strain ST1RCGLD-IPI vana gene cluster, partial sequence AB663321: Enterococcus cecorum orf1, orf2, vanr, vans, vanh, vana, vanx, vany, vanz genes, complete cds Sequence analysis of van A from VREF2 (gene bank submission ID ) CGGATTTGGTCAGTCATGCAGCTTGTACTTCATCTATGTGCGCGACTCATGAGCAGCTTTCGCTGCGCG GTGGTTGTTTTCTTGTTAGCGTGATAAGAAGTGTGGTCAGGGATCCTTGCGTAGCCTGCAGCTATTGTC AAGTTAGGAATATTGTAGTACAGCTACTTCTAATGCTCAACGGCTTGCATTGGACCATCGTTGACATA CATCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCTTTTGGGTTATTAATAAAGATGATAGGCCGG TGGCAGCTACGTTTACCTATCCTGTTTTTGTTAAGCCGGCGCGTTCAGGCTCATCCTTCGGTGTGAAAA AAGTCAATAGCGCGGACGAATTGGACTACGCAATTGAATCGGCAAGACAATATGACAGCAAAATCTT AATTGAGCAGGCTGTTTCGGGCTGTGAGGTCGGTTGTGCGGTATTGGGAAACAGTGCCGCGTTAGTTG TTGGCGAGGTGGACCAAATCAGGCTGCAGTACGGAATCTTTCGTATTCATCAGGAAGTCGAGCCGGA AAAAGGCTCTGAAAACGCAGTTATAACCGTTCCCGCAGACCTTTCAGCAGAGGAGCGAGGACGGATA CAGGAAACGGCAAAAAAAATATATAAAGCGCTCGGCTGTAGAGGTCTAGCCCGTGTGGATATGTTTT ACAGATAACGGCGCCATTGTACAACGGAGGGGGTGACTTTGACCCGGTGTTCCGAAATGAAATCAGA TATGCCGCATGTGTGCGGTCTTTTTATTGCAGTAATAATGCCGCTATTGTCAACGTACCGTTTTTAGCC AGCTAACAGATGATCCGGTACGTCTCAACTGTGTAAGTTTATTCACCATTTGATAGATCGTAGGTTCC CGAGTTGATTTTAGTCGGGTAAACGCGCATTTTCCAATG

14 Appendices 227 Phylogenetic analysis of van gene sequence of vnacomycin resistant E.Faecium (VREF4) VREF4 FJ GQ AB JN JN JN JN JN JN JN VERF4: Isolated Van Gene 4 JN207931: Enterococcus faecalis strain VRE16 VanA ligase (vana) JN207928: Enterococcus faecium strain VRE12 VanA ligase (vana) JN207932: Enterococcus faecalis strain VRE28 VanA ligase (vana) JN207933: Enterococcus faecalis strain VRE44 VanA ligase (vana)

15 228 JN207929: Enterococcus faecium strain VRE49 VanA ligase (vana) JN207930: Enterococcus faecium strain VRE56 VanA ligase (vana) JN029954: Enterococcus faecium strain CNM232_09 ST17 vancomycin resistance protein A (vana) FJ545640: Staphylococcus haemolyticus VanA (vana) GQ273978: Staphylococcus aureus strain ST1RCGLD-IPI vana gene cluster, partial sequence AB663321: Enterococcus cecorum orf1, orf2, vanr, vans, vanh, vana, vanx, vany, vanz genes, complete cds Sequence analysis of vanb from VREF4 (gene bank submission ID ) GCGCGATGAGTGTCACTTTGTGTTGATTACTTGTGCCGCGAACAACGAATATGAAATAAACCATGTTG ATGTAGCATTTTCAGCTTTGCATGGCAAGTCAGGTGAAGATGGATCCATACAAGGTCTGTTTGAATTG TCCGGTATCCCTTTTGTAGGCTGCGATATTCAAAGCTCAGCAATTTGTATGGACAAATCGTTGACATA CATCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCTTTTGGGTTATTAATAAAGATGATAGGCCGG TGGCAGCTACGTTTACCTATCCTGTTTTTGTTAAGCCGGCGCGTTCAGGCTCATCCTTCGGTGTGAAAA AAGTCAATAGCGCGGACGAATTGGACTACGCAATTGAATCGGCAAGACAATATGACAGCAAAATCTT AATTGAGCAGGCTGTTTCGGGCTGTGAGGTCGGTTGTGCGGTATTGGGAAACAGTGCCGCGTTAGTTG TTGGCGAGGTGGACCAAATCAGGCTGCAGTACGGAATCTTTCGTATTCATCAGGAAGTCGAGCCGGA AAAAGGCTCTGAAAACGCAGTTATAACCGTTCCCGCAGACCTTTCAGCAGAGGAGCGAGGACGGATA CAGGAAACGGCAAAAAAAATATATAAAGCGCTCGGCTGTAGAGGTCTAGCCCGTGTGGATATGTTTT TACAAGATAACGGCGCCATTGTACAGAGA

16 Appendices SrRNA gene typing: Dendrogram obtained from sample 2 Sequence analysis of sample 2 GGAATGAAACTTCGTCTAGATCATCAACATCGGCGATTATAGACACAGTAAACCTCTGGACT TTTTTTT AAAAATATAGGGCCCCTTAAAAAATTTCTAGAAAATTTTTTACCGCGGCGAGCTGACCCCCC CTTTAAA AACGATTCCGGCTTCATGCTGCC CAAATTGCACCCTGCATTCCGAACTGAGATAGCTTTAAGAG GATTAG CTTAGCCTCGCGACTTCGCAACT TCGTTGTACTTCCCATTGTAGCACGTGTGTAGCCCAGGTCA ATAAGG GGCATGATGATTTGACGTCATCC CCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGT TGCCCAA CTGAATGATGGCAACTAACAATA AAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCAC CGACAC GAGCTGACGACAACCATGCACC CACCTGTCACTTTGCCCCCGAAGGGGAAGCTCTATCTCTAG GAGTGGT CAATGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACC CGCTTG TGCGGGCCCCCGTAATTCCTTTGAGTTTCACCCTTGCAGTCGTACTCCCCAGGCGGAGTGCTT TAATGCT TTACTGCAGGACTGAAGGGCGG GAACCCTCCAACACTTTAGCACTCATCGTTTACGGCGTGGC CCAACGA GGGTATCTAACCCTGTTTGCTCCCACGCTTTTGAGCCTCAGCGGCATTTACTATGCCAGAAGC CGGCCTT CACGCGGGTGTTCGTCCATATATCTACGCATTCAGCGCTGCAGATGGATTCCGATGCTCCTCT TTCTGGA CTCAGTCACCCGAGTTTCCAAGGACCCGCGCGGAGATGAACGGGGCTTTACGAGACAGACTG GATGAA ACTGCTGACTCGCTTTACAGCAG GATAATGCAGAAAACGATTGACTACTAATATATATCGTCG GGGTGCA GTGTACGATATTTACCCGTTGACTCTCGTGATAGTATCCGCCAG

17 230 Dendrogram obtained from sample3 Sequence analysis of sample 3 GTCATAAACGATTTCGTGTGCGTAAATCTCGGTCACAAACTAAAAGGGACCAGAA ATGACTCCTGAAG AGAACTCAAAGAGGGGGGGCCGGGTTCTTTACCCATGAGAAGGGGATACGCTTGC CATTTTGGTGCTA ATACCTTATAACAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTACGGGTGT TCGCTGATGGATG GACCCGCTTGCATTAGCTAGTGGGTGAGGTAACGGCTCACCAAGGCCACGATGCA ATAGCCGACCTGA GAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCATACGGGAGGCA AGCAGTAGGGAA TCTTCGGCAATGTACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTT TTTCGGAGCGGGA AACTTTGTTGGTAGAGAAGAACAGGGTGAGAGTGACTGTCATCCGTTGACGGATC CTAACAAAAAAGC CACAGCTGACTAAGTGCTAACAGCCGCTGTAATACGTAGGTGGAAAGCGTGTTCC CGGATTTATTGGGC GAAAGCAAGGCGAGGGTTTCTTAGTTGATGTGAAGGACCGGTCAACGGGGGAGTG GTTATTGGAGAGT GAAACTTGGGTGCAGAG

18 Appendices 231 Dendrogram obtained from sample k. Sequence analysis of sample K GGATTTTCTTGGCGATGATCACA ACTCTACAACTTATAGAGACACGTAAACACTAGTAAAACT TCTCGTG ATGTGTCGGGCCCGAGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGA ATTACTA GCGATTCCGGCTTCATGCAGGCG GAGTTGCAGCCTGCATCCGAACTGAGAGAAGCTTTAAGAG GATTTGC ATGACCTCGCGGTCTAGCGACTC CGTTGTACTTCCCATTGTAGCACGTGTGTAGCCCAGGTCAT TAAGGG GCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCGCTAGAGTG GCCCAAC TAAATGATGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACG GACACG AGCTGACGACAACCATGCACCA ACCTGTCACTTTGTCCCCGAAGGGAAAGCTCTATCTCTAGA AGTGGTC AAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACC CGCTTGT GCGGGCCCCCGTCAATTCCTTTG GAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTT TAATGC GTTTGCTGCAGCACTGAAGGGCG GGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCGTG GACTACC AGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGGAGCCTCAGCGTCAGTTACAGACAGAGA AGCCGC CTTCGCCACTGGTGTTCCTTCCATTATATCTACGCATTTCACCGCTAACACATGGAATTCCAC CTCTCTC TTCTGCACTCAGGTCTCCAAGTT TCCAAATGACCCTCCCGGTGGAGCCGGGGCTTCACATCAGA ACTAAG AAACGGCCCTGGCGCTTCGCCTT TACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTAT TACCGCG GCCTGCTGACACGTAAGTTAGCC CGTTGCTTCTGGGTAAGATACCGTGCAGGGGACGTTCAGT TACTAAC GTCCTGTGTGCCTTCCTCGACGAGCA

19 232 Dendrogram obtained from sample c1. Sequence analysis of sample C1 TGACGTTAATTCTTATACTTCTCTCGGCTACATTGTGTACGACTTATAGTGAGAGGG GGCTAACTCGGG GGACCTTGTGAGAACTTATAGAGCGGGGGGTAAAACCCCCGGGAACGTATTCAAA AACATCGTGCTGA TCCGCGATTACTAGCGATTCCGGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGA AACTGAGAGAAGC TTTAAGAGATTAGCTTAGCCTCGCGACTTCGCGACTCGTTGTACTTCCCATTGTAGC CACGTGTGTAGCC CAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCA ACCGGCAGTCTTG CTAGAGTGCCCAACTGAATGATGGCAACTAACAATAAGGGTTGCGCTCGTTGCGG GGACTTAACCCAA CATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTTTGCCCCCGAA AGGGGAAGCTCTA TCTCTAGAGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGA AATTAAACCACAT GCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGT TACTCCCCAGGCG GAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAG GCACTCATCGTTTA CGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCCACGCTTTCGAGCCTC CAGCGTCAGTTAC AGACCAGAGAGCCGCTTCGCCACTGGTGTTCCTCCATATATCTACGCATTTCACCG GCTACACATGGAA TTCCACTCTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCCCCGGTTG GAGCCGGGGGCTT TCACATCAGACTTAAGAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACA AACGCTTGCCACC TACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGATACC CGTCAAGGGATGA ACAGTTACTCTCATCCT TTGTTCTTCTCTAACAACAGAGTTTTACGATCCGAAAACCT TTCTTCACTCACG CGGCGTTGCTCGGTCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGCCTCCC CGTAGGAGTTTGG GCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTCGGCTATGCATCGTG GGCCTTGGTGAGC CGTTACCTCACCAACTAGCTAATGCACCGCGGGTCCATCCATCAGCGACACCCGAA AAGCGCCTTTCAA ATCAAAACCATGCGGTTTCGATTGTTATACGGTATTAGCACCTGTTTCCAAGTGTT TATCCCCTTCTGAT GGGCAGGTTACCCACGTGTTACTCTTCCGTTCGCCAGTGCTCGGTATCAAACTGTG GCCGCCACCGTAT AAGATTTGTGAGCCAGTCAACTATAGCGCAAGTAATTTTCCGTATTGG

20 Appendices 233 FACKLAM AND COLLINS IDENTIFICATION SCHEME A. Identification of Group 1 Enterococci Reactions (% positive) for each species E.avium E.raffinosus E.maldoratus E.pseudoavium Arabinose Raffinosus Glucose Inuline Melibiose Trehalose Hippurate Growth at 10 0 C Tellurite tetrazolium VP gp D Ag

21 234 FACKLAM AND COLLINS IDENTIFICATION SCHEME B. Identification of group 2 Enterococci Reactions (% positive ) for each species E.faecalis E.solitarius E.gallinarum E.faecium E.casseliflavus E.mundtii Arabinose Sorbitol Lactose Motility Pigment Glucose Inuline Melibiose Trehalose Hippurate Growth at 10 0 C Tellurite tetrazolium VP gp D Ag

22 Appendices 235 FACKLAM AND COLLINS IDENTIFICATION SCHEME C. Identification of group 3 Enterococci Reactions (% positive ) for each species E.durans E.hirae E.faecalis varient Sucrose Raffinose Pyruvate Glucose Inuline Melibiose Trehalose Hippurate Growth at 10 0 C Tellurite tetrazolium VP gp D Ag