TABLE 4.

Strain composition of the ARDRA clusters standing for selected representatives of the FAME analysisaa

ARDRA cluster or single strainDairy farm isolates in clusterFarm/sample/Temp (°C)bARDRA identificationcStrain selected for 16S rRNA gene sequencing (accession no.)FASTA interpretation (accession no.)dG + C content (mol%)bProposed consensus identification
Clusters
    1R-6640 R-7412 R-7750 R-77514/MI17/55 13/RM14/55 16/RM17/55 16/RM17/55Related to Ureibacillus thermosphaericusR-7750 (AY373318)99.7% Ureibacillus thermosphaericus DSM10633T (AB101594) 99.7% Ureibacillus thermosphaericus P-11T     (X90640)NDUreibacillus thermosphaericus
    2R-6785 R-74287/KV13/37 7/KV21/37No identificationR-7428 (AY422987)95.9% Virgibacillus carmonensis LMG 20946T (AJ316302) 95.7% Virgibacillus necropolis LMG 19488T     (AJ315056)37.4Virgibacillus sp. nov. A
    3R-66784/KV5/55Related to Bacillus smithiiR-7413 (AY422985)95.7% Bacillus methanolicus C1 (X64465)33.8Bacillus sp. nov. B
R-69628/KV15/5595.6% Bacterium LMG 18435 (AJ250318)
R-741313/RM14/5595.2% Bacillus sporothermodurans M215T (U49079)
    4R-65111/MI3/55Bacillus smithiiR-7170 (AY373319)99.7% Bacillus smithii DSM 4216T (Z26935)NDBacillus smithii
R-65151/MI3/5597.0% Bacillus smithii DSM 4216T (X60643)
R-715911/KV18/55
R-717011/RM12/55
R-748414/KV23/55
R-782016/SI34/55
    5R-7409 R-7440 R-7499 R-776414/KV25/55 13/MI53/55 14/SI28/55 16/MI59/55Related to unknown Bacillus sp.R-7499 (AY373320)99.8% uncultured bacterium pPD10 (AF252322) 99.6% uncultured compost bacterium 4-50     (AB034714) 99.0% Bacillus thermoamylovorans CNCM I-    1873T (L27478)36.9Bacillus thermoamylovoranse
    6R-734912/SI21/37Bacillus circulansNDBacillus circulans
R-803114/MI63/37
    7R-64501/MI3/37Bacillus oleroniusNDBacillus oleronius
R-66914/KV8/37
R-777016/MI61/37
    8R-67103/KV4/55Bacillus sporothermoduransNDBacillus sporothermodurans
    9R-64841/MI6/55Related to Bacillus smithiiR-6488 (AY397764)99.6% Bacillus sp. TAT112 (AB066341; E63292)34.3Bacillus sp. nov. C
R-64861/RM1/5599.5% Bacillus sp. TAT105 (AB066342; E63291)
R-64881/RM1/5599.5% Bacillus sp. 115898 (AF071858)
R-6650 R-6739 R-71654/RM4/55 4/MI20/55 11/RM12/5593.7% Bacillus thermoamylovorans CNCM I-1378T (L27478)
    10R-6476 R-6490 R-65031/MI6/55 1/RM1/55 1/MI3/55Unidentified thermophilic Bacillus sp.R-7748 (AY373321)99.9% uncultured Geobacillus sp. Td (AJ564615) 99.7% Bacillus pallidus DSM 3670T (Z26930) 98.6% “Bacillus thermoalkalophilus” DSM 6866     (Z26931)38.8Bacillus pallidusf
R-69849/SI15/55
R-69879/KV16/55
R-717611/RM12/55
R-733912/KV19/55
R-741113/RM14/55
R-774816/RM17/55
    11R-6707 R-74333/KV4/55 13-KV22/55Related to Geobacillus thermoglucosidasius and Geobacillus kaustophilusR-6707 (AY397770)99.0% Geobacillus toebii SK-1T (AF326278) 98.0% Geobacillus thermoglucosidasiusATCC     43742T (AB021197) 98.0% Geobacillus sp. R-7653 (AY397769) 97.9% Geobacillus caldoxylosilyticus S1812T     (AF067651)42.6Geobacillus sp.
R-765315/KV26/55R-7653 (AY397769)99.9% Geobacillus caldoxylosilyticus Tj (AJ564618)43.5Geobacillus sp.
99.5% Geobacillus caldoxylosilyticus S1812T (AF067651)
98.0% Geobacillus toebii SK-1T (AF326278)
98.0% Geobacillus thermoglucosidasius ATCC 43742T (AB021197)
98.0% Geobacillus sp. R-6707 (AY397770)
    12R-64511/MI3/37Bacillus subtilisNDBacillus subtilis groupg
R-67083/KV4/55
R-69367/KV13/37
    13R-67194/KV6/55Bacillus licheniformisNDBacillus licheniformis
    14R-6760 R-77945/RM5/37 16/KV30/37Related to Bacillus galactosidilyticusR-6760 (AJ535639)99.9% low-G+C-content gram-positive bacterium M51 (AB116129) 98.1% Bacillus galactosidilyticus LMG 17892T     (AJ535638)39.2Bacillus sp. nov. Dh
    15R-65211/MI3/55Related to “Bacillus carotarumR-6521 (AY382192)i97.7% Bacillus sp. 112442 JS2 (AF071857)40.8Bacillus sp. nov. Ej
97.7% Bacillus farraginis R-6540T (AY443036)
97.3% Bacillus fordii R-7190T (AY443039)
97.2% Bacillus sp. R-6930 (AY382191)
97.1% Bacillus fortis R-6514T (AY443038)
R-65381/MI3/55R-6538 (AY443035)99.2% Bacillus farraginis R-8039 (AY443034)NDBacillus farraginisk
98.9% Bacillus farraginis R-6540T (AY443036)
    16R-6558 R-71574/KV5/20 11/KV18/37Related to “Bacillus carotarumR-6558 (AY422986)99.7% Bacillus flexus IFO 15715T (AB021185) 98.3% Bacillus megaterium IAM 13418T     (D16273)37.9Bacillus flexus
    17R-6539 R-6540 R-6735 R-7346 R-77891/MI3/55 1/MI3/55 3/SI5/55 12/KV19/37 16/KV30/55Related to “Bacillus carotarumR-6540 (AY443036)99.4% Bacillus farraginis R-6915 (AY443037) 98.9% Bacillus farraginis R-6538 (AY443035) 96.3% Bacillus fortis R-6514T (AY443038)43.7Bacillus farraginisk
R-69157/KV13/37R-6915 (AY443037)99.2% Bacillus farraginis R-6540T (AY443036)NDBacillus farraginisk
R-714811/SI18/5595.8% Bacillus fortis R-6514T (AY443038)
R-734312/KV19/55
    18R-64381/MI3/37No identificationR-7190 (AY443039)99.3% Bacillus sp. 112442 JS2 (AF071857)41.9Bacillus fordiik
R-667414/MI20/3797.7% Bacillus fortis R-6514T (AY443038)
R-719011/RM12/37
R-739913/KV22/37
R-779516/KV30/37
    19R-6470 R-6761 R-67671/RM1/37 5/RM5/37 5/RM5/20No identificationR-6767 (AY422988)99.4% Low G + C Gram-positive bacterium M54 (AB116132) 99.3% Bacillus sp. LMG 19636 (AF329473) 95.3% Virgibacillus picturae LMG 19492T     (AJ3I5060)35.4Virgibacillus sp. nov. F
    20R-6466 R-80301/SI1/37 17/KV31/37Related to Paenibacillus glucanolyticusR-6466 (AY397766)98.8% Paenibacillus sp. LMG 20245 (AJ316315) 94.6% Paenibacillus sp. R-7204 (AY382190) 94.4% Paenibacillus favisporus (GMP01T     (AY208751) 94.3% Paenibacillus cineris LMG 18439T     (AJ575658)NDPaenibacillus sp. nov. G
    21R-6435 R-6472 R-7193 R-71911/MI3/37 1/RM1/37 11/RM12/37 11/RM12/37Related to Paenibacillus lautusR-6472 (AY257870)98.8% Paenibacillus lactis MB 1871T (AY257868) 97.1% Paenibacillus sp. SB45-2B (AF395029) 97.1% Paenibacillus lautus JCM 9073T (AB073188)51.7Paenibacillus lactisl
    22R-7160 R-781811/KV18/55 16/SI34/55Related to Geobacillus thermoglucosidasius and Geobacillus kaustophilusR-7160 (AY397768)99.5% Bacillus sp. E2 (AB089215) 99.3% Geobacillus stearothermophilus BGSC     9A21 (AY297092) 99.1% Bacillus vulcani 3s-1T (AJ293805) 99.0% Geobacillus thermocatenulatus DSM 730T     (Z26926) 99.0% Geobacillus theroleovorans DSM 5366T     (Z26923) 98.7% Geobacillus stearothermophilus DSM 22T     (AJ294817) 97.4% Geobacillus thermodenitnficans DSM 465T     (Z26928)51.0Geobacillus sp.
    23R-6469 R-6547 R-80331/RM1/37 2/SI4/55 17/MI62/55Related to Brevibacillus thermoruberR-6469 (AY397767)98.7% Brevibacillus sp. 96452 (AF227853) 98.5% Brevibacillas brevis DSM 30T (AB101593) 98.0% Brevibacillus formosus NRRL NRS-863T     (D78460)52.7Brevibacillus sp.
    24R-67384/MI20/55Brevibacillus brevisNDBrevibacillus brevis
    25R-6982 R-7201 R-78159/RM10/55 11/RM12/37 16/KV29/37Related to Brevibacillus borstelensisR-7201 (AY373322)99.7% Brevibacillus borstelensis LMG 15536 (AF378230) 99.6% Brevibacilus brostelensin NRRL NRS-818T     (D78456) 97.5% Brevibacillus sp. R-7745 (AY397774) 97.4% Brevibacillus sp. R-6774 (AY422983) 96.9% Brevibacillus invocatus LMG 18962T     (AF378232)51.8Brevibacillus borstelensis
    26R-69568/MI35/37Related to BrevibacillusR-7745 (AY397774)99.1% Brevibacillus sp. R-6774 (AY422983)50.5Brevibacillus sp.
R-715511/KV18/37    agri98.8% Brevibacillus invocatus LMG 18962T (AF378232)
R-7418 R-774513/RM14/37 16/RM17/3797.4% Brevibacillus brevis DSM 30T (AB101593) 97.4% Brevibacillus borstelensis NRRL NRS-    818T (D78456)
97.0% Brevibacillus centrosporus NRRL NRS-664T (D78458)
97.0% Brevibacillus formosus NRRL NRS-863T (D78460)
R-6774 R-70455/MI23/37 10/M140/37R-6774 (AY422983)99.6% Brevibacillus invocatus LMG 18962T (AF378232)49.1Brevibacillus sp.
99.1% Brevibacillus sp. R-7745 (AY397774)
97.4% Brevibacillus borstelensis LMG 15536 (AF378230)
97.1% Brevibacillus brevis DSM 30T (AB101593)
    27R-7032 R-71529/KV16/55 11/KV18/37Related to Brevibacillus choshinensisR-7152 (AY397773)96.4% Brevibacillus borstelensis LMG 15536 (AF378230)52.0Brevibacillus sp. nov. H
96.2% Brevibacillus sp. R-7745 (AY397774)
96.2% Brevibacillus thermoruber DSM 7064T (Z26921)
    28R-6958 R-74018/MI35/37 13/KV22/37Aneurinibacillus aneurinilyticusNDAneurinibacillus aneurinilyticus
    29R-7443 R-763713/KV21/55 15/RM16/55Aneurinibacillus thermoaerophilusNDAneurinibacillus thermoaerophilus
    30R-6638 R-67623/RM3/37 5/RM5/37Virgibacillus proomiiR-6762 (AY373323)98.8% Virgibacillus proomii LMG 12370T (AJ012667) 96.9% Virgibacillus pantothenticus IAM 11061T (D16275)36.4Virgibacillus proomii
Single strains
    R-64431/MI3/37Paenibacillus thiaminolyticusNDPaenibacillus thiaminolyticus
    R-65071/MI3/55No identificationR-6507 (AY397765)99.4% bacterium strain 31293 (AF227859)57.9Paenibacillus sp. nov. I
99.3% bacterium strain 31295 (AF227835)
92.1% Paenibacillus azoreducens CM1T (AJ272249)
    R-67827/RM8/37No identificationR-6782 (AY422984)99.4% low G + C gram-positive bacterium HTA437 (AB002642)43.9Bacillus barbaricus
99.3% low G + C gram-positive bacterium HTA506 (AB002643)
97.3% Bacillus barbaricus VII-B3-A2T (AJ422145)
    R-69287/KV13/37No identificationR-6928 (AY397772)99.0% Paenibacillus sp. MB 2039 (AY257871)NDPaenibacillus sp. nov. J
92.8% Paenibacillus thiaminolyticus IFO 15656T (AB073197)
92.7% Paenibacillus larvae subsp. pulvifaciens DSM 3615T (AB073204)
92.7% Paenibacillus larvae subsp. larvae DSM 7030T (AB073205) 92.7% Paenibacillus popilliae ATCC 14706T     (AB073198)
    R-69307/SI12/37Related to Bacillus coagulansR-6930 (AY382191)97.5% Bacillus fortis R-6514T (AY443038) 97.2% low G + C gram-positive bacterium M52     (AB116130)NDBacillus sp. nov. K
    R-72041/RM12/37No identificationR-7204 (AY382190)95.7% Paenibacillus sp. LMG 20245 (AJ316315)NDPaenibacillus sp. nov. L
94.5% Paenibacillus daejeonensis AM141T     (AF391124)
94.4% Paenibacillus illinoisensis JCM 9907T     (AB073192)
    R-748715/RM16/37Related to Paenibacillus maceransR-7487i     (AY397771)96.4% Paenibacillus macerans IAM 12467T     (AB073196)NDPaenibacillus sp. nov. M
93.7% Paenibacillus lautus JCM 9073T     (AB073188)
    R-765215/KV26/55No identificationR-7652 (AY382189)99.3% uncultured synthetic wastewater bacterium     tmbr11-6 (AF309811)55.0Paenibacillus sp. nov. N
98.6% uncultured synthetic wastewater bacterium     tmbr11-29 (AF309809)
94.4% Paenibacillus kobensis DSM 10249T     (AB073363)
93.9% Bacillus chitinolyticus IFO 15660T     (AB021183)
  • a The dairy farm isolates in clusters 8, 12, 13, and 24 grouped together with the type strains B. sporothermodurans LMG 17894T, B. subtilis subsp. subtilis LMG 7135T, B. licheniformis LMG 12363T, and Brevibacillus brevis LMG 16703T, respectively, simultaneously analyzed in this study. When appropriate, the FASTA search interpretation of the determined 16S rRNA gene sequence is given. The proposed identification is given in the last column and is further discussed in the text.

  • b Abbreviations: KV, cattle feed (fodder); MI, milking installation; RM, raw milk; SI, silage (green crop); ND, not determined; Temp, isolation temperature.

  • c Similarity coefficients of at least 90% were considered an acceptable ARDRA identification (47). Values between 80 and 90% or below 80% are represented as “related to” or “no identification,” respectively.

  • d The highest similarity is mentioned, as are relevant scores higher or equal to 97%. Values lower than 97% are given only when they are either the highest score itself or the highest scores with type strains of recognized species.

  • e A DNA-DNA reassociation value of 90% was found between strains R-7499 and B. thermoamylovorans LMG 18084T.

  • f A DNA-DNA reassociation value of 85% was found between strains R-7748 and B. pallidus LMG 19006T.

  • g The phylogenetic B. subtilis group contains the following species: B. atrophaeus, B. amyloliquefaciens, B. mojavensis, B. subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. vallismortis.

  • h A DNA-DNA reassociation value of 33% was found between strains R-6760 and B. galactosidilyticus LMG 17892T (19).

  • i For strains R-6521 and R-7487, only a smaller stretch of 950 and 813 bp, respectively, of 16S rRNA gene sequence was obtained.

  • j An extended polyphasic study excluded R-6521 from the species B. farraginis, B. fortis, and B. fordii (38).

  • k The new species B. farraginis, B. fortis, and B. fordii were described in the course of this study (38).

  • l P. lactis was described as a new species following a polyphasic study including these strains (36).