TABLE 4.
Strain composition of the ARDRA clusters standing for selected representatives of the FAME analysisaa
| ARDRA cluster or single strain | Dairy farm isolates in cluster | Farm/sample/Temp (°C)b | ARDRA identificationc | Strain selected for 16S rRNA gene sequencing (accession no.) | FASTA interpretation (accession no.)d | G + C content (mol%)b | Proposed consensus identification |
|---|---|---|---|---|---|---|---|
| Clusters | |||||||
| 1 | R-6640 R-7412 R-7750 R-7751 | 4/MI17/55 13/RM14/55 16/RM17/55 16/RM17/55 | Related to Ureibacillus thermosphaericus | R-7750 (AY373318) | 99.7% Ureibacillus thermosphaericus DSM10633T (AB101594) 99.7% Ureibacillus thermosphaericus P-11T (X90640) | ND | Ureibacillus thermosphaericus |
| 2 | R-6785 R-7428 | 7/KV13/37 7/KV21/37 | No identification | R-7428 (AY422987) | 95.9% Virgibacillus carmonensis LMG 20946T (AJ316302) 95.7% Virgibacillus necropolis LMG 19488T (AJ315056) | 37.4 | Virgibacillus sp. nov. A |
| 3 | R-6678 | 4/KV5/55 | Related to Bacillus smithii | R-7413 (AY422985) | 95.7% Bacillus methanolicus C1 (X64465) | 33.8 | Bacillus sp. nov. B |
| R-6962 | 8/KV15/55 | 95.6% Bacterium LMG 18435 (AJ250318) | |||||
| R-7413 | 13/RM14/55 | 95.2% Bacillus sporothermodurans M215T (U49079) | |||||
| 4 | R-6511 | 1/MI3/55 | Bacillus smithii | R-7170 (AY373319) | 99.7% Bacillus smithii DSM 4216T (Z26935) | ND | Bacillus smithii |
| R-6515 | 1/MI3/55 | 97.0% Bacillus smithii DSM 4216T (X60643) | |||||
| R-7159 | 11/KV18/55 | ||||||
| R-7170 | 11/RM12/55 | ||||||
| R-7484 | 14/KV23/55 | ||||||
| R-7820 | 16/SI34/55 | ||||||
| 5 | R-7409 R-7440 R-7499 R-7764 | 14/KV25/55 13/MI53/55 14/SI28/55 16/MI59/55 | Related 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) | 36.9 | Bacillus thermoamylovoranse |
| 6 | R-7349 | 12/SI21/37 | Bacillus circulans | ND | Bacillus circulans | ||
| R-8031 | 14/MI63/37 | ||||||
| 7 | R-6450 | 1/MI3/37 | Bacillus oleronius | ND | Bacillus oleronius | ||
| R-6691 | 4/KV8/37 | ||||||
| R-7770 | 16/MI61/37 | ||||||
| 8 | R-6710 | 3/KV4/55 | Bacillus sporothermodurans | ND | Bacillus sporothermodurans | ||
| 9 | R-6484 | 1/MI6/55 | Related to Bacillus smithii | R-6488 (AY397764) | 99.6% Bacillus sp. TAT112 (AB066341; E63292) | 34.3 | Bacillus sp. nov. C |
| R-6486 | 1/RM1/55 | 99.5% Bacillus sp. TAT105 (AB066342; E63291) | |||||
| R-6488 | 1/RM1/55 | 99.5% Bacillus sp. 115898 (AF071858) | |||||
| R-6650 R-6739 R-7165 | 4/RM4/55 4/MI20/55 11/RM12/55 | 93.7% Bacillus thermoamylovorans CNCM I-1378T (L27478) | |||||
| 10 | R-6476 R-6490 R-6503 | 1/MI6/55 1/RM1/55 1/MI3/55 | Unidentified 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) | 38.8 | Bacillus pallidusf |
| R-6984 | 9/SI15/55 | ||||||
| R-6987 | 9/KV16/55 | ||||||
| R-7176 | 11/RM12/55 | ||||||
| R-7339 | 12/KV19/55 | ||||||
| R-7411 | 13/RM14/55 | ||||||
| R-7748 | 16/RM17/55 | ||||||
| 11 | R-6707 R-7433 | 3/KV4/55 13-KV22/55 | Related to Geobacillus thermoglucosidasius and Geobacillus kaustophilus | R-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) | 42.6 | Geobacillus sp. |
| R-7653 | 15/KV26/55 | R-7653 (AY397769) | 99.9% Geobacillus caldoxylosilyticus Tj (AJ564618) | 43.5 | Geobacillus 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) | |||||||
| 12 | R-6451 | 1/MI3/37 | Bacillus subtilis | ND | Bacillus subtilis groupg | ||
| R-6708 | 3/KV4/55 | ||||||
| R-6936 | 7/KV13/37 | ||||||
| 13 | R-6719 | 4/KV6/55 | Bacillus licheniformis | ND | Bacillus licheniformis | ||
| 14 | R-6760 R-7794 | 5/RM5/37 16/KV30/37 | Related to Bacillus galactosidilyticus | R-6760 (AJ535639) | 99.9% low-G+C-content gram-positive bacterium M51 (AB116129) 98.1% Bacillus galactosidilyticus LMG 17892T (AJ535638) | 39.2 | Bacillus sp. nov. Dh |
| 15 | R-6521 | 1/MI3/55 | Related to “Bacillus carotarum” | R-6521 (AY382192)i | 97.7% Bacillus sp. 112442 JS2 (AF071857) | 40.8 | Bacillus 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-6538 | 1/MI3/55 | R-6538 (AY443035) | 99.2% Bacillus farraginis R-8039 (AY443034) | ND | Bacillus farraginisk | ||
| 98.9% Bacillus farraginis R-6540T (AY443036) | |||||||
| 16 | R-6558 R-7157 | 4/KV5/20 11/KV18/37 | Related to “Bacillus carotarum” | R-6558 (AY422986) | 99.7% Bacillus flexus IFO 15715T (AB021185) 98.3% Bacillus megaterium IAM 13418T (D16273) | 37.9 | Bacillus flexus |
| 17 | R-6539 R-6540 R-6735 R-7346 R-7789 | 1/MI3/55 1/MI3/55 3/SI5/55 12/KV19/37 16/KV30/55 | Related to “Bacillus carotarum” | R-6540 (AY443036) | 99.4% Bacillus farraginis R-6915 (AY443037) 98.9% Bacillus farraginis R-6538 (AY443035) 96.3% Bacillus fortis R-6514T (AY443038) | 43.7 | Bacillus farraginisk |
| R-6915 | 7/KV13/37 | R-6915 (AY443037) | 99.2% Bacillus farraginis R-6540T (AY443036) | ND | Bacillus farraginisk | ||
| R-7148 | 11/SI18/55 | 95.8% Bacillus fortis R-6514T (AY443038) | |||||
| R-7343 | 12/KV19/55 | ||||||
| 18 | R-6438 | 1/MI3/37 | No identification | R-7190 (AY443039) | 99.3% Bacillus sp. 112442 JS2 (AF071857) | 41.9 | Bacillus fordiik |
| R-6674 | 14/MI20/37 | 97.7% Bacillus fortis R-6514T (AY443038) | |||||
| R-7190 | 11/RM12/37 | ||||||
| R-7399 | 13/KV22/37 | ||||||
| R-7795 | 16/KV30/37 | ||||||
| 19 | R-6470 R-6761 R-6767 | 1/RM1/37 5/RM5/37 5/RM5/20 | No identification | R-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) | 35.4 | Virgibacillus sp. nov. F |
| 20 | R-6466 R-8030 | 1/SI1/37 17/KV31/37 | Related to Paenibacillus glucanolyticus | R-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) | ND | Paenibacillus sp. nov. G |
| 21 | R-6435 R-6472 R-7193 R-7191 | 1/MI3/37 1/RM1/37 11/RM12/37 11/RM12/37 | Related to Paenibacillus lautus | R-6472 (AY257870) | 98.8% Paenibacillus lactis MB 1871T (AY257868) 97.1% Paenibacillus sp. SB45-2B (AF395029) 97.1% Paenibacillus lautus JCM 9073T (AB073188) | 51.7 | Paenibacillus lactisl |
| 22 | R-7160 R-7818 | 11/KV18/55 16/SI34/55 | Related to Geobacillus thermoglucosidasius and Geobacillus kaustophilus | R-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) | 51.0 | Geobacillus sp. |
| 23 | R-6469 R-6547 R-8033 | 1/RM1/37 2/SI4/55 17/MI62/55 | Related to Brevibacillus thermoruber | R-6469 (AY397767) | 98.7% Brevibacillus sp. 96452 (AF227853) 98.5% Brevibacillas brevis DSM 30T (AB101593) 98.0% Brevibacillus formosus NRRL NRS-863T (D78460) | 52.7 | Brevibacillus sp. |
| 24 | R-6738 | 4/MI20/55 | Brevibacillus brevis | ND | Brevibacillus brevis | ||
| 25 | R-6982 R-7201 R-7815 | 9/RM10/55 11/RM12/37 16/KV29/37 | Related to Brevibacillus borstelensis | R-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) | 51.8 | Brevibacillus borstelensis |
| 26 | R-6956 | 8/MI35/37 | Related to Brevibacillus | R-7745 (AY397774) | 99.1% Brevibacillus sp. R-6774 (AY422983) | 50.5 | Brevibacillus sp. |
| R-7155 | 11/KV18/37 | agri | 98.8% Brevibacillus invocatus LMG 18962T (AF378232) | ||||
| R-7418 R-7745 | 13/RM14/37 16/RM17/37 | 97.4% 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-7045 | 5/MI23/37 10/M140/37 | R-6774 (AY422983) | 99.6% Brevibacillus invocatus LMG 18962T (AF378232) | 49.1 | Brevibacillus sp. | ||
| 99.1% Brevibacillus sp. R-7745 (AY397774) | |||||||
| 97.4% Brevibacillus borstelensis LMG 15536 (AF378230) | |||||||
| 97.1% Brevibacillus brevis DSM 30T (AB101593) | |||||||
| 27 | R-7032 R-7152 | 9/KV16/55 11/KV18/37 | Related to Brevibacillus choshinensis | R-7152 (AY397773) | 96.4% Brevibacillus borstelensis LMG 15536 (AF378230) | 52.0 | Brevibacillus sp. nov. H |
| 96.2% Brevibacillus sp. R-7745 (AY397774) | |||||||
| 96.2% Brevibacillus thermoruber DSM 7064T (Z26921) | |||||||
| 28 | R-6958 R-7401 | 8/MI35/37 13/KV22/37 | Aneurinibacillus aneurinilyticus | ND | Aneurinibacillus aneurinilyticus | ||
| 29 | R-7443 R-7637 | 13/KV21/55 15/RM16/55 | Aneurinibacillus thermoaerophilus | ND | Aneurinibacillus thermoaerophilus | ||
| 30 | R-6638 R-6762 | 3/RM3/37 5/RM5/37 | Virgibacillus proomii | R-6762 (AY373323) | 98.8% Virgibacillus proomii LMG 12370T (AJ012667) 96.9% Virgibacillus pantothenticus IAM 11061T (D16275) | 36.4 | Virgibacillus proomii |
| Single strains | |||||||
| R-6443 | 1/MI3/37 | Paenibacillus thiaminolyticus | ND | Paenibacillus thiaminolyticus | |||
| R-6507 | 1/MI3/55 | No identification | R-6507 (AY397765) | 99.4% bacterium strain 31293 (AF227859) | 57.9 | Paenibacillus sp. nov. I | |
| 99.3% bacterium strain 31295 (AF227835) | |||||||
| 92.1% Paenibacillus azoreducens CM1T (AJ272249) | |||||||
| R-6782 | 7/RM8/37 | No identification | R-6782 (AY422984) | 99.4% low G + C gram-positive bacterium HTA437 (AB002642) | 43.9 | Bacillus barbaricus | |
| 99.3% low G + C gram-positive bacterium HTA506 (AB002643) | |||||||
| 97.3% Bacillus barbaricus VII-B3-A2T (AJ422145) | |||||||
| R-6928 | 7/KV13/37 | No identification | R-6928 (AY397772) | 99.0% Paenibacillus sp. MB 2039 (AY257871) | ND | Paenibacillus 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-6930 | 7/SI12/37 | Related to Bacillus coagulans | R-6930 (AY382191) | 97.5% Bacillus fortis R-6514T (AY443038) 97.2% low G + C gram-positive bacterium M52 (AB116130) | ND | Bacillus sp. nov. K | |
| R-7204 | 1/RM12/37 | No identification | R-7204 (AY382190) | 95.7% Paenibacillus sp. LMG 20245 (AJ316315) | ND | Paenibacillus sp. nov. L | |
| 94.5% Paenibacillus daejeonensis AM141T (AF391124) | |||||||
| 94.4% Paenibacillus illinoisensis JCM 9907T (AB073192) | |||||||
| R-7487 | 15/RM16/37 | Related to Paenibacillus macerans | R-7487i (AY397771) | 96.4% Paenibacillus macerans IAM 12467T (AB073196) | ND | Paenibacillus sp. nov. M | |
| 93.7% Paenibacillus lautus JCM 9073T (AB073188) | |||||||
| R-7652 | 15/KV26/55 | No identification | R-7652 (AY382189) | 99.3% uncultured synthetic wastewater bacterium tmbr11-6 (AF309811) | 55.0 | Paenibacillus 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).