Phylogenetic analysis and characterization of bacterial sporeformer isolates
obtained from raw milk, pasteurized milk, and dairy farm environments
45569
Milk Quality Improvement Program
200 Stocking Hall
Ithaca, NY 14853
ph: 607-255-2894
rai6@cornell.edu
f: 607-254-4868
R. A. Ivy, M. L. Ranieri, N. H. Martin, H. den Bakker, B. M. Xavier, M. Wiedmann, and K. J. Boor
61
AT024
AT254
AT205
B.safensis
AT141
AT258
AT251
73
AT143
100
AT222
83
AT230
AT252
72
AT210
B.pumilis3
64
AT228
AT224
B.pumilis2
AT253
AT068
AT062
AT072
AT137
76
AT047
B.pumilis1
AT175
98
AT054
92
AT144
AT020
61
AT206
AT219
AT245
AT161
AT182
AT218
A - Bacillus
AT236
AT065
AT267
AT221
72
AT243
9 4 AT231
AT262
AT211
98
AT249
AT066
AT142
AT212
83
AT237
AT213
82
AT217
AT229
97
99
B - Paenibacillus
N= 108
AT050
AT098
AT079
AT016
AT002
98
AT001
AT006
AT009
AT173
91
AT215
AT208
AT169
AT227
AT282
AT269
AT271
100
Bacillus s.l.
73
N= 188
B.licheniformiss.l.2
63
The objective of this study was to identify prominent dairy-associated psychrotolerant sporeformers by:
1. Using rpoB sequencing to systematically identify prominent clades of dairy-associated sporeformers
2. Determining relevant phenotypic characteristics (i.e., growth in milk during refrigerated storage and galactosidase activity) for representatives of prominent clades
67
100
76
100
91
96
100
74
99
99
90
68
AT073
AT017
AT165
AT089
N= 46
AT183
AT087
85
100
0
N= 55
74
III
AT166
66
AT197
AT104
AT168
AT109
87
AT202
AT163
61
N= 27
90
AT008
84
AT159
100
62
AT139
AT160
92
21
P. graminis 2
12
P. lautus
P. glucanolyticus
P. rhizosphaerae
AT111
Paenibacillus sp. 7
AT239
8
AT076
AT105
P. cf. peoriae
AT203
100
AT23
Paenibacillus sp. 6
AT067
N= 24
AT179
IV
IX
63
B.muralis
AT057
AT014
B. psychrosachharalyticus
AT101
P.aenibacillus sp. 8
AT113
AT048
AT274
B.clausii
AT093
AT189
AT184
60
N= 5
AT083
AT45
AT112
P. amylolyticus s.l.
N= 101
AT080
AT102
AT233
X
2
AT022
V
0
AT264
93
0
AT198
AT100
62
85
74
AT275
AT259
P. xylanilyticus s.l.
Paenibacillus sp. 9
Paenibacillus sp. 10
AT005
-2
AT095
100
AT094
N= 46
AT058
97
AT156
AT149
Paenibacillus
3
7
10
14
17
21
N= 12
AT276
AT192
Viridibacillus spp.
P. castanae
Paenibacillus sp. 11
N= 7
Time at
6o
72
H7-594
-
30
141
H8-512
-
Bacillus subtilis
17
65
H7-432
-
Bacillus weihenstephanensis
50
3
F4-079
-
Viridibacillus spp.
46
17
F4-143
-
Paenibacillus amylolyticus s.l.
96
23
F4-561
+
Paenibacillus cf. peoriae
24
157
H8-551
wp
Paenibacillus graminis 1
23
45
H3-335
+
Paenibacillus graminis 2
23
39
H8-025
+
Paenibacillus odorifer 1
463
15
F4-129
+
Paenibacillus odorifer 3
36
260
R5-738
+
Paenibacillus c.f. xylanilyticus
11
100
H8-287
+
•
Identification of Paenibacillus-specific targets will facilitate
the development of technologies to prevent the
introduction of psychrotolerant sporeformers into
pasteurized milk
C (day)
XI
Paenibacillus sp. 12
0.1
Figure 1. Maximum likelihood phylogenetic tree of rpoB allelic types (AT) of Bacillus spp. (A) and Paenibacillus spp. (B) isolated from pasteurized milk (red wedge) raw milk
(blue wedge) and dairy farm environments (green wedge). Numerical node labels values represent the percentage of bootstrap replications that supported the respective
node. Only bootstrap values greater than 60 are shown. Species IDs are based on 16S sequencing. The sensu lato (s.l.) notation is used for clades that contained isolates
matching (i.e. > 98% sequence similarity) more than one type strain 16S sequence. The confer (cf.) designation is used to denote isolates that are similar (97-98% sequence
similarity) to more than one 16S type sequence.
Alignment, tree construction, and species identification. rpoB sequences were trimmed and
aligned. An rpoB maximum likelihood (ML) phylogenetic tree was constructed using the rapid
maximum likelihood algorithm RAxML with rapid bootstrapping (100 bootstrap replicates). For
species identification, partial 16S rDNA sequences obtained for each unique rpoB allelic type were
compared against type-strain 16S rDNA sequences using the “Seqmatch” function the Ribosomal
Database Project (RDP) database (http://rdp.cme.msu.edu/).
Cold Growth. A single colony was inoculated into 5 ml of BHI broth. Serial dilutions of the culture
were performed and 1 mL was transferred into 9 mL of sterile Skim Milk Broth (SMB) for a final
inoculum level of ~102 CFU/ml. SMB samples were plated immediately and after 6, 10, 13 17,
20, and 24 days of incubation at 6°C.
-galactosidase (-gal) activity. Cultures were streaked onto BHI agar with and without an overlay
of 100 l of a solution of bromo-chloro-indolyl-galactopyranoside (X-gal; 40 g/ml) and incubated
at 32C for 24 h. Blue colonies on the plates containing X-gal were indicative that the isolate was
positive for -galactosidase activity.
52
Bacillus safensis
β-galactosidase activity can be used as a preliminary screen
for Paenibacillus, but cannot reliably discriminate
Paenibacillus from Bacillus.
0.1
Materials and Methods
Bacillus pumilis 1
AT100
AT250
AT114
AT111
AT261
100
+/-
•
AT260
AT028
AT023
N= 9
-
F4-073
Though both Bacillus spp. and Paenibacillus spp. are
commonly isolated from dairy systems, a small number of
Paenibacillus species account for the majority of
psychrotolerant sporeformers in pasteurized milk
AT15
AT049
91
H8-493
1
•
AT39
4
AT099
AT029
135
AT157
6
AT108
N= 9
24
181
AT170
AT157
AT086
AT288 Bacillussp. 5
AT191 B. circulans
Bacillus aerophilus s.l.
Bacillus licheniformis s.l. 1
Conclusions
AT159
AT199
Bacillussp. 4
β-gal activityb
P. macerans
AT187
AT266
B - Paenibacillus
10
VIII
P. lactis
AT214
99
FSL ID a
Cornell Food Safety Lab isolate identifiation. Additional isolate information available at
www.pathogentracker.net
b Indicates whether every representative isolate tested from this group was positive (+) or negative (-) for
β-gal activity or whether the group had both positive and negative representatives (+/-) or had at least one
isolate that showed weakly positive (wp) activity
Paenibacillus sp. 4
83
N= 8
Representative AT
a
Paenibacillus sp. 3
AT138 P. cookii
AT238
83
AT150
Bacilluss.l. sp. 1
9 9 AT034
AT071
AT171
Lysinibacillusspp.
AT177
AT188
AT136
AT053 Bacilluss.l. sp. 2
AT242
Bacilluss.l. sp. 3
AT162
AT133
AT286
100
AT119
100
AT147
99
AT283
Psychrobacillusspp.
AT285
8 8 AT280
AT284
AT130
100
AT131
AT152 Paenisporosarcina quisquiliarum
17
Paenibacillus sp. 5
AT117
B.cf.horikoshii
AT256
AT226
Solibacillusspp.
AT220
AT126
AT145
Bacilluscf. cecembensis
AT146
14
Paenibacillus. sp. 2
AT204
AT193
Oceanobacillussp.
10
Time at 6o C (day)
-2
AT200
100
AT064 Bacillussp. 2
AT153 Bacillussp. 3
AT209
AT118
B. cf. firmus
AT247
AT121
7
VII
AT060
AT185
100
100
Bacillussp. 1
3
AT039
100
100
AT196
AT096
61
99
AT270 B. barbaricus
AT225 B. gibsonii
AT055
86
AT216
AT287
100
0
P. graminis 1
AT195
63
87
98
100
AT272
AT279
AT281
100
AT178
AT103
70
AT051
AT084
97
AT17
AT044
AT045
Bacillussp. 6
AT268
2
N= 8
Paenibacillus sp. 1
AT3
6 4 AT201
66
B.licheniformiss.l.3
B.subtiliss.l.4
100
69
AT141
AT186
6 2 AT134
6 4 AT132
AT257
AT090
AT003
B.weihenstephanensis
AT075
88
AT273
AT097
97
AT128
B.mycoides
AT148
AT129
AT061
AT246
73
AT194
B. cereuss.l. 1
AT059
AT120
60
AT125
7 7 AT158
AT092
AT277
B.cereuss.l. 2
AT154
AT278
86
4
AT042
AT052
B.licheniformiss.l.1
AT056 B. cf. farraginis
Objectives
AT077
AT063
79
89
AT20
AT164
AT041
88
II
91
100
AT1
AT107
100
100
6
AT035
B.subtiliss.l.3
AT116
AT244
AT207
AT223
AT240
AT241
AT1
N= 463
AT038
N= 23
AT158
8
P. odorifer 1
AT040
AT174
According to the International Dairy Foods Association, the U.S. per annum fluid milk purchases total 6
billion gallons (6). As much as 20% of this is discarded prior to consumption, due in part to microbial
spoilage (7). Psychrotolerant, or “cold-thriving”, Gram-positive sporeformers have the potential to
survive conventional pasteurization and can grow during refrigerated storage, resulting in off flavors and
curdling in the final product.
Bacillus and Paenibacillus have been identified as the prominent genera of Gram-positive sporeformers
in pasteurized fluid milk (9). While both Bacillus spp. and Paenibacillus spp. have been traced from dairy
farm environments, through processing systems, to pasteurized milk (9), Bacillus spp. are predominantly
detected early during the shelf-life of pasteurized milk, whereas Paenibacillus has been shown to
predominate late in shelf-life (9). Therefore, though both species are present directly after processing,
Paenibacillus spp. are likely to predominate in refrigerated pasteurized fluid milk. Currently, due to a lack
of information on the population structure and genotypes of dairy-associated sporeformers, no methods
exist to differentiate Bacillus from Paenibacillus.
AT115
AT010
AT036
B.subtiliss.l. 1
B.megaterium
81
VI
AT180
AT004
AT265
AT033
AT011
66
AT046
AT088
74
AT025
AT032
AT031
99
AT135
AT015
AT007
96
99
Introduction
Isolate collection and selection. Gram-positive sporeformer isolates were collected from
several studies, which employed standard methods for the examination of dairy products,
(3-5, 8, 10). Spore treatments (80C for 12 min) were conducted to eliminate vegetative
cells. Colonies representing each visually distinct morphology were streaked for isolation on
BHI agar. 1288 total isolates were cataloged.
rpoB sequencing. Molecular subtyping of all isolates was performed based on the DNA
sequence for a 632-nucleotide (nt) fragment (nt 2455 to 3086) of the rpoB gene. Briefly, the
rpoB fragment was amplified using PCR primers that were previously described (1) and PCR
conditions detailed by Durak et al. (2). rpoB PCR products were purified using the QIAquick
PCR Purification Kit and sequencing was performed at Cornell University’s Life Sciences Core
Laboratory Center (Ithaca, N.Y.).
AT assignment. A unique rpoB allelic type (AT) was assigned to a gene sequence that
differed from any previously obtained sequence by one or more nucleotides. Isolates with
different ATs were considered to represent different subtypes. 283 unique rpoB ATs were
identified.
AT021
83
97
100
10
AT181
I
AT074
AT019
B.subtiliss.l. 2
# Isolates
AT091
AT027
74
91
100
N= 7
AT013
AT018
90
AT248
AT255
AT085
AT151
P. odorifer 2
AT012
Clade ID
A - Bacillus
12
AT110
AT082
AT081
100
94
88
AT030
AT167
90
64
84
Table 1. Frequency of isolation and β-galactosidase activity of rpoB clades isolated more than ten times
N= 36
P. odorifer 3
AT026
B.aerophiluss.l.
AT176
AT190
AT172
AT135
AT069
AT232
AT234
AT260
AT078
Log (cfu/ml)
The presence of psychrotolerant endospore-forming bacteria represents a major challenge to extending
the shelf life of pasteurized dairy products. The objective of this study was to identify prominent
phylogenetic groups of dairy-associated aerobic sporeformers (i.e., isolates from raw and pasteurized
milk, and dairy farm environments) and characterize representative isolates for phenotypes relevant to
cold growth in milk. All isolates (n = 1288) were classified within the family Bacillaceae. Frequently
isolated clades consisted of Bacillus spp. (n = 467; e.g., B. licheniformis s.l., B. pumilis, and B.
weihenstephanensis), genera formerly classified as Bacillus (n = 84; e.g., Viridibacillus spp.) and
Paenibacillus spp. (n = 737; e.g., P. odorifer, P. graminis, and P. amylolyticus). Only two out of nine
isolates representing prominent non-Paenibacillus subtypes [as determined by rpoB allelic typing (AT)]
grew to > 4 log(CFU/ml) in skim milk broth (SMB) at 6C, whereas all but two out of Paenibacillus
representative isolates grew to > 4 log(CFU/ml) in SMB at 6C. Though most Paenibacillus isolates were
positive for β-galactosidase activity at 32°C and most non-Paenibacillus were negative, isolates
representative of Bacillus licheniformis s.l. AT1 (13% of non-Paenibacillus isolates) showed varying βgalactosidase activity. Therefore β-galactosidase activity alone cannot reliably distinguish Paenibacillus
from other Bacillaceae. Our study confirmed that Paenibacillus spp. are the predominant psychrotolerant
sporeformers in fluid milk and provided molecular subtype organization and phenotypic characteristics of
prominent clades of aerobic sporeformers. This study, thus, contributes to the understanding of fluid
milk bacterial ecology and will facilitate the development of sporeformer prevention methods aimed at
extending the shelf life of pasteurized dairy foods.
AT263
62
63
AT070
AT124
AT140
AT106
AT122
AT235
Log (cfu/ml)
Abstract*
Figure 2. Growth of isolates representing prominent rpoB allelic types of non-Paenibacillus
Bacillaceae (a) and Paenibacillus (b) in skim milk broth at 6C. Bacillus clades tested were B.
licheniformis s.l. 1 (AT001), B. weihenstephanensis (AT003), Viridibacillus spp. (AT17), B. pumilis
1(AT20), B. aerophilus s.l. (AT135), B. safensis (AT141), and B. cereus s.l. 1 (AT158). Paenibacillus
clades tested were P. odorifer 1 (AT15), P. amylolyticus s.l. (AT23 and AT111), P. graminis 2 (AT39),
P. graminis 1 (AT45), P. xylanilyticus s.l. (AT100), P. cf. peoriae (AT157), P. lautus (AT159), and P.
odorifer 3 (AT260).
Results and Discussion
Prominent dairy-associated Bacillus clades. B. pumilis (Group I) B.
licheniformis s.l. (Group II), B. cereus s.l., or B. weihenstephanensis (Both in
Group III), together, represented 337 out of 685 (49%) of Bacillus isolates in
our study (Fig 1a). Group IV consists of isolates identified as belonging to
genera formerly classified as Bacillus (i.e., Bacillus sensu lato), including
Viridibacillus spp., which accounted for 46 isolates (Fig 1a).
Representatives from prominent Paenibacillus clades grow in milk during
refrigeration, whereas, with the exception of B. weihenstephanensis,
representatives from prominent Bacillus clades do not. Representative isolates
of the B. weihenstephanensis [allelic type (AT) 3], and Viridibacillus (AT17) were
the only non-Paenibacillus isolates to reach > 4.0 log CFU/ml by 21 d (Fig 2a),
while all but two representative Paenibacillus isolates (AT17 and AT100) reached
4.0 log CFU/ml by 21 d (Fig 2b).
Prominent dairy associated Paenibacillus clades. Within Paenibacillus
isolates (n = 737), six major clades, each consisting of a single species ID
accounted for 677 (92%) of Paenibacillus isolates. These clades are P. odorifer
1-3, P. graminis, P. cf. peoriae and P. amylolyticus s.l. Therefore, a relatively
small number of species and clades represent the majority of dairyassociated aerobic sporeformers.
Most Paenibacillus isolates were positive for β-gal activity, whereas most
Bacillus clades were not. β-gal is required for the metabolism of the milk
carbohydrate lactose. In general, representative isolates of Bacillus spp. were
negative for β-gal activity, whereas representative Paenibacillus isolates were
positive (Table 1). However, representatives of the most frequently isolated
Bacillus species (B. licheniformis) showed varying activity (Table1).
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Acknowledgements
The contributions of the staff of the Milk Quality Improvement Program (MQIP) at Cornell University are
acknowledged. This work is supported by the New York State Milk Promotion Advisory Board through the
New York State Department of Agriculture and New York State dairy farmers