Multilocus sequence type profiles of Bacillus cereus isolates from infant formula in China

Accepted Manuscript Multilocus sequence type profiles of Bacillus cereus isolates from infant formula in China Yong Yang, Xiaofeng Yu, Li Zhan, Jiancai Chen, Yunyi Zhang, Junyan Zhang, Honghu Chen, Zheng Zhang, Yanjun Zhang, Yiyu Lu, Lingling Mei PII:

S0740-0020(16)30181-2

DOI:

10.1016/j.fm.2016.09.007

Reference:

YFMIC 2616

To appear in:

Food Microbiology

Received Date: 7 March 2016 Revised Date:

9 September 2016

Accepted Date: 11 September 2016

Please cite this article as: Yang, Y., Yu, X., Zhan, L., Chen, J., Zhang, Y., Zhang, J., Chen, H., Zhang, Z., Zhang, Y., Lu, Y., Mei, L., Multilocus sequence type profiles of Bacillus cereus isolates from infant formula in China, Food Microbiology (2016), doi: 10.1016/j.fm.2016.09.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Multilocus sequence type profiles of Bacillus cereus isolates from infant formula in China

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Yong Yang , Xiaofeng Yu , Li Zhan , Jiancai Chen , Yunyi Zhang , Junyan Zhang , Honghu

3

Chena, Zheng Zhanga, Yanjun Zhanga, Yiyu Lua, Lingling Meia* a

4 5

b

a

a

a

a

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a

Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang 310051, China

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b

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*Corresponding author.

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Prof. Lingling Mei

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Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention

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College of Food Science, Shihezi University, Shihezi, Xinjiang 832001,China

No. 3399, Binsheng Rd., Hangzhou, 310051, China

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Tel: 086-571-87115397, Fax: 086-571-87115279 E-mail: [email protected]

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Abstract: Bacillus cereus sensu stricto is an opportunistic foodborne pathogen. The

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multilocus sequence type (MLST) of 74 B. cereus isolated from 513 non-random infant

14

formula in China was analyzed. Of 64 sequence types (STs) detected, 50 STs and 6 alleles

15

were newly found in PubMLST database. All isolates except for one singleton (ST-1049),

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were classified into 7 clonal complexes (CC) by BURST (n-4), in which CC1 with core

17

ancestral clone ST-26 was the largest group including 86% isolates, and CC2, 3, 9, 10 and

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13 were first reported in China. MLST profiles of the isolates from 8 infant formula brands

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were compared. It was found the brands might be potentially tracked by the variety of STs,

20

such as ST-1049 of singleton and ST-1062 of isolate from goat milk source, though they

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could not be easily tracked just by clonal complex types of the isolates.

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Keywords: Bacillus cereus, MLST, clonal complex, infant formula, surveillance

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1. Introduction

Bacillus cereus sensu lato is a Gram-positive spore-forming bacterial group, consisting

of

B. anthracis, B. cereus, B. cytotoxicus, B. mycoides, B. pseudomycoides, B.

thuringiensis, and B. weihenstephanensis. It has a great diversity of phylogenesis and

27

occupies widespread ecological niches (Ceuppens et al. 2013). B. cereus sensu stricto (B.

28

cereus in short) is an opportunistic foodborne pathogen, produces toxins such as cereulide,

29

cytotoxin K, hemolysin BL (HBL) and non-hemolytic enterotoxin (NHE), and causes food

30

poisoning or even severe human infectious diseases (Rowan and Anderson, 1998; 1

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Ehling-Schulz et al, 2005; Arnesen et al., 2008; Bottone, 2010). It was suggested that B.

32

cereus should be controlled and might be a suitable microbiological safety indicator for food

33

products, especially for infant formula (De Jonghe et al., 2008; Lund, 2015). Infant formula is considered as one of high risk foods on account of its high protein

35

contents and its vulnerable consumers. Great attention has been paid to infant formula

36

safety around the world, especially after melamine incidence in China (Newell et al., 2010).

37

The surveillance of B. cereus along infant formula production chains was carried out (Carlin,

38

2011). It was discovered that B. cereus isolates mostly occurred in infant formula with other

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thermophilic

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stearothermophilus (Becker et al., 1994; Bartoszewicz et al., 2008; Haughton et al., 2010;

41

Sadiq et al., 2015). B. cereus might come from farm lands, endure ultrahigh-temperature

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(UHT) pasteurization and concentration, survive from spray drying tower and appear in final

43

products (McAuley et al., 2014). Toxin genes such as ces, cytK, hbl and nhe were detected

44

in B. cereus isolates from lots of food (Svensson et al., 2007; Lücking et al., 2013; Hwang

45

and Park, 2015; Organji et al., 2015). It was suggested that the B. cereus pathogens should

46

be intensively monitored in infant formula (Shaheen et al., 2006; Di Pinto et al., 2013).

species

such

as

B.

licheniformis

and

Geobacillus

M AN U

SC

spore-forming

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34

Multilocus sequence type (MLST) is a housekeeping gene-based molecular typing

48

technique, first developed for Neisseria meningitidis typing (Maiden et al., 1998; Spratt,

49

1999; Urwin and Maiden, 2003). It is not only a portable and comparable method for

50

characterizing bacteria at a nucleic acid molecule level, but also a powerful tool for

51

pathogenic microorganism tracing and genetic evolution exploration (van Belkum, 2003;

52

Cardazzo et al., 2008; Pérez-Losada et al., 2013). MLST has been intensively used to type

53

over 70 bacterial species, 7 eukaryotes, 1 bacteriophage (Lactococcus lactis 936-like

54

bacteriophages) and 4 plasmids (Chan et al., 2001; Cooper and Feil, 2004; Maiden, 2006).

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Since the MLST scheme for B. cereus sensu lato was built in 2004, the genetic diversity

56

of clinical and foodborne isolates was extensively studied with MLST (Helgason et al., 2004;

57 58 59 60 61

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Vassileva et al., 2006; Cardazzo et al. 2008; Hoffmaster et al., 2008). However, the MLST of B. cereus sensu stricto isolates from infant formula were not systematically estimated both in China and abroad. The exploration of molecular character of B. cereus in infant formula is in great need for foodborne disease prevention and control (Ehling-Schulz and Messelhäusser, 2013).

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The aim of this study was to provide the first overview of MLST profiles of B. cereus

63

isolates from infant formula in China. The results would enrich database of B. cereus in

2

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PubMLST (http://pubmlst.org/bcereus) and might be used in tracking of infant formula

65

brands in the future.

66

2. Materials and methods 2.1. Infant formula samples and B. cereus isolates

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74 B. cereus isolates in this study were isolated from 513 non-random infant formula

69

samples of 23 brands in Chinese market between 2012 and 2013 by using mannitol, yolk

70

and polymyxin agar plate (MYP) (LUQIAO，Beijing, China) (GB 4789.14, 2014), and

71

re-identified by both morphological observation and biochemical test with VITEK 2 Systems

72

and BCL TEST KIT (bioMerieux, Inc, USA).

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2.2. Genome DNA extract

SC

68

Each strain was streaked on nutrient agar plates and incubated at 37℃ for 16-18 h.

75

5-10 colonies were collected into 200ul sterilized purified water, centrifuged at 12,000 rpm

76

for 5 min. The precipitate was used to extract genome DNA with Bacterial DNA kit (OMEGA,

77

bio-tek) according to operating instruction.

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2.3. Housekeeping gene amplification

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Seven housekeeping genes glp, gmk, ilv,pta, pur, pyc and tpi were to be sequenced

80

according to MLST scheme for B. cereus in PubMLST (http://pubmlst.org/bcereus/

81

info/primers.shtml). PCR amplification conditions for the genes were briefly showed in table

82

1 (Hoffmaster et al., 2008). PCR kit (TaKaRa, China), Mastercycler proS (Eppendorf,

83

Germany), Electrophoresis and Gel Imaging System (BIO-RAD, Milan, Italy) were used in

84

the study. All primers were synthesized by Sangon, China.

85

Table 1

86

The PCR amplifying conditions for housekeeping genes of B. cereus.

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Gene

glpF

gmk

Protein

PCR primers

(ºC)

glycerol

uptake

a

F-GCGTTTGTGCTGGTGTAAGT

facilitator protein

a

guanylate

F-ATTTAAGTGAGGAAGGGTAGG

kinase,

(putative) ilvD

Annealing temp.

dihydroxy-acid

59

R-CTGCAATCGGAAGGAAGAAG 56

R-GCAATGTTCACCAACCACAA F-CGGGGCAAACATTAAGAGAA 3

58

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dehydratase ilvD_2*b

R-GGTTCTGGTCGTTTCCATTC F-AGATCGTATTACTGCTACGG

58

R-GTTACCATTTGTGCATAACGC phosphate acetyltransferase pur

phosphoribosyl-amin o-imidazole

F-GCAGAGCGTTTAGCAAAAGAA R-TGCAATGCGAGTTGCTTCTA F-CTGCTGCGAAAAATCACAAA

SC

pyruvate carboxylase

56

R-CTCACGATTCGCTGCAATAA

carboxamide pycA

56

RI PT

pta

F-GCGTTAGGTGGAAACGAAAG

57

tpi

triosephosphate isomerase

Recycling

step 1: 95℃ 3min.

procedure step 2: 94℃,30S.

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R-CGCGTCCAAGTTTATGGAAT

F-GCCCAGTAGCACTTAGCGAC

58

R-CCGAAACCGTCAAGAATGAT

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step 3: annealing temperature (above), 60~90S. step 4: 72℃, 60S.

EP

step 2-4, 30 cycles. step 5: 72℃,7min.

88 89 90 91

a. F- and R- were referred to forward and reverse primers with direction from 5' to 3',

AC C

87

respectively.

b.ilvD_2* was alternative to ilvD as B. cereus was emetic toxin producing strain. In this

study, if the amplification of ilvD was failed, then ilvD_2 was used. 2.4. Housekeeping gene sequencing

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PCR product was purified using the kit SK1131 (Sangon, China) according to

93

manufacturer’s instructions, then 10 ng purified DNA was sequenced in 3730XL Genetic

94

Analyser (Applied Biosystems, California, USA) with PCR forward primer firstly. The reverse 4

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primer was used when the forward primer did not work and the gmk (504bp) was sequenced

96

with both forward and reverse primers in case of sequencing data distortion.

97

2.5. Database interrogation for allele sequence and ST identification The allele numbers were provided after submission of housekeeping gene sequences

99

to PubMLST. If there were no exact allele sequences for submitted sequences, novel allele

100

numbers would be assigned. The multilocus sequence type of each isolate was the

101

arrangement of 7 housekeeping gene allele numbers. If there was no same arrangement

102

for submitted ST, a new ST number would also be assigned. All novel allele sequences

103

and STs detected in the study has been submitted to PubMLST database, and endowed

104

new numbers.

SC

105

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98

2.6. Clonal complexes clustering

Online BURST (based upon related sequence types) algorithm, simple and speedy for

107

epidemiological surveillance disregarding much of the evolutionary information contained in

108

the nucleotide sequence, was used for analysis (Urwin and Maiden, 2003; Vassileva et al.,

109

2006). The isolates in this study together with isolates of B. cereus sensu stricto in

110

PubMLST database were clustered into separate clonal complexes by BURST (n-4, 3 or

111

more matches) (Didelot et al., 2009). The collection places of the isolates were adopted as

112

their geographical distribution regions (origins) in tracking analysis.

113

3. Results and Discussion

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3.1. The MLST profile diversity of B. cereus isolates from infant formula in China

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The MLST profiles of 74 isolates were shown in table 2. Of the 74 isolates subtyped, 64

116

STs were obtained and 50 STs were first found and assigned to new ST numbers from 1029

117

to 1077, and 1079, with corresponding ID from 1776 to 1824, and 1753. Six novel alleles

118

including pta-232, pur-216, pur-217, pyc-181, tpi-183 and tpi-184 were detected. It indicated

120 121 122

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that the MLST profiles of B. cereus isolates from infant formula in China were highly diverse (Barker et al., 2005; Vassileva et al., 2007; Hoffmaster et al., 2008; Didelot et al., 2009).

3.2. The clonal complexes of B. cereus isolates from infant formula in China Based on genetic, prevalent and microbiological criteria, distinct STs in MLST datasets

123

were classified into separate clonal complexes by BURST (n-4), which was an unweighted

124

similarity comparison method other than genetic comparison method of Split-tree (Urwin

125

and Maiden, 2003). 64 STs were grouped into 7 of 23 clonal complexes (table 3). The

126

largest clonal complex 1 (CC1) containing 55 STs was sub-grouped into 9 sub-clonal 5

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complexes (figure 1). ST-26 was a well-known cosmopolitan ST distributed worldwide, and

128

was a key ancestral clone in B. cereus (Tourasse et al., 2011). It was verified that ST-26

129

was also the most popular ancestral clone of the B. cereus isolates from infant formula in

130

China, since 86% STs (55/64) belonging to CC1 were originated from it. Five ST-1038, 1048, 1050, 1057 and 1062 were belonged to CC2, 3, 9, 10 and 13,

132

respectively. These 5 CCs are first reported in China, however it was uncertain whether they

133

were indigenous or exotic because the milk powder or whey powder used in infant formula

134

in China were mostly imported from other countries after the melamine incident. The ST-978,

135

1033 and 1075 in CC15 were only detected in China and the ancestral clone ST-978 was

136

isolated from seawater in Fujian province of China. It was speculated that the CC15

137

members could be disseminated around circumjacent ocean countries. The singleton

138

ST-1049 could not be grouped into any clonal complex by BURST (n-4), and the origin of

139

milk powder was uncertain.

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131

140

Table 3

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The clonal complexes of 64 STs classified by BURST (n-4). Clonal complex

ST

(CC)

4,24,26*a,31,32,92,118,144,205,371,380,795,

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CC1

Origin

Cosmopolitan

869,1029-1032,1034-1037,1039-1047,1051-10 56,1058-1061,1063-1074,1076,1077,1079

CC3

(12, 202) ,1048

UK, USA

(125) ,1038

USA

CC10

(160), 1062

Japan, Italy, UK

CC13

(375), 1057

Italy

CC15

978*, 1033,1075

China

1049

?

singleton

142 143

Japan, USA Canada, Italy, Pakistan,

AC C

CC9

b

(365*) ,1050

EP

CC2

c

Note: a. Potential ancestral STs were marked by asterisk (*). b. STs enclosed in brackets were the reference STs, not isolates in this study. 6

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c. Its origin was uncertain.

147 148 149 150 151 152

Fig.1. The image of clonal complex 1 (CC1) and its 9 sub-clonal complexes.

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3.3. Comparison of MLST profiles between isolates from different brands Considering that the STs of isolates in infant formula might vary with different milk

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145

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SC

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144

sources used by manufacturers (Hall et al., 2012), it made sense to dissect isolate ST presence from different brands. Comparison of MLST profiles between the 18 isolates from 8 brands with definite milk sources were listed in table 4. Most STs were included in CC1, and only ST-1049 from brand 2, ST-1033, ST-1075

153

from brand 3, and ST-1062 from brand 6 were included in singleton, CC15 and CC10,

154

respectively. It indicated that the brands or milk sources of infant formula could not be easily

155

tracked only by isolate clonal complex types. However, if the singleton ST-1049 was specific

7

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156

to New Zealand, and ST-1062 (in CC10) was specific to goat milk in China, they might be

157

used as a specific tracking tool. All STs of isolates were not overlapped in 8 brands except for ST-24 detected in 3

159

brands, and two isolates of A141 and A243 from brand 1 had same ST-1077, which

160

indicated that a variety of STs might be potentially used to track which brands the strains

161

isolated from (Quested et al., 2010). Both ST-1033, and ST-1075 were detected in brand 3

162

from New Zealand, which supported the speculation that the CC15 members could be

163

disseminated around circumjacent ocean countries.

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In conclusion, the MLST profiles of the isolates from infant formula in China were highly

165

diverse. A larger number of infant formula samples from different countries, brands and

166

batches over a longer time-frame will be collected to verify whether the variety of STs could

167

be used to track origins of the isolates in next study.

168

Table 4

169

The comparison of MLST profiles between the isolates from 8 brands.

24(A73)

1056(A284)

32(B101)

1058(A316)

Clonal complex

Milk source

CC1

China

New Zealand

TE D

1

ST (isolate)

M AN U

Brand

SC

164

1050(A159)

1029(A9)

1054(A279)

1032(A33)

1055(A282)

EP

795(A329)

1076(B258)

1041(A56)

1077(A124,A131）

AC C

1039(A51)

2

3

144(A10)

1065(A8)

CC1

1030(A16)

1066(B232)

*Singleton

1045(A118)

1067(B233)

1047(A122)

1049*(A157)

24(A58)

1027(B82)

26(A320)

1051(A243)

8

CC1

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1075(B135)

CC15

24(A114)

1031(A17)

CC1

Netherlands

118(B281)

1052(A257) Ireland

4

5

1063(B133)

CC1

6

1062(A342)

CC10

1074(B7)

CC1

7

1065(B160)

CC1

8

1071(B259)

CC1

RI PT

1033(A34)

China

SC

(goat milk)

Table 2

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The MLST profiles of B. cereus isolates from infant formula in China. Isolateb

MLST

gmk

ilv

pta

pur

pyc

tpi

ST

31

5

19

3

91

1029

63

5

36

3

4

1030

111

11

9

104

8

1031

A9

3

2

1777

A16

22

2

1778

A17

14

8

1779

A33

117

4

123

118

43

6

3

1032

1780

A34

81

53

8

193

113

93

80

1033

1781

A39

13

8

122

107

38

12

88

1034

1782

A339

3

2

31

5

9

3

4

1035

1783

A44

5

2

122

5

39

3

91

1036

TE D

1776

EP

glp

AC C

IDa

M AN U

170

1784

A46

11

9

14

12

16

14

180

1037

1785

A49

58

34

31

48

16

20

26

1038

1786

A51

3

2

31

5

32

3

4

1039

1787

A52

3

2

31

5

52

3

4

1040

1788

A56

3

2

31

5

12

3

4

1041

1789

A57

43

26

59

42

19

41

63

1042

9

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A59

19

2

70

60

181

3

43

1043

1791

A115

12

8

8

11

4

12

8

1044

1792

A118

13

9

14

12

12

14

180

1045

1793

A119

11

2

63

5

36

3

4

1046

1794

A122

3

9

18

220

20

19

7

1047

1795

A123

22

7

7

2

7

10

13

1048

1796

A157

85

56

59

64

64

85

26

1049

1797

A159

65

1

93

214

51

37

166

1050

1798

A243

3

2

63

5

36

3

55

1051

1799

A257

47

28

31

26

2

36

7

1052

1800

A278

19

2

1801

A279

19

2

1802

A282

19

2

1803

A284

22

2

1804

A285

110

56

1805

A316

3

23

1806

A319

24

1807

A332

3

1808

A336

43

1809

A342

73

5

16

3

55

1053

31

5

16

3

55

1054

63

5

19

3

55

1055

18

5

36

19

7

1056

9

102

112

96

26

1057

122

152

38

43

88

1058

22

14

37

34

14

180

1059

26

59

17

19

126

55

1060

26

59

42

39

41

63

1061

43

224

60

64

63

32

1062

AC C

TE D

21

EP

M AN U

SC

RI PT

1790

1810

B133

22

9

18

220

20

19

7

1063

1811

B159

12

8

28

14

11

12

10

1064

1812

B160

3

2

31

5

19

3

2

1065

1813

B232

19

2

31

5

19

3

91

1066

1814

B239

19

4

31

5

43

46

91

1067

1815

B239

6

40

41

59

61

60

3

1068

10

ACCEPTED MANUSCRIPT

B251

70

8

65

11

9

12

48

1069

1817

B252

13

2

8

5

19

3

7

1070

1818

B259

3

4

63

4

204

6

4

1071

1819

B264

6

4

8

5

36

6

181

1072

1820

B279

3

2

63

5

19

3

4

1073

1821

B7

67

2

63

5

216*c

3

4

1074

1822

B135

179

53

117

232*

113

93

80

1075

1823

B258

19

2

21

5

36

156

183*

1824

A124

40

9

14

12

217*c

181*c

7

1077

1753

A280

14

8

43

11

9

104

184*c

1079

A287

13

8

A58

12

8

A73

12

8

A114

12

8

A322

12

8

A74

3

2

A320

3

B282

3

SC

c

1076

11

11

12

7

4

9

14

11

12

10

24

9

14

11

12

10

24

9

14

11

12

10

24

9

14

11

12

10

24

31

5

16

3

4

26

2

31

5

16

3

4

26

2

31

5

16

3

4

26

A50

21

2

19

5

16

18

2

31

A317

5

4

3

4

15

6

16

32

AC C

TE D

8

EP

M AN U

c

RI PT

1816

B101

5

4

3

4

15

6

16

32

A321

6

4

42

4

16

6

3

92

B281

47

28

14

26

2

36

7

118

A10

67

2

63

5

36

3

4

144

A330

67

2

63

5

36

3

4

144

A76

19

2

21

5

19

3

2

205

11

ACCEPTED MANUSCRIPT

21

2

19

5

32

18

2

371

A259

3

2

116

5

36

3

4

380

A329

51

2

21

5

19

3

2

795

A132

19

2

59

5

19

3

2

869

A77

81

53

117

193

113

93

80

978

B82

174

5

29

49

191

154

80

1027

A8

3

2

31

5

19

3

2

1065

A131

40

9

14

12

217

181

7

1077

SC

RI PT

A286

Notes: a. ID was only assigned to isolates with new STs by PubMLST. So there were

173

some isolates with no ID in this ID column.

174 175

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b. In front of isolate number, A or B was added. “A-” indicated the strain was collected in 2012, “B-” indicated the strain was collected in 2013.

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c. Novel alleles found in this study were marked with asterisk (*).

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Acknowledgements

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This

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no.2015M570525),

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(LY13C200008), Major Science and Technology Special Projects of Zhejiang, China

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(2013C03045-1), and Public Welfare Project of Zhejiang Province (2015C37058). We thank

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Prof. Jay Gee (CDC, USA) for advice on this paper.

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Highlights: The MLST of 74 B. cereus isolates from infant formula in China was analyzed. Of 64 STs obtained, 50 STs and 6 alleles were newly found in PubMLST database. The isolates were classified into 7 clonal complexes and one singleton by BURST (n-4). A variety of STs of the isolates might be used in milk sources or brands tracking.