Downloaded from · 7/30/2015 · 7KHJHQHVIRUEHWD ODFWDPDVH -lac WHWUDF\FOLQHUHVLVWDQFH tet 3 DQGOLQFRP\FLQ UHVLVWDQFHSURWHLQ lmr % KDYHEHHQSUHYLRXV O\LGHQWLILHGLQ C. butyricum W\SH(LVRODWHV

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1

Laboratory investigation of the first case of C. butyricum type E botulism in the United 1

States 2

Janet K. Dykes#, Carolina Luquez, Brian H. Raphael, Loretta McCroskey, and Susan E. 3

Maslanka 4

5

Enteric Diseases Laboratory Branch, Centers for Disease Control and Prevention, 6

Atlanta, Georgia, USA 7

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Running title: First US C. butyricum type E botulism case 9

10

#Corresponding author. 11

Mailing address: Centers for Disease Control and Prevention, 1600 Clifton Road, MS G-12

29, Atlanta, GA 30329. Telephone: 404-639-3625. Fax: 404-639-4290. E-mail: 13

[email protected] 14

15

16

17

JCM Accepted Manuscript Posted Online 5 August 2015J. Clin. Microbiol. doi:10.1128/JCM.01351-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract 18

We report here the laboratory investigation of the first known case of botulism in 19

the United States caused by C. butyricum type E. This investigation demonstrates the 20

importance of extensive microbiological examination of specimens which resulted in the 21

isolation of this organism. 22

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Botulism is a rare but acute paralytic disease caused by botulinum neurotoxin 26

(BoNT). Botulism can occur when contaminated food is ingested (foodborne) or when 27

BoNT-producing clostridia colonize wounds or the intestine. Infant botulism occurs 28

through intestinal colonization by BoNT-producing clostridia, and the subsequent in situ 29

production of BoNT in infants (1). Diagnosis is based on clinical presentation (1, 2). In 30

the United States, Botulism Immune Globulin Human (Intravenous) (BIG-IV) is 31

available for treatment of infant botulism through the California Department of Public 32

Heath (CDPH) Infant Botulism Treatment and Prevention Program (IBTPP) (2). 33

Laboratory confirmation is performed at the Centers for Disease Control and Prevention 34

(CDC) and state public health laboratories (1, 2). 35

C. botulinum types A and B are the serotypes most frequently associated with 36

infant botulism worldwide, accounting for ~98% of reported infant botulism cases (2). 37

Infant botulism caused by C. butyricum type E was first reported in 1987 (3). Since then, 38

this organism has been linked to infant botulism cases in Italy, Japan, the Republic of 39

Ireland and England (3, 4, 5, 6). Additionally, C. butyricum type E has been associated 40

with foodborne botulism in China, Italy, and India (7, 4, 8). We report here the laboratory 41

investigation of the first case in the United States of botulism caused by C. butyricum 42

type E. 43

Stool collected from a 7-day-old infant with suspected botulism was submitted to 44

the Centers for Disease Control and Prevention (CDC) National Botulism Laboratory for 45

laboratory testing. The sample was tested for BoNT by mouse bioassay (1). Association 46

for Assessment and Accreditation of Laboratory Animal Care guidelines and 47

institutionally approved protocols for the ethical use of laboratory animals were followed. 48

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The amount of BoNT type E detected in the stool was 126,400 mLD50/gm (mLD50 = 50% 49

mouse lethal doses) which is >150 times higher than previously reported for type E infant 50

botulism cases (4). The stool sample was cultured for the growth of BoNT-producing 51

clostridia by standard laboratory methods (9). McClung Toabe egg yolk agar (EYA) 52

plates were streaked for isolation from stool, and from enriched broth cultures. Plates 53

were incubated for 2-3 days; broth cultures were incubated for 4-5 days. All broth 54

cultures and plates were incubated in anaerobic conditions at 35°C. 55

Plate growth of stool and stool culture was heavily mixed with lipase-positive 56

(Lip+) colonies and lipase-negative/ lecithinase-negative (Lip–/Lec–) colonies. Because 57

both C. botulinum (Lip+) and C. butyricum (Lip–/Lec–) are known to produce BoNT 58

type E, both Lip+ (N=11) and Lip–/Lec– (N=25) colonies were selected for further 59

culture and characterization. BoNT type E was detected in the cultures of two Lip–/Lec– 60

isolates. Phenotypic characteristics (Table 1) and 16S sequence data were consistent with 61

C. butyricum. This strain was designated as CDC51208. 62

Eleven Lip+ colonies and 23 Lip–/Lec– colonies were negative for BoNT by 63

mouse bioassay and/or for the presence of bont/E by PCR. Further investigations were 64

conducted to identify the associated non-toxigenic bacteria that impeded rapid 65

identification of C. butyricum type E. A representative Lip+ colony was identified as C. 66

sporogenes by biochemical (TABLE 1) analysis. A representative non-toxic Lip– /Lec– 67

colony was identified as C. tertium by biochemical (TABLE 1) and 16S sequence 68

analysis. 69

C. tertium is an aerotolerant anaerobe common in the fecal flora of infants (10, 70

11) that can be readily distinguished from the obligate anaerobe C. butyricum by growth 71

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in aerobic conditions. Further, C. tertium produces oval terminal spores, whereas C. 72

butyricum produces subterminal spores (10). Out of the 25 Lip-/Lec- isolates examined, 73

23 isolates showed growth on blood agar in aerobic conditions and/or terminal spores by 74

gram stain suggesting that the majority of these isolates were C. tertium. Direct stool and 75

enrichment cultures were heat-shocked (80°C for 15 min.) and alcohol-treated (equal 76

parts sample and 100% ethanol for 1 hour) to eliminate vegetative growth and select for 77

clostridia. However, these methods were not useful in eliminating these associated non-78

toxin bacteria from the stool culture. 79

Strain CDC51208 was characterized by molecular analyses. The bont/E gene 80

nucleotide sequence was determined using previously reported primers that amplified 81

overlapping regions of the gene (12) and submitted to GenBank under accession number 82

KP455988. Of the eleven currently recognized bont/E subtypes, only E4 and E5 are 83

known to be produced by C. butyricum (13). The nucleotide sequence of bont/E in strain 84

CDC51208 clustered with other bont/E4 sequences (FIG 1). All previously reported 85

strains containing bont/E4 are associated with botulism cases in Italy. C. butyricum 86

strains containing bont/E5 are associated with a foodborne outbreak and soil samples 87

from China (12). C. butyricum strain CDC51208 was compared, by pulsed-field gel 88

electrophoresis (PFGE) (14), to two available C. butyricum type E strains (5262, 5520) 89

from the first and second Italian infant botulism cases, respectively (3). Although the 90

bont/E sequences of strains 5262, 5520, and CDC51208 were identical, the SmaI and 91

Xho1 PFGE profiles of strains 5262 and 5520 (>93% similar to each other) were distinct 92

from CDC51208 (<66% similar) (FIG 2A). These results indicate that while the toxin 93

gene sequence is stable among the strains examined, differences in the genomic 94

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background of these strains appear to be associated with their origin (i.e. CDC51208 95

isolated in the US was genetically distinct from the Italian strains 5520 and 5262). 96

Identification of antimicrobial resistance genes provided another way to 97

characterize and compare strain CDC51208 to C. butyricum type E isolates from Italy. 98

The genes for beta-lactamase (β-lac), tetracycline resistance (tet(P)) and lincomycin 99

resistance protein (lmrB) have been previously identified in C. butyricum type E isolates 100

from Italy (15). Using primer sequences reported by Franciosa et al. (15), we searched for 101

these three genes by PCR in strain CDC51208, and for comparison, in strains 5262 and 102

5520. Amplification for β-lac and tet(P) was observed in all three C. butyricum type E 103

strains (FIG 2B). β-lac has previously been identified in C. butyricum type E clinical 104

isolates from Italy and China, but was not found in soil isolates from China (15). The 105

presence of β-lac in strain CDC51208 lends additional support for the presence of this 106

gene among clinical isolates. Although lmrB was present in strains 5262 and 5520, it 107

was not detected in strain CDC51208 (FIG 2B). Interestingly, this gene also was not 108

detected in any isolates containing bont/E5 isolated in China (15), suggesting that the 109

presence of lmrB is variable among C. buytrcium type E strains. 110

To our knowledge, this is the first case of botulism caused by C. butyricum type E 111

in the United States, and the first identification of a strain containing bont/E4 outside of 112

Italy. The isolation of C. butyricum type E in the U.S. suggests that this strain may be 113

more widely distributed than previously recognized. The presence of numerous 114

nontoxigenic Lip+ and Lip-/Lec+ bacteria in this infant stool and the rarity of C. 115

butyricum type E significantly complicated the identification of this rarely identified 116

cause of infant botulism. Increased awareness of these concomitant strains in infant stool 117

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may help investigators more readily identify C. butyricum type E in suspected infant 118

botulism cases. 119

120

This publication was supported by funds made available from the Centers for 121

Disease Control and Prevention, Office of Public Health Preparedness and Response. The 122

findings and conclusions in this report are those of the authors and do not necessarily 123

represent the official position of the Centers for Disease Control and Prevention. 124

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References 125

1. Centers for Disease Control and Prevention. 1998. Botulism in the United States 126

1899-1996: handbook for epidemiologists, clinicians and laboratory workers. Centers 127

for Disease Control and Prevention, Atlanta, Ga. 128

2. Koepke R, Sobel J, Arnon SS. 2008. Global Occurrence of Infant Botulism, 1976 - 129

2006. Pediatrics. 122:e73-e82. 130

3. Aureli P, Fenicia L , Pasolini B, Gianfranceschi M, McCroskey LM, Hatheway 131

CL. 1986. Two cases of type E infant botulism caused by neurotoxigenic Clostridium 132

butyricum in Italy. J. Infect. Dis. 154:207-211. 133

4. Fenicia L, Anniballi F, Aureli P. 2007. Intestinal toxemia botulism in Italy, 1984-134

2005. Eur. J. Clin. Microbiol. Infect. Dis. 26:385-394. 135

5. Abe Y, Negasawa T, Momma C, Oka A. 2008. Infantile botulism caused by 136

Clostridium butyricum type E toxin. Pediatr. Neurol. 38:55-57. 137

6. Shelley EB, O’Rourke D, Grant K, McArdle E, Capra L, Clarke A, McNamara 138

E, Cunney R, McKeown P, Amar CFL, Cosgrove C, Fitzgerald M, Harrington 139

P, Garvey P, Grainger F, Griffin J, Lynch BJ, McGrane G, Murphy J, Ni 140

Shuibhne N, Prosser J. 2014. Infant botulism due to C. butyricum type E toxin: a 141

novel environmental association with pet terrapins. Epidemiol. Infect. 13:1-9. 142

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Yamakawa K, Oguma K, Sakaguchi Y, Nakamura S. 2000. Genetic analysis of 144

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8. Chaudhry R, Dhawan B, Kumar D, Bhatia R, Gandhi JC, Patel RK, Purohit 148

BC. 1998. Outbreak of suspected Clostridium butyricum botulism in India. Emerg 149

Infect Dis. 4:506-507. 150

9. Maslanka SE, Solomon HM, Sharma S, and Johnson EA. Clostridium botulinum 151

and its Toxins. In: Tororello ML, Dowes FP, Doores S, Ito K, Salfinger Y, eds. 152

Compendium of Methods for the Microbiological Examination of Foods. American 153

Public Health Association. 2013: eBook ISBN: 978-0-87553-022-2. 154

10. Holdeman LV, Cato EP Moore WEC (ed.). 1977. Anaerobe laboratory manual, 4th 155

ed., p. 79-106. Virginia Polytechnic Institute and State University, Blacksburg, VA. 156

11. Tonooka T, Sakata S, Kitahara M, Hanai M, Ishizeki S, Takada M, Sakamoto 157

M, Benno Y. 2005. Detection and quantification of four species of the genus 158

Clostridium in infant feces. Microbiol. Immunol. 49:987-992. 159

12. Hill K K, Smith TJ, Helma CH, Ticknor LO, Foley BT, Svensson RT, Brown 160

JL, Johnson EA, Smith LA, Okinaka RT, Jackson PJ, Marks JD. 2007. Genetic 161

diversity among botulinum neurotoxin-producing clostridial strains. J. Bacteriol. 162

189:818-832. 163

13. Rossetto O, Pirazzini M, Montecucco C. 2014. Botulinum neurotoxins: genetic, 164

structural and mechanistic insights. Nat. Rev. Microbiol. 12:535-49. 165

14. Luquez C, Joseph LA, Maslanka SE. Molecular Subtyping of Clostridium 166

botulinum by Pulsed-Field Gel Electrophoresis. In: Jordan K, Dalmasso M, eds. Pulse 167

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15. Franciosa G, Scalfaro C, Di Bonito P, Vitale M, Aureli P. 2011. Identification of 170

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Clostridium butyricum type E strains. PLoS ONE 6:1-10. 172

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TABLE 1 Characteristics of reference clostridia C. botulinum type E, C. sporogenes, C. 174

butyricum, C. tertium and 3 phenotypically distinct isolates from infant stool. 175

176

Organism BoNT

typea

Lipaseb Aerobic

growthb

Nitrate

reductionb

Spore

formationc

Reference clostridiad

C. botulinum E + - - ST

C. sporogenes NT + - - ST

C. butyricum NT - - - ST

C. tertium NT - + +- T

Isolates from infant

Toxigenic Lip-Lec-

(CDC51208)

E - - - ST

Non-toxigenic Lip-Lec- NT - + + T

Non-toxigenic Lip+ NT + - - ST

a NT, non-toxic. 177

b +, positive; -, negative; +-, most strains positive, some strains negative. 178

c ST, subterminal; T, terminal. 179

d Species characteristics as described in the VPI Anaerobe Laboratory Manual 180

(Holdeman1977). 181

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Figure legends 183

FIG 1. Neighbor joining phylogenetic tree of bont/E sequences. The bont/E sequence of 184

strain CDC51208 and representative variants retrieved from GenBank were analyzed. 185

The accession numbers of reprehensive bont/E sequences are: E1, AM695756; E2, 186

EF028404; E3, AM695753; E5, AB037704; E6, AM695752; E7, JX424537; E8, 187

JN695730; E9, JX424534; E10, KF861922; E11, KF861875; BL 5520, AB039264; BL 188

5262, AB088207. 189

190

FIG 2. Molecular characterization of C. butyricum type E strains 5520, 5262 and 191

CDC51208. (A) SmaI and XhoI pulsed-field gel electrophoresis (PFGE) patterns, with 192

dendograms constructed by unweighted pair group method with averages (UPGMA) 193

analysis. PFGE patterns were compared by the Dice coefficient with a tolerance window 194

of 1.5% and an optimization of 1.5%. (B) PCR products (~400bp) for B-lac, tet(P), and 195

lmrB. Products were run on 1% agarose gel and stained with ethidium bromide. Results 196

for strains 5520 and 5562 are consistent with previous findings by Franciosa et al. (15) 197

198

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