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Assessment of Whole Genome Mapping in a well-defined 1 outbreak of Salmonella enterica Serotype Saintpaul 2

P.D. Fey1*, P.C. Iwen1, E.B. Zentz2, A.M. Briska2, J.K. Henkhaus2, K.A. 3 Bryant1, M.A. Larson1, R.K. Noel1, and S.H. Hinrichs1 4

1Department of Pathology and Microbiology, University of Nebraska Medical 5 Center, Omaha, NE. USA 68198-5900. 2OpGen, Inc. Gaithersburg, MD 20878. 6

7 Running Header: Whole Genome Mapping of Salmonella Saintpaul 8 9 Keywords: Bacterial typing, Salmonella, Molecular epidemiology, Food-borne 10 outbreaks. 11 12 *Corresponding author: 13 Paul D. Fey, Ph.D. 14 Department of Pathology and Microbiology 15 University of Nebraska Medical Center 16 Omaha, NE. 68198-5900 17 pfey@unmc.edu 18 (402) 559-2122 19 (402) 559-5900 (fax) 20 21 22 23

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.01320-12 JCM Accepts, published online ahead of print on 20 June 2012

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Abstract: 24 We investigated the use of Whole Genome Mapping and PFGE with isolates 25 from an outbreak of Salmonella enterica serotype Saintpaul. PFGE and Whole 26 Genome Mapping were concordant on 22 of 23 isolates. Whole Genome 27 Mapping is a viable alternative tool for the epidemiological analysis of Salmonella 28 food-borne disease investigations. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

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For epidemiological analyses of Salmonella, the use of pulsed field gel 47 electrophoresis (PFGE) has been employed which relies on genetic 48 polymorphisms within restriction enzyme recognition sites and large scale 49 insertions, deletions, and rearrangements that disrupt the restriction fragment 50 profile (10). In conjunction with the PulseNet tracking system, PFGE has been 51 highly effective in assessing the epidemiological relationship of various 52 Salmonella isolates associated with outbreaks of food-borne disease (1, 2). 53 However, in some serotypes, such as Salmonella enterica Serotype Enteritidis, it 54 is difficult to interpret PFGE data due to genetic homogeneity (13). Therefore, 55 other methodologies have been utilized to assess the epidemiological 56 relatedness of Salmonella isolates including determination of variable number of 57 tandem repeats (VNTR) or multi-locus sequence typing (3). 58 An alternative molecular approach to global genomic analysis, termed Whole 59 Genome Mapping, holds significant potential for the characterization of bacteria. 60 Whole Genome Maps have been used to assist in the closing of entire genomic 61 sequences and, in addition, have been used to ascertain epidemiological 62 relationships of Escherichia coli O157:H7 (4-7, 9, 11, 12, 14, 15). 63 A recent outbreak in 2009 involved infection with Salmonella serotype 64 Saintpaul linked to consumption of alfalfa sprouts (2). This investigation resulted 65 in 228 cases of salmonellosis detected in 13 states occurring in two distinct 66 waves of disease as described by the Centers for Disease Control and 67 Prevention. Twenty-three Salmonella Saintpaul isolates obtained from this well-68 defined outbreak provided the opportunity to evaluate Whole Genome Mapping 69

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as an epidemiological tool as compared to PFGE. 70 Forty-three Salmonella strains were obtained from the Nebraska Public Health 71 Laboratory to include 28 Salmonella Saintpaul (Table 1), six Salmonella 72 Typhimurium (10556, 10633, 10684, 10702, 10803, and 11022), three 73 Salmonella Newport (10532, 10722, and 11023), two Salmonella Enteritidis 74 (10454, 10712), two Salmonella Heidelberg (10495 and 10773), and one each of 75 Salmonella Stanley (100700) and Salmonella Paratyphi B (11005). 76 Preparation of chromosomal DNA suitable for PFGE was performed 77 essentially as described by Ribot et al (10). DNA was digested with XbaI and 78 analyzed using BioNumerics software (version 4.0, Applied Maths, Sint-Martens-79 Latem, Belgium). PFGE demonstrated that all S. Saintpaul isolates obtained 80 from the first and second wave of illness (see Table 1) were highly related 81 (Figure 1). Isolate 10600 contained one additional band of low molecular weight, 82 which was determined to be of plasmid origin (data not shown), in comparison to 83 the other S. Saintpaul isolates (Figure 1). In contrast, the three S. Saintpaul 84 isolates obtained prior to the first wave of illness (8995, 9495, and 10013) and 85 the two isolates obtained after the second wave of illness (10946 and 11028) 86 were distinguishable from the outbreak group as assessed by PFGE. Two of 87 these S. Saintpaul isolates 10013 and 11028 were indistinguishable from one 88 another; although 10013 was isolated prior to the outbreak and 11028 was 89 isolated following the second wave of illness. When other Salmonella serotypes 90 from the repository were evaluated, all were found to be clearly distinguishable 91 from the S. Saintpaul outbreak strains. Interestingly, each of four pairs of 92

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isolates (10803 and 11022, S. Typhimurium; 10633 and 10804, S. Typhimurium; 93 10773 and 10495, S. Heidelberg; and 10712 and 10454, S. Enteritidis) had 94 indistinguishable PFGE patterns and thus may have been epidemiologically 95 related. 96 High molecular weight DNA for each Salmonella isolate was prepared directly 97 from isolated colonies using the OpGen Sample Preparation Kit (OpGen, Inc., 98 Gaithersburg, MD) and Agencourt® Genfind® v2 Kit (Beckman Coulter, FL). 99 Whole Genome Maps were produced using the Argus™ Whole Genome 100 Mapping System. NcoI was chosen as the most appropriate restriction enzyme 101 using software (Enzyme Chooser, OpGen, Gaithersburg, MD) that identifies 102 enzymes which result in a 6-12 kb average fragment size and no single 103 restriction fragment larger than 80 kb. To determine the resolution of Whole 104 Genome Mapping, a well-characterized and sequenced isolate of Francisella 105 tularensis subsp. tularensis was used (GenBank accession number CP001633) 106 (8). Two restriction enzyme digests (NcoI, NdeI) of the isolate were performed 107 and compared. Comparison of the control DNA sequences of Francisella 108 tularensis demonstrated that the Whole Genome Mapping was capable of 109 correctly placing a 2 kb restriction fragment, therefore the resolution was 110 experimentally determined to be at least 2 kb, however, no fragments less than 111 3kb were used to characterize the Salmonella isolates. 112 Analysis of the 43 Salmonella isolates by Whole Genome Mapping was 113 performed and results were compared to the PFGE (Figure 2). Using this 114 approach, all S. Saintpaul strains from the outbreak were determined to be 115

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indistinguishable by Whole Genome Mapping. The outbreak pattern included 116 isolate 10600 in which an additional plasmid band had been seen using PFGE 117 (Figure 1). All five pre- and post-outbreak isolates were distinguishable from the 118 outbreak isolates; 10013 and 11028 were considered indistinguishable in 119 concordance with PFGE results. As expected, all non-S. Saintpaul Whole 120 Genome Maps were distinct from the S. Saintpaul outbreak isolates. In 121 concordance with PFGE results, isolate pairs 10803/11022 (S. Typhimurium), 122 10633/10684 (S. Typhimurium) and 10495/10773 (S. Heidelberg) were 123 indistinguishable as assessed by Whole Genome Mapping. However, S. 124 Enteritidis isolates 10454 and 10712 were divergent by Whole Genome Mapping 125 in contrast to the PFGE result. 126 Whole Genome Mapping generates a finely detailed restriction enzyme map 127 that has the added advantage of providing the order of the fragments in the 128 genome. However, questions have arisen as to whether the increased resolution 129 of Whole Genome Mapping would generate an excessively large number of 130 fragments that would exceed the ability to link the variations to one serotype or 131 outbreak strain. Results from this study demonstrate that molecular relationships 132 determined by Whole Genome Mapping are equivalent to those from PFGE 133 generated data. Therefore Whole Genome Mapping has potential value as an 134 epidemiological analysis tool for investigation of outbreaks of disease caused by 135 Salmonella. Further work is required to determine whether Whole Genome 136 Mapping can be utilized as an epidemiological tool with other bacteria of 137 epidemiological importance such as Staphylococcus aureus and difficult to type 138

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bacteria such as Salmonella serotype Enteritidis and Clostridium difficile. 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161

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References 162 163 1. Centers for Disease Control and Prevention. 2009. Multistate outbreak 164

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231 232 233 234 235 236 237 238 239 240 241 242

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Table 1. Listing of Salmonella Saintpaul isolates used in this study in relation to 243 the temporal periods described for the outbreaka. 244 Time period Datesa S.Saintpaul isolate 245 ________________________________________________________________ 246 247 Prior to outbreak Before 2009 8995, 9495, 248 10013 249 250 1st wave of illness 1 February through 10520, 10521, 251

15 March 2009 10522,10523, 252 10582, 10586, 253

10587, 10600, 254 10601, 10612, 255 10614, 10621, 256 10644, 10645, 257 10646, 10648, 258 10658, 10660 259 260 2nd wave of illness 16 March through 10680, 10690, 261 18 April, 2009 10695, 10701, 262 10741 263 264 Post outbreak After 18 April 2009 10946, 11028 265 266 aDates were based on a CDC report defining the outbreak epidemic curve (2). 267 268 269 270 271 272 273 274 275 276 277

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Figure Legends: 278 Figure 1: PFGE analysis of Salmonella isolates used in study. 279 Figure 2: Whole Genome Mapping analysis of Salmonella isolates used in study. 280 281