Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype

  • Brian H Raphael1Email author,

    Affiliated with

    • Matthew Lautenschlager1,

      Affiliated with

      • Suzanne R Kalb1,

        Affiliated with

        • Laura I T de Jong2,

          Affiliated with

          • Michael Frace1,

            Affiliated with

            • Carolina Lúquez1,

              Affiliated with

              • John R Barr1,

                Affiliated with

                • Rafael A Fernández2 and

                  Affiliated with

                  • Susan E Maslanka1

                    Affiliated with

                    BMC Microbiology201212:245

                    DOI: 10.1186/1471-2180-12-245

                    Received: 24 August 2012

                    Accepted: 30 October 2012

                    Published: 31 October 2012

                    Abstract

                    Background

                    Clostridium botulinum strains that produce botulinum neurotoxin type E (BoNT/E) are most commonly isolated from botulism cases, marine environments, and animals in regions of high latitude in the Northern hemisphere. A strain of C. botulinum type E (CDC66177) was isolated from soil in Chubut, Argentina. Previous studies showed that the amino acid sequences of BoNT/E produced by various strains differ by < 6% and that the type E neurotoxin gene cluster inserts into the rarA operon.

                    Results

                    Genetic and mass spectral analysis demonstrated that the BoNT/E produced by CDC66177 is a novel toxin subtype (E9). Toxin gene sequencing indicated that BoNT/E9 differed by nearly 11% at the amino acid level compared to BoNT/E1. Mass spectrometric analysis of BoNT/E9 revealed that its endopeptidase substrate cleavage site was identical to other BoNT/E subtypes. Further analysis of this strain demonstrated that its 16S rRNA sequence clustered with other Group II C. botulinum (producing BoNT types B, E, and F) strains. Genomic DNA isolated from strain CDC66177 hybridized with fewer probes using a Group II C. botulinum subtyping microarray compared to other type E strains examined. Whole genome shotgun sequencing of strain CDC66177 revealed that while the toxin gene cluster inserted into the rarA operon similar to other type E strains, its overall genome content shared greater similarity with a Group II C. botulinum type B strain (17B).

                    Conclusions

                    These results expand our understanding of the global distribution of C. botulinum type E strains and suggest that the type E toxin gene cluster may be able to insert into C. botulinum strains with a more diverse genetic background than previously recognized.

                    Keywords

                    Botulism Mass spectrometry Genomics Whole genome sequencing

                    Background

                    There are 7 serotypes (types A-G) of botulinum neurotoxins (BoNT) and types A, B, E or F are the most frequent causes of botulism in humans. Strains of Clostridium botulinum producing BoNT/E share similar metabolic characteristics including the inability to digest proteins such as gelatin, casein, or meat. These non-proteolytic strains are psychrophilic with the ability to grow at refrigeration temperatures [1]. In rare cases, strains of Clostridium butyricum have been shown to produce BoNT/E [2].

                    Clostridium botulinum type E strains can be isolated from various marine environments and cases of botulism due to BoNT/E typically occur in Canada, Alaska, Northern Europe, and Japan [3]. A total of 56 cases of type E botulism were reported to the Centers for Disease Control and Prevention between 2001–2010 and 87.5% of these cases occurred in Alaska (http://​www.​cdc.​gov/​nationalsurveill​ance/​botulism_​surveillance.​html). Type E botulism has also occurred in the lower 48 states including various outbreaks associated with smoked fish from the Great Lakes [4, 5]. A recent outbreak of botulism in birds and fish in the Great Lakes region was attributed to genetically distinct strains of C. botulinum type E and the organism was also found in lake sediment [6]. A case of infant botulism occurred in Illinois in 2007 although the source of spores in this case could not be determined [7].

                    Genetic analysis of 16S rRNA sequences from various C. botulinum strains reveals the presence of distinct phylogenetic groups (I-IV) [8, 9] which correspond to previously recognized metabolic differences. All Group II strains are non-proteolytic and include type E strains and some type B and type F strains. Nucleotide sequencing of various toxin genes has demonstrated the presence of amino acid variation within genes encoding a single toxin serotype and these variants are identified as toxin subtypes [9, 10]. Among type E strains, a total of 8 such subtypes (E1-E8) have been identified [11]. These subtypes differ at the amino acid level by up to 6%.

                    The genes encoding BoNT/A-G are found in toxin gene clusters that also encode several nontoxic proteins and regulatory proteins. The gene encoding BoNT/E is found within a toxin gene cluster that includes ntnh (nontoxic nonhemagglutinin), p47, and orfX1-3[12, 13]. Hill et al. [13] demonstrated that the bont/E toxin gene cluster inserted into the rarA operon. The transposon-associated gene, rarA, likely plays a role in this insertion event in which the gene is split into small and large fragments that flank the toxin gene cluster [13]. Remarkably, an intact rarA gene is also located within the toxin gene cluster and the nucleotide sequences of the intact and split genes were shown to differ by phylogenetic analysis. Moreover, the split rarA gene fragments can be pasted together to form a gene with a nucleotide sequence with similarity to the gene found in the Group II C. botulinum type B strain 17B. In another study, the intact and split rarA genes were detected across a panel of 41 type E strains [11].

                    In this study, we characterized a previously unreported C. botulinum type E strain isolated in 1995 from soil in Chubut, Argentina. This represents the first report of a type E strain (CDC66177) originating from the Southern hemisphere. We further show evidence that this strain produces a unique type E toxin subtype and that the genetic background of this strain is highly divergent compared to other type E strains.

                    Results and discussion

                    Phylogenetic analysis of bont/E in C. botulinum strains

                    The nucleotide sequence of the entire bont/E gene was determined for each of the 16 C. botulinum type E strains examined in this study. Previous studies have identified several bont/E subtypes [912]. Nucleotide sequences of bont/E determined in this study were compared along with representatives of other reported bont/E subtypes (Figure 1). It is important to note that in some cases strain names used in previous reports may not refer to identical strains examined in this study with a similar name. For instance, the CDC reference strain labeled “Alaska” harbored a gene encoding a subtype E2 toxin and is unlikely to be related to the genome-sequenced strain Alaska E43 (Genbank accession number: NC_010723) which encodes a subtype E3 toxin. Another strain labeled “Minnesota” was distinguished from a strain with the same name reported by Macdonald et al. [11]. The CDC Minnesota strain harbored an E7 subtype-encoding gene while the strain examined by Macdonald et al. [11] encoded an E3 subtype toxin.
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-12-245/MediaObjects/12866_2012_1859_Fig1_HTML.jpg
                    Figure 1

                    Dendrogram of bont/E nucleotide sequences. Shown is a neighbor-joining tree of bont/E nucleotide sequences with bootstrap values (based on 100 replications) and genetic distance (bar) shown. BoNT/E subtypes (E1-E9) encoded by clusters of genes are also shown. Accession numbers for bont/E genes not sequenced in this study are indicated with an asterisk.

                    Strain CDC66177 harbored a significantly divergent bont/E gene which formed a unique clade when compared to other bont/E genes. Comparison of the translated amino acid sequence of this gene with the gene encoding BoNT/E1 in strain Beluga indicated that the sequences differed by ~11%. Since previous comparisons of BoNT/E subtypes resulted in differences of up to 6% amino acid sequence variation, the BoNT/E produced by strain CDC66177 can be considered a unique subtype (E9) [10, 11]. Comparison of the amino acid sequence of BoNT/E9 with representatives of BoNT/E subtypes E1-E8 demonstrated that the most divergent region of the toxin was located in the last ~200 residues (Figure 2) which corresponds to the C-terminal part of the heavy chain (Hc-C) that is involved with binding to neuronal cells [14]. BLAST analysis of this region indicated < 75% amino acid sequence identity with other BoNT/E sequences.
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-12-245/MediaObjects/12866_2012_1859_Fig2_HTML.jpg
                    Figure 2

                    Comparative analysis of representative BoNT/E subtypes. Shown is a similarity plot comparing representative BoNT/E subtype amino acid sequences to BoNT/E9 (from strain CDC66177). The most divergent region of the amino acid sequence is shaded. Sequences from representative strains examined in this study or accession numbers retrieved from Genbank are compared in the plot as follows: E1, Beluga; E2, Alaska; E3, CDC40329; E4, AB088207 E5, AB037704; E6, AM695752; E7, Minnesota; E8, JN695730.

                    BLAST analysis of the 16S rRNA nucleotide sequence from strain CDC66177 shared > 99.8% identity with strains Alaska E43 and 17B indicating that the strain clusters with other Group II C. botulinum strains [9].

                    Mass spectrometric analysis of BoNT/E produced by strain CDC66177

                    Since the BoNT/E produced by strain CDC66177 appeared to be a previously unreported toxin subtype, the enzymatic light chain activity of the toxin was assessed in culture supernatants generated from the strain. The light chain of BoNT/E cleaves the synaptosomal-associated protein, SNAP-25, and the Endopep-MS method was used to measure this activity upon a specific peptide substrate mimic of SNAP-25 (IIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKT). Endopep-MS analysis revealed that the toxin cleaved the peptide substrate for BoNT/E in the expected location, resulting in products with peaks at m/z 1136.8 and 2924.2 [15] (Figure 3A).
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-12-245/MediaObjects/12866_2012_1859_Fig3_HTML.jpg
                    Figure 3

                    Mass spectral analysis of BoNT/E9. Panel A shows the products of endopeptidase cleavage of a type E specific peptide substrate detected by mass spectrometry. Peaks indicating the cleavage of the substrate by the toxin are marked with asterisks. Panel B illustrates the sequence coverage of BoNT/E9 amino acid sequence determination (in red font) of tryptic digestion products using mass spectrometry.

                    BoNT/E9 extracted from culture supernatants of strain CDC66177 was subjected to tryptic digestion and the products were analyzed by mass spectrometry to confirm that the toxin's amino acid sequence was indeed unique based on the predicted translation of the DNA sequence. The amino acid sequence of BoNT/E9 was determined with 94.5% coverage (Figure 3B).

                    DNA microarray analysis of strain CDC66177

                    A Group II C. botulinum subtyping DNA microarray [16] was used to evaluate gene content in a panel of 21 Group II strains from the CDC culture collection. Briefly, this array featured 495 probes targeting ~15% of the annotated genes in the C. botulinum type E strain Alaska E43 and 5 additional probes targeting genes present on the bont/B-encoding plasmid (pCLL) in C. botulinum type B strain 17B. Genomic DNA isolated from 15 type E strains (not including CDC66177) hybridized with 90.5% of the probes on this array while DNA isolated from type B strains (N=4) and type F strains (N=2) hybridized with 71.9% and 71.0% of the probes, respectively. Genomic DNA from strain CDC66177 hybridized with 66.8% of the probes present on the array.

                    Comparison of the profile of present or absent genes demonstrated the presence of two clusters of strains (Figure 4). Cluster 1 consisted entirely of type E strains. Interestingly, strain CDC66177 grouped with cluster 2 which included the Group II type B and type F strains examined in this study.
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-12-245/MediaObjects/12866_2012_1859_Fig4_HTML.jpg
                    Figure 4

                    Microarray analysis of Group II C. botulinum strains. Microarray hybridization profiles of Group II type B, E, and F strains were compared with a UPGMA dendrogram. Type E strains are shown in red, type B strains are shown in blue, and type F strains are shown in green. Cluster 1 consists entirely of type E strains, however, strain CDC66177 groups with Cluster 2.

                    Southern hybridization of the split rarA gene in strain CDC66177

                    In order to determine if the toxin gene cluster in CDC66177 inserted into the rarA operon as described for other type E strains [11, 13], we performed Southern hybridization using a probe that binds to the larger split rarA gene fragment in type E strains or the intact rarA gene in the type B strain 17B. Genomic DNA isolated from CDC66177, Beluga, and 17B was digested with XbaI and hybridized with the probe. The presence of XbaI sites flanking the intact rarA gene in strain 17B generated a ~2.8 kb fragment that hybridized the rarA probe shown in Figure 5. A ~7.4 kb fragment hybridized with the rarA probe in DNA isolated from strain Beluga. These results were expected based on analysis of the C. botulinum type E strain Beluga genome sequence (Genbank accession number: ACSC01000002) which demonstrated the presence of separate XbaI sites flanking the larger split rarA than found at the corresponding intact rarA gene in strain 17B (Genbank accession number: NC_010674). The rarA probe hybridized a similar size fragment in several other type E strains examined (data not shown). Unexpectedly, a ~1.7 kb band was hybridized by the probe using DNA isolated from strain CDC66177 suggesting the possibility that the regions flanking the toxin gene insertion in this strain were not similar to those of other type E strains.
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-12-245/MediaObjects/12866_2012_1859_Fig5_HTML.jpg
                    Figure 5

                    Southern hybridization of the rarA operon. Schematic representations of the regions surrounding the rarA operon are shown. The intact rarA gene in strain 17B or the split rarA fragments in strain Beluga are shaded. The probe used in the accompanying Southern blot (lane 1, 17B; lane 2, Beluga; and lane 3, CDC66177) targeted either the intact rarA gene in strain 17B or the larger rarA fragment (indicated by an asterisk) in strain Beluga. XbaI restriction sites are indicated by a red line and expected fragment sizes are shown.

                    Whole genome shotgun sequencing of strain CDC66177

                    Since the region flanking the rarA operon in strain CDC66177 was suspected to be unlike that of other type E strains, whole genome shotgun sequencing of this strain was performed using the PacBio SMRT sequencer. An ~3.85 Mb draft sequence consisting of 120 contigs was assembled (Genbank accession number: ALYJ00000000). Analysis of this sequence revealed that the toxin gene cluster inserted into the rarA operon (Figure 6). The nucleotide sequence of the bont/E gene extracted from the genome sequence data was identical to that determined previously by Sanger sequencing. The nucleotide sequence of a ~7.9 kb region starting at alaS and extending through CLH_1119 (relative to Alaska E43) was similar to that found in strain 17B but differed from the sequences found in strains Alaska E43 and Beluga.
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-12-245/MediaObjects/12866_2012_1859_Fig6_HTML.jpg
                    Figure 6

                    Organization of the toxin gene cluster and surrounding regions in CDC66177. The arrangement of genes in the toxin gene cluster and surrounding regions of strain CDC66177 is compared to that of Alaska E43. The toxin gene cluster of strain CDC66177 is located within the rarA operon similar to the arrangement in strain Alaska E43. Regions I and II (indicated by green font) contain putative insertion sequences and the location of split and intact rarA genes are shown. XbaI restriction sites (indicated by red lines) flanking the larger split rarA gene (indicated by an asterisk) are shown. The nucleotide sequence between alaS and the larger split rarA gene of the indicated strains was used to generate the neighbor-joining tree shown.

                    As shown in Figure 6, the regions between orfX3 and the larger split rarA fragment (region I) and between the smaller split rarA fragment and bont/E (region II) contain insertion sequences that are likely involved with transposon-mediated mobility of the toxin gene cluster [13]. It is notable that regions I and II differ in size and nucleotide sequence between strains Alaska E43 and CDC66177. In order to determine if the nucleotide sequences of these regions are strain-specific, we also performed an alignment of these regions with strain Beluga. Interestingly, region I in strain Beluga differed from both CDC66177 and Alaska E43 while region II was identical to that found in Alaska E43. While the mechanism of toxin gene cluster insertion into the rarA operon is unclear, the sequence similarity in region II between strains Beluga and Alaska E43 suggests at least a partial similarity in the origin of the recombination event that results in the insertion of the toxin gene cluster. However, strain CDC66177 lacks similarity to either strain Beluga or Alaska E43 at either region suggesting that the recombination event resulting in the insertion of the toxin gene cluster in strain CDC66177 originated differently compared to strains Beluga or Alaska E43.

                    Analysis of the genome sequence data explains the unexpected ~1.7 kb band hybridized by the rarA probe in strain CDC66177. The presence of an XbaI site within the toxin gene cluster of both CDC66177 and Alaska E43 and an additional site downstream of the larger rarA fragment in strain CDC66177 yield an ~1.7 kb fragment. Notably the genome sequence of strain 17B also demonstrates the presence of a XbaI site downstream of the intact rarA gene. Similar to other type E toxin gene clusters, strain CDC66177 contains an intact rarA gene that does not hybridize the rarA probe used in our studies. BLAST analysis of this gene demonstrated 98% nucleotide similarity with the gene present in Alaska E43.

                    Since the bont/E gene in strain CDC66177 displayed significant divergence compared to other reported bont/E genes, we compared the nucleotide sequences of the remaining toxin gene cluster components (ntnh, p47, orfX1-3) to those found in Alaska E43 and Beluga (Table 1). While these genes are nearly identical in Alaska E43 and Beluga, the genes in CDC66177 ranged from 88.2-96.9% nucleotide identity compared to those in Alaska E43 and/or Beluga.
                    Table 1

                    Pairwise alignment of toxin gene cluster components

                    Gene

                    % Nucleotide Identity

                    Alaska E43/CDC66177

                    Beluga E/CDC66177

                    Alaska E43/Beluga E

                    orfX3

                    94.9

                    94.9

                    100

                    orfX2

                    91.1

                    91.1

                    99.5

                    orfX1

                    94.9

                    94.9

                    100

                    p47

                    88.2

                    88.2

                    100

                    ntnh

                    96.8

                    96.9

                    99.9

                    bont/E

                    93.9

                    94.1

                    99.3

                    In order to further investigate the genomic sequence of strain CDC66177, the average nucleotide identity (ANI) of this strain was compared to Alaska E43 and Beluga. Briefly, 1,020 nucleotide fragments of the query genome were compared to the subject genome using BLAST to determine the ANI value [17]. Richter and Rosselló-Móra [17] proposed an ANI of 95-96% as the boundary of considering two genomes as belonging to a single bacterial species. While comparison of the genomes of strains Alaska E43 and Beluga resulted in an ANI > 97%, comparison of strain CDC66177 with Alaska E43 and Beluga resulted in ANI values between 93-94% (Table 2). Interestingly, comparison of strain CDC66177 with 17B displayed > 98% ANI while comparison of either Alaska E43 or Beluga with 17B resulted in ANI values < 94%. Importantly, only the strain 17B chromosomal sequence was used in these calculations. However, ANI calculations were based on the entire CDC66177 genome sequence since it is unknown if any of the contigs represent mobile elements such as plasmids. Notably, all three strains (Alaska E43, Beluga, and CDC66177), share nearly identical 16S rRNA sequences and clearly cluster with Group II C. botulinum (data not shown).
                    Table 2

                    Average nucleotide identity (ANI) of genomic sequences

                    Subject Sequence

                    Query Sequence

                    % ANI

                    Beluga

                    CDC66177

                    93.58

                    Beluga

                    17B

                    93.41

                    Beluga

                    Alaska E43

                    97.91*

                    CDC66177

                    Beluga

                    93.50

                    CDC66177

                    17B

                    98.91*

                    CDC66177

                    Alaska E43

                    93.73

                    17B

                    Beluga

                    93.53

                    17B

                    CDC66177

                    98.97*

                    17B

                    Alaska E43

                    93.67

                    Alaska E43

                    Beluga

                    97.78*

                    Alaska E43

                    CDC66177

                    93.63

                    Alaska E43

                    17B

                    93.50

                    The following genome sequences were used in the ANI analysis: Beluga, accession number: ACSC00000000 (4.0 Mb); CDC66177, accession number: ALYJ00000000 (3.85 Mb); 17B, accession number: NC_010674.1 (3.85 Mb); Alaska E43, NC_010723.1 (3.66 Mb).

                    * ANI values ≥ 96% are marked with an asterisk.

                    Our analysis of the genetic diversity of type E strains using a DNA microarray was limited to those isolated from botulism cases. Therefore, we considered the possibility that strain CDC66177 was genotypically divergent since it was isolated from an environmental source. We performed an in silico analysis of multilocus sequence typing (MLST) alleles from selected type E strains (representing isolates from soil and/or sediment, different MLST clades, and different BoNT/E subtypes) reported by Macdonald et al. [11]. These alleles were compared with alleles extracted from the genome sequences of strains 17B and CDC66177. Not surprisingly, strains 17B and CDC66177 formed a separate clade when concatenated MLST alleles were compared to other type E strains (Figure 7).
                    http://static-content.springer.com/image/art%3A10.1186%2F1471-2180-12-245/MediaObjects/12866_2012_1859_Fig7_HTML.jpg
                    Figure 7

                    In silico analysis of MLST alleles. Concatemers of MLST alleles for each strain were aligned with CLUSTALW and a UPGMA tree is shown. The scale represents number of differences. Strains isolated from soil and/or sediment sources are indicated with an asterisk. Strain CDC66177 clusters with strain 17B and separately from other type E strains.

                    Conclusions

                    In a previous study [18], botulinum toxin-producing clostridia were isolated from 23.5% of soil samples collected in Argentina. The distribution of toxin serotypes reported from the Southern region of Argentina included types A, B, and F. In this study, we characterized a previously unreported C. botulinum type E strain (CDC66177) isolated in 1995 from soil collected in Chubut, Argentina. This region is located at a latitude of approximately 43°S which is located as far from the equator as the Great Lakes are located in the Northern hemisphere. While strain CDC66177 was isolated from soil in proximity to the Atlantic Ocean, it is notable that no cases of type E botulism have been reported in Argentina. This is the first known report of the isolation of this strain and extends the known global distribution of C. botulinum type E.

                    While the strain CDC66177 produces a novel BoNT/E subtype, the toxin was shown to cleave a peptide substrate in the same location as other BoNT/E subtypes. It remains to be determined if the toxin produced by this strain varies in its neuronal cell receptor compared to other BoNT/E subtypes. Finally, the presence of bont/E in the rarA operon of a strain with genetic similarity to strain 17B raises the intriguing possibility of a bivalent non-proteolytic strain expressing BoNT/E encoded by a chromosomally located gene and BoNT/B encoded by a plasmid (such as pCLL found in 17B).

                    Methods

                    Bacterial strains used in this study

                    Bacterial strains used in this study are listed in Table 3. Strain CDC66177 was isolated in 1995 from soil collected in Dolavon, Chubut, Argentina (located approximately 58 km from the Atlantic Ocean). The soil sample was originally collected in 1993 in an urbanized area next to a perennial shrub (Ligustrum sinense). All C. botulinum strains were grown in Trypticase Peptone Glucose Yeast Extract Broth (TPGY) at 35°C under anaerobic conditions.
                    Table 3

                    Bacterial strains used in this study

                    Strain

                    bontsubtype

                    Source

                    Location

                    Year Isolated

                    bontAccession Number

                    Beluga†

                    E1

                    Fermented whale

                    Alaska

                    1982

                    GQ244314

                    CDC41648

                    E1

                    Seal flipper

                    Alaska

                    1996

                    JX424539

                    CDC42747

                    E1

                    Stool

                    Alaska

                    1997

                    JX424540

                    CDC42840

                    E1

                    Stool

                    Alaska

                    1997

                    JX424536

                    CDC47437

                    E1

                    Stool

                    Alaska

                    1992

                    JX424545

                    CDC5247

                    E2

                    Fermented seal flipper

                    Alaska

                    1984

                    EF028404

                    Alaska†

                    E2

                    Unknown

                    Unknown

                    Unknown

                    JX424535

                    CDC52256

                    E3

                    Stool

                    Illinois

                    2007

                    GQ294552

                    CDC59470‡

                    E3

                    Stink eggs

                    Alaska

                    2004

                    JX424544

                    CDC59471‡

                    E3

                    Stool

                    Alaska

                    2004

                    JX424542

                    CDC59498

                    E3

                    Stink head

                    Alaska

                    2004

                    JX424543

                    CDC42861

                    E3

                    Seal

                    Alaska

                    1997

                    JX424541

                    CDC40329

                    E3

                    Fish

                    Alaska

                    1995

                    JX424538

                    VH

                    E3

                    Unknown

                    Unknown

                    Unknown

                    GQ247737

                    Minnesota†

                    E7

                    Unknown

                    Unknown

                    Unknown

                    JX424537

                    CDC66177

                    E9

                    Soil

                    Argentina

                    1995

                    JX424534

                    CDC38597

                    B4

                    Blood sausage

                    Iceland

                    1983

                    JX437193

                    17B†

                    B4

                    Marine sediment

                    Pacific coast, US

                    1967

                    EF051570

                    CDC706

                    B4

                    Fermented salmon brine

                    Alaska

                    1977

                    JX437192

                    CDC30592

                    B4

                    Gastric fluid

                    Alaska

                    1985

                    JX437194

                    KA-173 (610B)

                    F6

                    Salmon

                    Columbia River, US

                    ~1966

                    GU213230

                    VPI7943

                    F6

                    Venison jerky

                    California

                    1966

                    GU213228

                    † Strain provided by J. Ferreira (FDA, Atlanta, GA).

                    ‡ Strains are associated with same botulism event.

                    DNA extraction, genetic analysis, and DNA microarray

                    Genomic DNA used in Sanger sequencing and DNA microarrays was extracted using the PureLink Genomic DNA kit (Life Technologies, Grand Island, NY). Neurotoxin and 16S rRNA gene sequences were determined using previously reported primers that amplified overlapping regions [9, 19]. Phylogenetic analysis was performed using CLUSTALX and the resulting phylogenetic tree was rendered using MEGA 5.05 [20]. Comparative analysis among representative BoNT/E subtypes was performed using SimPlot (http://​sray.​med.​som.​jhmi.​edu/​SCRoftware/​simplot/​) with a 200 amino acid window.

                    The Group II C. botulinum subtyping microarray was designed as described elsewhere [16]. Briefly, the microarray featured 495 probes representing genes distributed throughout the C. botulinum Alaska E43 genome sequence and 5 additional probes specific for pCLL which encodes the toxin gene cluster in strain 17B. Microarray spotting was performed by ArrayIt (Sunnyvale, CA) or onsite using an Omnigrid Micro microarrayer (Digilab, Holliston, MA). Genomic DNA was labeled with Cy5 random primers and hybridized to the array as previously described [21]. The log of the ratio of the mean fluorescence signal at 635 nm for triplicate probes compared to background fluorescence (locations spotted with buffer alone) was calculated. Log ratios ≥ 1.0 were considered positive and those < 0.5 were considered negative. Log ratios between 0.5 and < 1.0 were considered intermediate likely due to nucleotide sequence variation [21]. Hybridization profiles were converted to binary data by assigning 1 to positive probes and 0 to negative and intermediate probes. Profiles were compared using a UPGMA dendrogram generated with DendroUPGMA (http://​genomes.​urv.​cat/​UPGMA/​) and selecting the Jaccard coefficient. Microarray data were deposited in the Gene Expression Omnibus with series accession number GSE40271.

                    Southern hybridization

                    Genomic DNA was digested with XbaI for 1 h and run on a 1% TBE agarose gel. Alkaline transfer was performed using the TurboBlotter system (Whatman, Kent, ME). An 874 bp probe corresponding to the large rarA fragment was generated by PCR amplification with primers RarA-F and RarA-R (RarA-F, 5-GCAAGCACAACTGAAAATCCT-3; RarA-R, 5-CTCGGCTTTTGTXCAATTCATTAG-3) and labeled with the DIG DNA Labeling and Detection kit (Roche, Indianapolis, IN). Hybridization was carried out at 42°C in standard hybridization buffer (5X SSC, 0.1% N-laurylsarcosine, 0.02% SDS, 1% Blocking buffer (from DIG DNA Labeling and Detection kit).

                    Mass spectrometric analysis

                    Botulinum neurotoxin in culture supernatant CDC66177 was extracted and tested for light chain protease activity in a manner similar to that previously described [15], with the exception that 200 μL of culture supernatant was used for this study. Briefly, the neurotoxin was extracted from the culture supernatant using protein G beads coated with antibodies to BoNT/E. Following washing, the beads were then incubated for 4 h at 37°C with a peptide substrate known to be cleaved by BoNT/E in the presence of a reaction buffer. The reaction supernatant was then analyzed by MALDI-TOF mass spectrometry as described previously to determine the location of cleavage of the peptide substrate.

                    The reaction supernatant was then completely removed from the beads, and the toxin on the beads was digested and analyzed by LC-MS/MS essentially as described previously [22], with the exception that an Orbitrap Elite was used in place of the fourier transform magnetic trap. Briefly, the beads with toxin attached were digested with trypsin and then chymotrypsin. The resultant peptide mixtures were separated by nano-LC and mass analyzed on an Orbitrap Elite, generating MS/MS of the peptides. The MS/MS data were then searched against a database indexed for only Clostridium spp. for protein identification.

                    Whole genome sequencing and analysis

                    Genomic DNA was isolated from strain CDC66177 using the MasterPure kit (Epicenter, Madison, WI) with modifications previously described [23]. This DNA was further purified using a Genomic-tip 100/G column (Qiagen, Valencia, CA). One microgram of genomic DNA was sheared using a Covaris S2 ultrasonicator system to a mean size of 1 Kb. The sheared DNA was used to construct a SMRTbell sequencing library (Pacific Biosciences) according to manufacturer’s instructions. The SMRTbell library was then bound into SMRTbell-DNA polymerase complexes and loaded into zero-mode waveguides (ZMW) on 4 SMRTcells and sequenced using Pacific Biosciences C2 chemistry. This relatively small insert sized library was utilized to promote production of circular concensus reads (CCS) which retain higher accuracy base calls than the longer continuous length reads (CLR). Eight 45 min movies were recorded and processed, yielding ~305 K reads with a mean readlength of 2.9 Kbases and total of 889 Mbases of sequence. CCS reads (140 K reads) were then used to error correct the longer (165 K reads) CLR reads [24] utilizing the Pacific Biosciences analysis script BLASR and then the combined CCS/corrected CLR fastq format reads were imported into CLC Genomics workbench. Sequence reads were then trimmed of any remaining Pacific Biosciences hairpin adaptor sequences and quality trimmed to a base Q value of 20. The filtered reads were then assembled de novo using the CLC denovo assembler. The 188,898 input reads provided a draft assembly of a 3.85 Mb genome comprised of 119 contigs with an N50 value of 87,742 bases with an average coverage of 28X.

                    Annotation of the whole genome sequence was performed using RAST [25]. Pairwise alignments of various genes were made with EMBOSS Needle (http://​www.​ebi.​ac.​uk/​Tools/​psa/​emboss_​needle/​nucleotide.​html). ANI values were determined using the computer program JSpecies [17]. MLST loci from selected previously reported type E strains were obtained from Genbank [11]. These MLST loci were used to search for the corresponding alleles in the strain 17B genome sequence and the CDC66177 whole genome sequence using BLAST. Concatemers of the alleles for each strain were generated and a multiple sequence alignment was performed using CLUSTALW because the lengths of some alleles in strains 17B and CDC66177 differed due to insertion and/or deletions.

                    Declarations

                    Acknowledgements

                    Sanger sequencing was performed in the Genomics Unit within the Division of High Consequence Pathogens and Pathology at CDC. This publication was supported by funds made available from the Centers for Disease Control and Prevention, Office of Public Health Preparedness and Response. ML was supported by an Oak Ridge Institute for Science and Education fellowship. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

                    Authors’ Affiliations

                    (1)
                    Centers for Disease Control and Prevention
                    (2)
                    Area Microbiología, Universidad Nacional de Cuyo

                    References

                    1. Graham AF, Mason DR, Maxwell FJ, Peck MW: Effect of pH and NaCl on growth from spores of non-proteolytic Clostridium botulinum at chill temperature. Lett Appl Microbiol 1997, 24:95–100.PubMedView Article
                    2. McCroskey LM, Hateway CL, Fenicia L, Pasolini B, Aureli P: Characterization of an organism that produces type E botulinal toxin but which resembles Clostridium butyicum from the feces of an infant with type E botulism. J Clin Microbiol 1986, 23:201–202.PubMed
                    3. Horowitz BZ: Type E botulism. Clin Toxicol 2010, 48:880–895.View Article
                    4. Kautter DA: Clostridium botulinum in smoked fish. J Food Sci 1964, 29:843–849.View Article
                    5. Whittaker RL, Gilbertson RB, Garrett AS: Botulism, Type E. Ann Intern Med 1964, 61:448–454.PubMed
                    6. Hannett GE, Stone WB, Davis SW, Wroblewski D: Biodiversity of Clostridium botulinum type E associated with a large outbreak of botulism in wildlife from Lake Erie and Lake Ontario. Appl Environ Microbiol 2011, 77:1061–1068.PubMedView Article
                    7. Lúquez C, Dykes JK, Yu PA, Raphael BH, Maslanka SE: First report worldwide of an infant botulism case due to Clostridium botulinum type E. J Clin Microbiol 2010, 48:326–328.PubMedView Article
                    8. Collins MD, East AK: Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins. J Appl Microbiol 1998, 84:5–17.PubMedView Article
                    9. Hill KK, Smith TJ, Helma CH, Ticknor LO, Foley BT, Svensson RT, Brown JL, Johnson EA, Smith LA, Okinaka RT, Jackson PJ, Marks JD: Genetic diversity among botulinum neurotoxin-producing clostridial strains. J Bacteriol 2007, 89:818–832.View Article
                    10. Smith TJ, Lou J, Geren IN, Forsyth CM, Tsai R, Laporte SL, Tepp WH, Bradshaw M, Johnson EA, Smith LA, Marks JD: Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization. Infect Immun 2005, 73:5450–5457.PubMedView Article
                    11. Macdonald TE, Helma CH, Shou Y, Valdez YE, Ticknor LO, Foley BT, Davis SW, Hannett GE, Kelly-Cirino CD, Barash JR, Arnon SS, Lindström M, Korkeala H, Smith LA, Smith TJ, Hill KK: Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing. Appl Environ Microbiol 2011, 77:8625–8634.PubMedView Article
                    12. Chen Y, Korkeala H, Aarnikunnas J, Lindström M: Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype. J Bacteriol 2007, 189:8643–8650.PubMedView Article
                    13. Hill KK, Xie G, Foley BT, Smith TJ, Munk AC, Bruce D, Smith LA, Brettin TS, Detter JC: Recombination and insertion events involving the botulinum neurotoxin complex genes in Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains. BMC Biol 2009, 7:66.PubMedView Article
                    14. Schiavo G, Matteoli M, Montecucco C: Neurotoxins affecting neuroexocytosis. Physiol Rev 2000, 80:717–766.PubMed
                    15. Kalb SR, Garcia-Rodriguez C, Lou J, Baudys J, Smith TJ, Marks JD, Smith LA, Pirkle JL, Barr JR: Extraction of BoNT/A,/B,/E, and/F with a single, high affinity monoclonal antibody for detection of botulinum neurotoxin by Endopep-MS. PLoS One 2010, 5:e12237.PubMedView Article
                    16. Raphael BH: Exploring genomic diversity in Clostridium botulinum using DNA microarrays. Botulinum J 2:99–108.
                    17. Richter M, Rosselló-Móra R: Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A 2009, 106:19126–19131.PubMedView Article
                    18. Lúquez C, Bianco MI, de Jong LIT, Sagua MD, Arenas GN, Ciccarelli AS, Fernández RA: Distribution of botulinum toxin-producing clostridia in soils of Argentina. Appl Environ Microbiol 2005, 71:4137–4139.PubMedView Article
                    19. Lúquez C, Raphael BH, Maslanka SE: Neurotoxin gene clusters in Clostridium botulinum type Ab strains. Appl Environ Microbiol 2009, 75:6094–6101.PubMedView Article
                    20. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 2001, 28:2731–2739.View Article
                    21. Raphael BH, Joseph LA, McCroskey LM, Lúquez C, Maslanka SE: Detection and differentiation of Clostridium botulinum type A strains using a focused DNA microarray. Mol Cell Probes 2010, 24:146–53.PubMedView Article
                    22. Kalb SR, Baudys J, Rees JC, Smith TJ, Smith LA, Helma CH, Hill K, Kull S, Kirchner S, Dorner MB, Dorner BG, Pirkle JL, Barr JR: De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics. Anal Bioanal Chem 2012, 403:215–26.PubMedView Article
                    23. Raphael BH, Choudoir MJ, Lúquez C, Fernández R, Maslanka SE: Sequence diversity of genes encoding botulinum neurotoxin type F. Appl Environ Microbiol 2010, 76:4805–12.PubMedView Article
                    24. Bashir A, Klammer AA, Robins WP, Chin CS, Webster D, Paxinos E, Hsu D, Ashby M, Wang S, Peluso P, Sebra R, Sorenson J, Bullard J, Yen J, Valdovino M, Mollova E, Luong K, Lin S, Lamay B, Joshi A, Rowe L, Frace M, Tarr CL, Turnsek M, Davis BM, Kasarskis A, Mekalanos JJ, Waldor MK, Schadt EE: A hybrid approach for the automated finishing of bacterial genomes. Nat Biotechnol 2012, 30:701–7.PubMedView Article
                    25. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O: The RAST Server: rapid annotations using subsystems technology. BMC Genomics 2008, 9:75.PubMedView Article

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                    © Raphael et al.; licensee BioMed Central Ltd. 2012

                    This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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