A Family Outbreak of Type E Botulism Caused by Contaminated Vacuum-Packed Ambient-Stored Chili Chicken Feet in Zhangjiakou,China

Abstract

The epidemiological investigation and laboratory-based confirmation were performed on samples from a family botulism outbreak in Zhangjiakou, Hebei province, China. Forty-four samples, including 14 samples (leftover food, and swabs taken of both food packaging bags and dishes, and serum and vomitus of the victims) related to outbreak and 30 causative food products after outbreak, were collected and analyzed. Isolation, bacterial identification, toxin detection, and whole-genome sequencing of Clostridium spp. cultured from the latter samples and animal assays were performed. Mice injected with the cultures of the leftover chili chicken feet, together with the inner layer of its packaging bag, the plate for serving it, and supernatant of two patients' serum that demonstrated the typical signs of botulism. The polyvalent anti-botulinum neurotoxin (BoNT) and the monovalent anti-BoNT/E exhibited protective effects when administered to mice. Three Clostridium botulinum cultures were obtained and verified to be positive for BoNT/E. The whole genome analysis of the isolates revealed that the classic bont/e gene orfX cluster was found to be located on the chromosomes of all three isolates. Single nucleotide polymorphism analysis suggested that these might be from the same source. Our findings indicated that this botulism outbreak occurred following the ingestion of vacuum-packed chili chicken feet contaminated with BoNT/E produced by C. botulinum.

Introduction

Clostridium spp. are widely distributed in soil, marine, and freshwater sediments of fens, rivers, and lakes. Among these, Clostridium botulinum and rare strains of Clostridium butyricum and Clostridium baratii produce botulinum neurotoxins (BoNTs) (Mazuet et al., 2017; Camerini et al., 2019). Several distinct serotypes of the BoNT (denoted as A through G) have been described based on their antigenic properties, with types A, B, E, and F being responsible for cases of human botulism (Carter and Peck, 2015).

Botulism is a severe and life-threatening illness. In China, most cases result from the ingestion of preserved raw meat and homemade fermented soy and cereal contaminated with BoNT. The predominant BoNT serotypes varied geographically in China. The Xinjiang autonomous region has the highest recorded incidence of foodborne botulism, predominately type A toxin. In contrast, type B is the predominant serotype identified in other provinces (Shih and Chao, 1986).

In many countries, human type E botulism has been associated with consuming fish and aquatic mammals (Hyytiä et al., 1999; Horowitz, 2010; Leclair et al., 2013), but that is rare in China. The first case of type E botulism was reported in Panshi county of Jilin province, in northeast China in 1965, and homemade stinky tofu was implicated in the poisoning. Since then, most cases were found in northeast, northwest, and southwest China, far from lakes and oceans (Fu and Wang, 2008).

This study described the epidemiological and clinical investigation of a type E botulism outbreak in Zhangjiakou city, Hebei province, China. Also, the source for the contamination was tracked.

Materials and Methods

Ethics statement

Experiments related to both animals and human specimens were performed following the protocols approved by the Ethics Committee of the China National Centre for Food Safety Risk Assessment (CFSA) or the written informed consent from patients and their guardians. The CFSA office approved all experimental protocols of laboratory quality control. The management of experimental materials, equipment, and waste was carried out in compliance with the medical research regulations of the National Health Commission of the People's Republic of China.

Clinical data

In July 2019, a previously healthy family became intoxicated in a small village of Zhangjiakou city, Hebei province, China. Four individuals, including grandfather, grandmother, father, and grandson, gathered at home for dinner and the 14-year-old grandson developed nausea, began vomiting and feeling dizzy 10 h following this family meal. He was transferred to the county hospital and received treatment, including metabolic acidosis correction, intravenous infusion of cephalosporin, and fluids, after being diagnosed as gastroenteritis. Unfortunately, the grandson died 35 h after the appearance of his first symptoms.

Another three members also developed gastrointestinal symptoms and blurred vision subsequently, and they were admitted to the hospital in Beijing and diagnosed with botulism. All three members survived after being treated with polyvalent antineurotoxin administered on the basis that this episode was suspected to be an outbreak of botulism. The local Centre for Disease Control and Prevention was informed and responded rapidly. They visited the patients' home, conducted an epidemiological investigation, collected samples for further laboratory testing, and took the appropriate control measures, including controlling all foods with the same batches to those of being consumed by the victims. In addition, food samples were also collected from the manufactures, but all of them were negative on C. botulinum and BoNT.

Sample collection

A total of 44 samples, including 14 samples related to outbreak (4 leftover food consumed by patients, 4 swabs from the plastic bags of vacuum-packed food, 2 swabs of tableware, and 2 serum samples from patients) and 30 vacuum-packaged chili chicken feet with the lot number very closely to the causative foods at the retail level, were collected after outbreak, since the raw materials and final products with the same batch as the causative one has been used up or sold out. Clostridium isolation and BoNT testing were carried out following the methods issued by both Chinese government (China National Food Safety Standard GB 4789.12, 2016) and U.S. Food and Drug Administration (FDA) (Bacteriological Analytical Manual [BAM], Chapter 17 Clostridium botulinum 2001).

Sample enrichment

Four leftover food, 30 vacuum-packaged chili chicken feet, and 1 vomitus were diluted with equal volumes of gelatin phosphate buffer (GPB, pH 6.2, 2 g/L gelatin, and 4 g/L Na₂HPO₄). The swab samples from 4 food packaging plastic bags, 2 tableware items, and 1 trachea cannula were thoroughly vortexed in 10 mL of buffered peptone water.

A 2 mL aliquot of the sample was inoculated directly into two tubes of cooked meat medium (CMM; Beijing Land Bridge Company, China) and trypticase-peptone-glucose-yeast extract (TPGY; Beijing Land Bridge Company) that contained 10 mL enrichment broth.

All inoculated CMM and TPGY tubes were grown anaerobically in an anoxic jar (Anoxomat MARK II, Netherlands) for 5 days at 35 ± 1°C (CMM) and 28 ± 1°C (TPGY).

Isolation, identification of Clostridium, and bont-gene detection

A 1 mL of enrichment culture was mixed with an equal volume of absolute alcohol (Beijing Chemical Works, China) and incubated at room temperature for 1 h. A loopful of the mixture was then streaked onto Columbia blood agar (Beijing Land Bridge Company) and incubated anaerobically at 35 ± 1°C for 48 h.

Three putative colonies from the leftover read-to-eat (RTE) chili chicken feet, the inner swabbing of its plastic packaging bag, and swabbing of the plate for serving it were selected for gram staining and microscopic examination. 16S rRNA gene sequencing was performed following the recommended testing (Weisburg et al., 1991). The bont genes encoding neurotoxins A, B, E, and F by PCR were analyzed following the protocol described previously (FDA, BAM, Chapter 17 Clostridium botulinum 2001).

Determination of BoNTs in samples or cultures

Four leftover-food matrices, 2 serums, 1 vomitus and 30 vacuum-packaged chili chicken feet samples were diluted with equal volumes of GPB, respectively, and allowed to stand at ambient temperature for 30 min. The mixtures were centrifuged at 4000 × g (Hitachi, Japan) for 15 min, and the supernatant was filtered through a 0.45-μm membrane (Pall Corporation, USA).

All CMM or TPGY cultures after incubation in section 2.4 were centrifuged at 4000 × g for 15 min, and the supernatant was filtered through a 0.45-μm membrane.

All filtrates above were divided into three separate portions, respectively, for further treatment before the mouse bioassay (at least 1.8 mL for each group, denoted as groups I–III). In group I, the filtrates were heated at 100°C for 10 min. In group II, the filtrates were treated with 10% trypsin (Gibco, USA) at a ratio of 9 (1.8 mL supernatant or cultures): 1 (0.2 mL 10% trypsin) followed by incubation at 37°C for 1 h. No treatment was applied to group III.

Three female Institute Cancer Research (ICR) mice (Vitalriver Company, China) with a bodyweight ranging from 15 to 20 g in each group were intraperitoneally injected with an aliquot of 0.5 mL solution taken from group I, II, or III. In addition, BoNT/A (produced by type A C. botulinum, which was isolated from the causative food in China)-positive control group and 3 negative control groups, including GPB, CMM, and TPGY, were included. All mice were observed periodically over 96 h.

Typing of BoNT

Three isolates confirmed as C. botulinum from the leftover RTE chili chicken feet, the inner swabbing of its plastic packaging bag, and swabbing of the plate for serving it were purified. A single colony was inoculated into 10 mL TPGY and incubated anaerobically at 28 ± 1°C for 5 days. Each anti-BoNT, including hexavalent anti-BoNT A, B, C, D, E, F, and monovalent type-specific antitoxin to A, B, E, and F (Lanzhou Bio-product Institute Corporation, China), was prepared in 1 mL water, respectively. TPGY cultures were filtered through a 0.45-μm membrane. A 1 mL filtrate and 1 mL anti-BoNT were mixed and incubated at 37°C for 45 min. A 0.5 mL of mixture was injected into a mouse, each mixture for three mice repeat. BoNT/A-positive control and negative controls of GPB and TPGY were carried out. All mice were observed periodically for 96 h.

Genomic DNA purification and whole-genome sequencing

Genomic DNA of 3 C. botulinum isolates was extracted and purified using the Bacterial DNA Kit (OMEGA, USA) following the procedures recommended by the manufacturer. Whole-genome sequencing was carried out using the Pacific Biosciences RS II platform (Pacific Biosciences, USA; SMRTbell Template Prep Kit, Sequel binding and Internal Control Kit, SMRT Cell, Sequel Sequencing Kit were used) and Illumina NovaSeq PE150 platform (Illumina, USA; NEBNext^® UltraTM DNA Library Prep Kit was used). De novo assembly of the reads was performed using a hybrid assembly algorithm implemented in Allpaths-LG software (v44620; www.broadinstitute.org/software/allpaths-lg/blog/) (Gnerre et al., 2011).

Genomic analyses

The virulence factor database (www.mgc.ac.cn/VFs/main.htm) was employed to predict the presence of any known virulence factor in the genomes of sequenced strains with Basic Local Alignment Search Tool for Protein (BLASTP) and filtered with 75% identity and 95% match length (Altschul et al., 1990). The bont gene cluster and its flanking genes were identified by manual inspection and visualized by Mauve alignment of genomes within Geneious (Biomatters, Auckland, New Zealand). Parsnp (OSX64v1.2) from the Harvest suite of all 3 sequenced strains together with 98 available type E C. botulinum genomes downloaded from the National Centre for Biotechnological Information (NCBI) database (Supplementary Table S1) were used to construct the phylogenetic tree (Treangen et al., 2014). It was executed with the default options and Clostridium botulinum ATCC 9564 was used as reference.

ITOL v6 was used to visualize the tree (Letunic and Bork et al., 2016). Average Nucleotide Identity (ANI) analysis evaluated the genomic relationships using the recommended 95% identity for species identification (Goris et al., 2007; Varghese et al., 2015). Core genome single nucleotide polymorphism (SNP) analysis was employed using SAMtools (version 1.6) (Li et al., 2009), BWA (version 0.7.17) (Li and Durbin., 2009; 2010; Li, 2012), and GATK HaplotypeCaller (version 4.0.0) (Poplin et al., 2018) to understand the relationship between 3 isolates cultured from samples in this study.

Results

Isolation and identification of Clostridium

Three presumptive isolates of Clostridium were cultured from the leftover RTE chili chicken feet (coded ZJK-3), the inner swabbing of its plastic packaging bag (coded ZJK-8), and swabbing of the plate for serving it (coded ZJK-9). Other samples taken were negative for Clostridium (Table 1).

Table 1.

A Table Providing an Information Summary of Isolate Identification and Botulinum Neurotoxin Detection from Samples in This Study

Sample category	Sample source	Sample name	No. of samples	Isolates identification	BoNT detection (directly)	BoNT detection (culture)
Related to outbreak	Leftover foods	Pickled vegetables	1	—	—	—
		Tremella	1	—	—	—
		Vacuum-packaged RTE chili chicken feet	1	Clostridium botulinum	—	E
		Mashed garlic	1	—	—	—
	Food packaging materials	Plastic bag for RTE quail eggs packaging	1	—	—	—
		Plastic bag for RTE Spanish mackerel with black bean packaging	1	—	—	—
		Plastic bag for RTE chili chicken feet packaging	1	C. botulinum	—	E
		Plastic bag for RTE sausage packaging	1	—	—	—
	Tableware	Plate for serving RTE chili chicken feet	1	C. botulinum	—	E
	Tableware	Bowl for serving other food	1	—	—	—
	Patients	Grandson's vomitus	1	—	—	—
		Grandson's serum	1	/	E	/
		Grandson's trachea cannula	1	—	—	—
		Grandfather's serum	1	/	E	/
Related to food production line	Manufactures	Vacuum-packaged chili chicken feet at the retail level with the lot number closely to the causative foods	30	—	—	—
	Total		44	3	2	3

Note: “E” means BoNT/E positive; “—” means BoNT negative; “/” means not applicable.

The morphological appearance of all 3 isolates on Columbia blood agar included a white, semitransparent colony with irregular contours. The colonial diameter was much smaller than those of toxigenic isolates of serotypes A and B. When examined microscopically, the 3 isolates produced oval-shaped spores that seemed to be a tennis racket after gram-staining. 16S rRNA gene sequencing, and bont/e gene detection by PCR (Fig. 1) confirmed that all three isolates were type E toxin-producing C. botulinum and all were subjected to whole-genome sequencing.

FIG. 1.

A 1% agarose gel showing bont/e gene PCR detection.

BoNT identification in samples and cultures

The filtrates of CMM and TPGY cultures of leftover RTE chili chicken feet, swabs of its inner plastic packaging bag, and the plate used for serving it, and 2 patients' serum, were positive for BoNT. Moreover, the intraperitoneal injection of cultures or serum resulted in mouse death within 24 h. The typical signs of BoNT poisoning commenced with ruffling of the animal fur, followed by labored breathing (noted wasp waist when breathing), weakness of the limbs, and finally total paralysis with gasping for breath before succumbing. Moreover, mice injected with the same cultures or serum treated with trypsin died sooner than these not trypsinized.

However, neither poisoning symptom nor death was observed in mice intraperitoneally injected with the above-mentioned cultures or serum after heating. In addition, filtrates of TPGY cultures inoculated with 3 C. botulinum isolates obtained from samples related to chili chicken feet also gave rise to the same clinical signs and death of animals. All symptoms and death in mice were consistent with those observed in type A BoNT-positive control.

The serotyping results revealed that mice injected with monovalent anti-BoNT A, B, or F developed botulism symptoms and eventually died, while those injected with monovalent anti-BoNT E and polyvalent anti-BoNT survived.

Whole-genome sequencing of C. botulinum

Whole-genome sequences of the C. botulinum isolates coded ZJK-3, ZJK-8, and ZJK-9 were determined and submitted to GenBank. The genome sequence consisted of a single circular chromosome and two circular plasmids for Clostridium botulinum ZJK-3 and three circular plasmids for Clostridium botulinum ZJK-8 and ZJK-9. A summary of this genomic information and the accession numbers is shown in Table 2.

Table 2.

A Table Showing General Information Describing the Features of All 3 Sequenced Clostridium botulinum Genomes

Features	Clostridium botulinum ZJK-3			Clostridium botulinum ZJK-8				Clostridium botulinum ZJK-9
Features	Chromosome (Accession: CP070936)	pZJK3.1 (Accession: CP070937)	pZJK3.2 (Accession: CP070938)	Chromosome (Accession: CP070939)	pZJK8.1 (Accession: CP070940)	pZJK8.2 (Accession: CP070942)	pZJK8.3 (Accession: CP070941)	Chromosome (Accession: CP070943)	pZJK9.1 (Accession: CP070945)	pZJK9.2 (Accession: CP070946)	pZJK9.3 (Accession: CP070944)
Genome size (bp)	3,780,354	14,381	3358	3,821,175	14,381	3358	9886	3,783,352	14,381	3358	9886
G + C content (%)	27.4	27.8	31.4	27.4	27.8	31.4	27.5	27.4	27.8	31.4	27.5
No. of predicted coding sequences (CDS)	3423	16	3	3477	16	3	9	3423	16	3	9
No. of rRNA operons	34	0	0	34	0	0	0	34	0	0	0
No. of tRNA operons	76	0	0	76	0	0	0	76	0	0	0
No. of CRISPR regions	0	0	0	0	0	0	0	0	0	0	0
Filtered reads	13041			27145				28367
Mean coverage	284.99			473.04				486.59
N50 length (bp)	167,376			64,358				273,442
N90 length (bp)	33,933			15,242				62,150
Maximum length (bp)	419,699			226,785				794,420
Minimum length (bp)	1011			693				1218

BoNT, botulinum neurotoxin; CDS, coding sequence; CRISPR, clustered regularly interspaced short palindromic repeats; rRNA, ribosomal ribonucleic acid; RTE, read-to-eat; tRNA, transfer ribonucleic acid.

Genetic characterization of BoNT/E-producing bacteria

SNP analysis showed that the 3 isolates were nearly identical, with only 5, 7, and 8 SNP differences being recorded. The genes encoding virulence factor BoNT/E were located on the chromosome, and no virulence genes were identified on the sequenced plasmids.

Subtypes of bont/e-encoding genes in the isolates were analyzed at the nucleotide and amino acid levels, with a similar result. All 3 isolates were identified as subtype E3 with identity values higher than 99.9%. The bont/e gene was in the classic orfX cluster in all 3 isolates. The orfX cluster sequences and flanking regions matched the reference genomes of subtype E1 strain Clostridium botulinum NCTC 8226 and E3 strain Alaska E43 downloaded from the NCBI database (Fig. 2). This finding indicated that these genes were highly conserved with nucleotide identities >99.5%.

FIG. 2.

A detailed physical map showing the conserved orfX cluster containing bont/e gene (red arrowhead) mapped on the chromosomes of Clostridium botulinum ZJK-3; ZJK-8; ZJK-9 cultured from samples taken in the study along with the same locus identified in Clostridium botulinum NCTC 8226 and Alaska E43 downloaded from NCBI. NCBI, National Centre for Biotechnological Information.

A phylogenetic tree was generated based on the analysis of core-genome sequence alignment and is shown in Figure 3. The relationships among different subtypes were marked in the figure, and their relative similarity was inversely proportional to their distance. Furthermore, the 3 C. botulinum isolates were in the same clade. Four closest strains to them were Saroma, SAR, IFR18/112, and IFR18/132, which were subtype E1.

FIG. 3.

A single nucleotide polymorphism-based phylogenetic tree showing the relationships between the three study Clostridium botulinum ZJK-3, ZJK-8, and ZJK-9 compared with 98 type E C. botulinum strains obtained from NCBI. NCBI, National Centre for Biotechnological Information.

Furthermore, ANI analysis showed 3 C. botulinum isolates in this study with identities above 99.999%. Compared with other 98 C. botulinum strains, the identities ranging from 97.5% to 98.5% (Supplementary Table S2).

Discussion

The majority of human botulism cases reported are caused by BoNT/A and B types. In contrast, type E botulism, originally described in 1936, is rare (Gunnison et al., 1936). BoNT/E can be produced by phenotypically and genetically different Clostridium spp., including C. botulinum and some strains of C. butyricum (Meng et al., 1997). The type E C. botulinum strains can grow and produce toxins at 3°C (Peck, 2009; Brunt et al., 2020). Therefore, strains forming type E neurotoxin predominate in sediments, fish, and sea animals of the arctic and subarctic regions, such as Alaska, Nunavik, Quebec, or regions of northern Europe. Moreover, type E botulism has been noted as a substantial risk to humans in these regions (Hielm et al., 1998; Leclair et al., 2013). Most type E botulism cases in China were reported on the Qinghai-Tibet plateau or northeast inland regions of China located far from the sea.

Fermented soybean food and dried raw meat products were the primary causative foods leading to type E botulism (Fu and Wang, 2008). Hence, homemade, uncooked, processed, or poorly preserved foods stored for a long time, even at low temperatures, are recognized risk factors for food safety in these regions. Nevertheless, in this study, three type E C. botulinum isolates were detected from samples related to RTE vacuum-packaged chili chicken feet. Whole genome sequencing analysis showed that 3 isolates might be from the same source.

The epidemiological data showed that all vacuum-packaged foods in this outbreak were best before dates and purchased from two villages near patient's home. They are RTE foods without any heating before consumption. Whereas pickled vegetables were homemade food that dressing with salt in a bowl and let them stand for about 1 to 2 days before eating. Besides, tremella and mashed garlic were also homemade before dinner. The laboratory-based results indicated that this outbreak was caused by the contaminated vacuum-packaged chili chicken feet, which is popular for its hot and sour taste in China. The production process of chili chicken feet is complicated, with typical food preparation involving boiling the chicken feet in water for 25 min followed by transfer to cold water for cooling.

The cooled chicken feet are mixed with pickle juice made by boiling various spices in water; then, they are put in a container and stored at 8–15°C for 12–15 h. Some food flavorings (plant source) are subsequently added to the pickled chicken feet, stirred, bagged, and vacuum sealed. The vacuum-packaged chili chicken feet analyzed in this study had a pH 5, 2.25% NaCl, and 9-month shelf life when stored at room temperature. It was hypothesized that if the factory did not have a good environmental sanitation control, the contamination of C. botulinum in the chili chicken feet might happen during preparation, flavoring addition, or packaging steps.

As we know, type E C. botulinum are capable of growth and toxin production even at 3°C, pH 5.0–7.3, and 0.1% to 5.0% NaCl concentration (Graham et al., 1996). In our study, the pH 5 and 2.25% salinity of the final product and a nearly 6-month long-time storage (from production to consumption) at room temperature might be responsible for type E C. botulinum growth and toxin production.

Conclusions

This botulism outbreak was caused by the consumption of RTE vacuum-packaged chili chicken feet contaminated with BoNT/E produced by C. botulinum. Our report highlighted that the risk of botulism from processing contamination of commercially produced foods, especially vacuum-packaged foods, was of concern to public health. Pay close attention to environmental sources of botulinum neurotoxigenic clostridia contamination. Therefore, some necessary steps should be taken to reduce the exposure to pathogens and BoNTs production along the food processing chain. Such as the application of sanitisers and sterilization operation, shorten the shelf life or lower the storage temperature, monitoring incidents of botulism attributed to food, particularly those canned or vaccum-packaged foods.

Footnotes

Disclosure Statement

The authors declare that they have no competing interests.

Funding Information

This study was supported by Natural Science Foundation of Beijing Municipality, China (Grant No. 7212146).

Supplementary Material

Supplementary Table S1

Supplementary Table S2

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