Quantitative Prevalence,Phenotypic and Genotypic Characteristics of Bacillus cereus Isolated from Retail Infant Foods in China

Abstract

Bacillus cereus is an important foodborne pathogen, which can cause severe food poisoning. The aim of this study was (i) to evaluate the quantitative prevalence of B. cereus in retail prepackaged infant formula and ready-to-eat rice flour in China and (ii) to gain the basic information on pheno- and genotypic characteristics of B. cereus isolates. We found that 40 out of the 587 samples were positive for B. cereus. B. cereus in 3.5% of infant formula samples and 1.0% of rice flour samples outnumbered 100 Colony-Forming Units (CFU)/g. B. cereus level even attained 10³–10⁴ CFU/g in four infant formula samples and one rice flour sample. Furthermore, we identified the distribution patterns of toxin genes in B. cereus isolates. The results showed that 97.5% of B. cereus isolates harbored at least one enterotoxin gene. Antibiotic susceptibility tests revealed that all isolated B. cereus strains were resistant to penicillin and 50% of them were multidrug resistant. Thirteen new sequence types (STs) and four new alleles were identified via multilocus sequence typing. Clonal Complex (CC) ST-205 and CC ST-142 were predominant clonal complexes. Interestingly, we revealed the special relationship between STs of B. cereus isolates and the geographical distributions of infant food manufacturers for the first time. The data implied that B. cereus of different STs might have a distinct ecological niche in China. In view of relatively high contamination level of enterotoxin- producing B. cereus in a proportion of infant foods, especially in those suitable for the ≤6-month-old infant group, appropriate safety criteria and hygienic control measures for infant foods should be drafted in China to prevent B. cereus infection.

Introduction

B acillus cereus is a Gram-positive, spore-forming rod bacterium. It inhabits ubiquitously in natural environment due to strong survival ability under unfavorable conditions and high resistance of its endospore to various stresses (Bottone, 2010; Merzougui et al., 2014). Wide distribution and adhesive force of endospore enable B. cereus to contaminate a wide variety of foods (Schoeni and Wong, 2005; Senesi and Ghelardi, 2010).

As an important foodborne pathogen around the world, B. cereus was recorded as the fourth major cause of notified foodborne outbreaks in the European Union [European Food Safety Authority, European Centre for Disease Prevention and Control (EFSA, ECDC) 2015] and the second major causative agent in weak evidence foodborne outbreaks in France (Glasset et al., 2016). From 1998 through 2008, 1.74% of the reported foodborne outbreaks were caused by B.cereus in the United States (Bennett et al., 2013). In China, B. cereus was attributed to 6.8% of pathogenic microorganism-induced outbreaks from 1992 to 2001 (Liu et al., 2004). More than 3000 patients were affected by B. cereus infection from 2003 to 2008. Among them, around 2000 patients had been hospitalized (Mao et al., 2011). Furthermore, B. cereus is a health threat to the infant group, and 0.61% of bacteria-related acute diarrhea cases in infants were caused by B. cereus in Kosovo during a period of 7 years (Azemi et al., 2013). Although foodborne illness caused by B. cereus is usually self-limiting, it can be fatal in some outbreaks and sporadic cases (Dierick et al., 2005; Naranjo et al., 2011). B. cereus mainly causes two types of food poisoning: the diarrheal syndrome, characterized by abdominal pain and watery diarrhea symptoms, resulted from ingestion of vegetative cells of enterotoxin- producing B. cereus; and the emetic syndrome, characterized by vomiting symptom and attributed to intake of foods contaminated with a dodecadepsipeptide toxin cereulide secreted by B. cereus emetic strains (Senesi and Ghelardi, 2010).

The safety of foods for infants is all the time causing the world concern due to the specific consumer group with low immunity to pathogens. To date, several countries have made guidelines on B. cereus standard for infant foods (Hasell and Salter, 2003). However, there are still no associated safety criteria on B. cereus in various foods, including infant foods in China. Furthermore, little information on the contamination level and characteristics of B. cereus in infant foods has been reported. Therefore, the aim of this study was (i) to evaluate the quantitative prevalence of B. cereus in prepackaged infant foods, including infant formula and ready-to-eat rice flour, at retail in markets and (ii) to gain basic information on the characteristics of B. cereus isolates from infant foods.

Materials and Methods

Sampling and isolation of B. cereus

A total of 587 prepackaged infant foods were sampled from markets from 2013 to 2015, 401 of which were infant formulae from 65 brands and 186 of which were ready-to-eat rice flour from 53 brands. The infant formulae were suitable for different age groups, including 0–6, 0–12, and ≥6 months. The rice flour was for all aged ≥6 months. The number of B. cereus in each sample was tested using the direct plating method. Briefly, 25 g of each sample was suspended in 225 mL of PBS and homogenized for 2 min. The homogenate was 10-fold serially diluted in sterilized saline. The dilutions were separately spread on Mannitol–Egg Yolk–Polymyxin (MYP) agars in duplicate. Plates were incubated at 30°C for 24 h. Five suspect colonies on each plate were selected for identification. All suspect colonies were selected if less than five colonies grew on one plate. The Food and Drug Administration Bacteriological Analytical Manual (U.S. FDA, 2012) was followed to calculate B. cereus numbers and confirm B. cereus isolates. One confirmed colony isolated from each positive sample was stored at −70°C for further analysis.

Detection of toxin genes

The DNA of B. cereus isolates was extracted using the Bacteria DNA Extraction Kit (Omega). The primers were designed as previous reports, including ces (Ehling-Schulz et al., 2005), hblA (Zhou et al., 2008), hblC, hblD, nheA, nheB, nheC (Melnick et al., 2012), bceT (in't Veld et al., 2001), cytK1, cytK2 (Guinebretiere et al., 2006), and entFM (Ngamwongsatit, 2008). The polymerase chain reaction (PCR) system (25 μL) contained 50 ng of DNA template, 0.5 μL of each primer (10 μM), 0.125 U of Taq polymerase (TaKaRa, China), 2.5 μL of 10 × PCR buffer (Mg2⁺ free), 1.5 μL of MgCl2 (25 mM), and 2 μL of dNTP mixture (2.5 mM each). PCRs were performed as follows: 95°C for 2 min, 30 cycles of denaturation at 95°C for 30 s, annealing for 30 s, and elongation at 72°C for 1.5 min. The anneal temperature differed according to the Tm of primers. The PCR products were analyzed with electrophoresis on 1% agarose gel.

Antibiotic susceptibility tests

Antibiotic susceptibility of forty B. cereus isolates was determined by using the broth microdilution minimum inhibitory concentration (MIC) method following the Clinical and Laboratory Standards Institute (CLSI) guidelines (Clinical and Laboratory Standards Institute, 2015). Staphylococcus aureus strain ATCC 29213 was used as quality control strain. The MIC results were interpreted based on the breakpoint for Bacillus species advised in CLSI documents M45-A3 (Clinical and Laboratory Standards Institute, 2015), except for ceftriaxone, of which the breakpoint was according to CLSI documents M45-A2 (Clinical and Laboratory Standards institute, 2010). The strains that resist three or more types of antibiotics belonging to different antibiotic classes were defined as multidrug-resistant strains (Magiorakos et al., 2012).

Multilocus sequence typing analysis and phylogenic tree construction

Seven housekeeping genes glpF, gmk, ilvD, pta, pur, pycA, and tpi were amplified following the PubMLST scheme (http://pubmlst.org/bcereus/info/primers.shtml) (Jolley and Maiden, 2010) and sequenced in 3730XL Genetic Analyzer (Applied Biosystems). The nucleotide sequences were submitted to PubMLST database. Sequence type (ST) was designated to each B. cereus isolate. The clonal complex was determined using online eBURST V3 software (http://eburst.mlst.net/v3/mlst_datasets/) with the n value at 5. Minimum spanning tree was constructed by using BioNumerics 6.6 software based on multilocus sequence typing (MLST) analysis. The phylogenic tree was computed using MEGA 5.1 software based on the concatenated sequence (2829 bp) of seven housekeeping genes. Neighbor-joining method was applied as statistical method. The number of Bootstrap replications was 1000.

Results and Discussion

Quantitative prevalence of B. cereus in infant foods

The contamination level of B. cereus in infant formula and ready-to-eat rice flour suitable for different age groups at retail in China was estimated and the results are presented in Table 1. The percentage of positive samples (B. cereus ≥10 CFU/g) in infant formula was 8.2%, which was significantly higher than that in rice flour (3.8%) (p < 0.05). The data confirmed that milk products were more frequently contaminated by B. cereus as reported previously (Bartoszewicz et al., 2008; Merzougui et al., 2014). B. cereus is an opportunistic foodborne pathogen, and high-dose ingestion of vegetative cells or emetic toxin may result in gastroenteritis or/and vomiting syndrome. 10⁵–10⁸ cells or 8 μg of emetic toxin per kilogram of body weight was suggested to be the poisonous dose for adults (Paananen et al., 2002; Schoeni and Wong, 2005), whereas the poisonous dose for infants was still obscure. Epidemiological data suggested that infant foods contaminated by 10²–10⁴ CFU/g B. cereus could induce infant food poisoning (Duc et al., 2005; Ye, 2008). In our results, B. cereus in 3.5% of infant formula samples and 1.0% of rice flour samples outnumbered 100 CFU/g. Once infants were fed with these high contaminated foods, food poisoning might occur. The contaminated infant foods at retail raised a potential and big risk for the infant group.

Table 1.

Quantitative Prevalence of Bacillus cereus in Infant Formula and Ready-to-Eat Rice Flour

			B. cereus level (CFU/g), n (%)
Samples	N	Positive, n (%)	10–10²	10²–10³	10³–10⁴	ND (<10)
Infant formula	401	33 (8.2)	19 (4.7)	10 (2.5)	4 (1.0)	368 (91.8)
For 0–6 months	143	10 (7.0)	5 (3.5)	3 (2.1)	2 (1.4)	133 (93.0)
For 0–12 months	151	11 (7.3)	10 (6.6)	1 (0.7)	0 (0)	140 (92.7)
For ≥6 months	107	12 (11.2)	4 (3.7)	6 (5.6)	2 (1.9)	95 (88.8)
Ready-to-eat rice flour (for ≥6 months)	186	7 (3.8)	5 (2.7)	1 (0.5)	1 (0.5)	179 (96.2)
Total	587	40 (6.8)	24 (4.1)	11 (1.9)	5 (0.9)	547 (93.2)

ND, not detected.

Several countries have made safety criteria for B. cereus in infant foods. The acceptable level of B. cereus in infant foods is <10 CFU/g in Chile and <100 CFU/g in the Netherlands (Lv et al., 2002). Three-class sampling plans are adopted in Australia and New Zealand, n, c, m, M are set 5, 2, 10, 100 CFU/g for infant formula (Hasell and Salter, 2003). Although B. cereus in most of the infant food samples was <10 CFU/g in our study, B. cereus in 3.5% of infant formula samples and 1.0% of rice flour samples outnumbered 100 CFU/g, which exceeded the acceptable level of many countries. Moreover, B. cereus in four infant formula samples and one rice flour sample attained 10³–10⁴ CFU/g, even higher than the acceptable level for ready-to-eat foods (not for infants) advised in South Korea, Australia, and New Zealand (NSW Food Authority, 2009; Chon et al., 2015). 31.3% of the samples contaminated by high level of B. cereus (≥100 CFU/g) were suitable for the ≤6-month-old infant group. Our data suggested that appropriate safety criteria and hygienic control measures for B. cereus were necessary to improve the microbiological safety of infant foods in China.

Toxin gene profile of B. cereus isolates

The pathogenic mechanism for diarrhea and emetic types of food poisoning caused by B. cereus has been in research for a long time. Secreted hemolysin BL (HBL), nonhemolytic enterotoxin, necrotic enterotoxin CytK, enterotoxin FM (EntFM), and BceT are determined to be the major virulence factors for diarrheal disease (Granum and Lund, 1997; Schoeni and Wong, 2005). The emetic syndrome is caused by emetic toxin cereulide (Ehling-Schulz et al., 2005). Survey of the above enterotoxin encoding genes and essential synthesis gene of cereulide can assess the potential pathogenic ability of B. cereus isolates (Anderson Borge et al., 2001; Guinebretière et al., 2002; Kim et al., 2011; Glasset et al., 2016).

A total of 17 distribution patterns of toxin genes (Table 2) were determined. The predominant one was pattern II, which consisted of 22.5% of isolates. Strains belonging to this group possessed nine enterotoxin genes. 97.5% of isolates harbored at least one enterotoxin gene. The nheABC genes were present in 90% of isolates in agreement with reports that most of the B. cereus strains isolated from various food source samples contained nhe genes (Anderson Borge et al., 2001; Guinebretière et al., 2002; Kim et al., 2011). The detection rate of nheABC was also approximately close to that in isolated strains associated with foodborne outbreaks caused by B. cereus (Glasset et al., 2016). The occurrence frequency of hblCDA in B. cereus isolates was 52.5%. However, the detection rate in infant formula source and rice flour source strains was 45.5% and 85.7%, respectively, showing a large difference between the two types of infant foods. The detection rate in infant formula was consistent with the previous reports of infant formula or other milk products (Zhou et al., 2008; Hwang and Park, 2015). The detection rate in rice flour samples was similar to that of ready-to-eat foods, including vegetables, rice, and grain-based foods (Anderson Borge et al., 2001; Chon et al., 2015; Hwang and Park, 2015). Along with other researches, our results validated that the detection rate of hblCDA in B. cereus isolated from milk products was much lower than that from other types of foods.

Table 2.

Toxin Gene Distribution Profile in Bacillus cereus Isolates

Patterns	Ces	hblA	hblC	hblD	nheA	nheB	nheC	cytK1	cytK2	bceT	entFM	No. of strains (%)
I	+	−	−	−	+	+	+	−	+	+	−	1 (2.5)
II	−	+	+	+	+	+	+	−	+	+	+	9 (22.5)
III	−	+	+	+	+	+	+	+	−	+	−	1 (2.5)
IV	−	+	+	+	+	+	+	−	+	−	+	3 (7.5)
V	−	+	+	+	+	+	+	−	−	+	+	3 (7.5)
VI	−	+	+	+	+	+	+	−	−	+	−	1 (2.5)
VII	−	+	+	+	+	+	+	−	−	−	+	1 (2.5)
VIII	−	+	+	+	+	+	+	−	−	−	−	3 (7.5)
IX	−	−	−	−	+	+	+	−	+	+	+	2 (5)
X	−	−	−	−	+	+	+	−	+	−	+	3 (7.5)
XI	−	−	−	−	+	+	+	−	−	+	+	1 (2.5)
XII	−	−	−	−	+	+	+	−	−	−	+	4 (10)
XIII	−	−	−	−	+	+	+	−	−	+	−	1 (2.5)
XIV	−	−	−	−	+	+	+	−	−	−	−	2 (5)
XV	−	−	−	+	+	+	+	−	+	−	+	1 (2.5)
XVI	−	−	−	−	−	−	−	−	−	−	+	3 (7.5)
XVII	−	−	−	−	−	−	−	−	−	−	−	1 (2.5)

CytK1 and CytK2 are two different forms of CytK in B. cereus strains. CytK1 presents more severe hazards than CytK2 and is determined responsible for highly serious outbreaks (Fagerlund et al., 2004; Guinebretiere et al., 2006). Although cytK was detected in various types of foodborne or clinical B. cereus isolates (Anderson Borge et al., 2001; Guinebretière et al., 2002; Kim et al., 2011; Chon et al., 2015), distribution of both cytK1 and cytK2 was scarcely reported. To the best of our knowledge, we investigated the profile of the two genes in B. cereus isolates from infant foods for the first time. Furthermore, although 50% of B. cereus isolates possessed either cytK1 or cytK2, cytK1 was only detected in one strain, accounting for 2.5%. This significant difference of the existence frequency of cytK1 and cytK2 hinted that although most of the B. cereus isolates might be relatively mild when causing diarrhea, the possibility of severe food poisoning outbreak caused by cytK1-positive strain existed in China.

Our study identified ces only in one strain, which is consistent with the known fact that ces-positive B. cereus strains are hardly isolated from food source samples (Arslan et al., 2014; Chon et al., 2015).

Antibiotic susceptibility assay

The susceptibility of all B. cereus isolates to twelve types of antibiotics is shown in Table 3. All strains were resistant to penicillin, which was consistent with previous findings that B. cereus isolates from either food or clinical source were highly resistant to penicillin antibiotic (Park et al., 2009; Merzougui et al., 2014). Meanwhile, all strains presented were susceptible to imipenem, vancomycin, amikacin, and rifampicin. The resistance rate of B. cereus to different cephem antibiotics varied according to our results along with previous reports. High resistance of more than 95% was present to ceftiofur and cefepime. However, ceftriaxone and cefotetan showed 47.5% and 30% resistance rate, which was relatively low compared to ceftiofur and cefepime (Park et al., 2009; Merzougui et al., 2014; Cui et al., 2016). High resistance rate of 82.5% was also found to trimethoprim/sulfamethoxazole. All B. cereus isolates were grouped into 8 antibiotic resistance patterns (Fig. 1.), including 20 isolates (50%) that were multidrug resistant. Eighty percent of the multidrug-resistant strains were resistant to penicillin, ceftriaxone, and trimethoprim/sulfamethoxazole. We even identified one strain that was resistant to six types of antibiotics.

FIG. 1.

Characterizations of Bacillus cereus isolates obtained in infant foods in China. The phylogeny tree was computed by MEGA 5.1 software with the neighbor-joining statistical method. The number of Bootstrap replications was set 1000. The numbers in brackets meant the serial number of antibiotic resistance profile patterns. PCN, penicillin; CTRX, ceftriaxone; E, erythrocin; TE, tetracycline; DA, clindamycin; SXT, trimethoprim/sulfamethoxazole.

Table 3.

Antibiotic Susceptibility of Bacillus cereus Isolates

Antibiotics (%)	PCN	CTRX	IPM	VA	AK	E	TE	CIP	DA	SXT	C	RFP
R	40 (100)	19 (47.5)	0 (0)	0 (0)	0 (0)	1 (2.5)	4 (10)	0 (0	3 (7.5)	33 (82.5)	0 (0)	0 (0)
I	0 (0)	21 (52.5)	0 (0)	0 (0)	0 (0)	9 (22.5)	1 (2.5)	2 (5)	28 (70)	0 (0)	8 (20)	0 (0)
S	0 (0)	0 (0)	40 (100)	40 (100)	40 (100)	30 (75)	35 (87.5)	38 (95)	9 (22.5)	7 (17.5)	32 (80)	40 (100)

R, resistant; I, intermediate; S, susceptible; PCN, penicillin; CTRX, ceftriaxone; IPM, imipenem; VA, vancomycin; AK, amikacin; E, erythrocin; TE, tetracycline; CIP, ciprofloxacin; DA, clindamycin; SXT, trimethoprim/sulfamethoxazole; C, chloramphenicol; RFP, rifampicin.

MLST and phylogeny analysis

High genetic diversity was present in B. cereus isolates (Table 4). Thirty-seven different STs were generated by using MLST, and the Simpson's diversity index (DI) attained 0.996 (Hunter and Gaston, 1988). Thirteen new STs were designated from ST 1314 to ST1326. Furthermore, four new alleles, including tpi AT206, tpi AT207, tpi AT208, and glpF AT252, were identified.

Table 4.

The Multilocus Sequence Typing Information of Bacillus cereus Isolates from Infant Foods

Isolate no.	ST	Clonal complex	Lineage	Source	Province, City or region ^a	Country ^a
BC01	1027	—	I	Infant formula	Guangdong	China
BC02	1063	—	II	Infant formula	—	Netherland
BC03	1075	—	I	Infant formula	Guangdong	China
BC04	1072	—	I	Infant formula	—	Netherland
BC05	1076	ST-205	I	Infant formula	Inner Mongolia	China
BC06	1071	—	I	Infant formula (goat)	Shaanxi	China
BC07	1323^b	—	I	Infant formula	—	Australia
BC08	1073	ST-205	I	Infant formula	—	Ireland
BC09	118	—	II	Infant formula	Guangdong	China
BC10	26	—	I	Infant formula	Shandong	China
BC11	1120	ST-205	I	Infant formula	Shandong	China
BC12	1074	—	I	Infant formula	Shaanxi	China
BC13	1314^b	—	I	Infant formula	Heilongjiang	China
BC14	1064	—	II	Infant formula	Jiangxi	China
BC15	1065	ST-205	I	Infant formula (goat)	Shaanxi	China
BC16	1067	—	I	Infant formula	—	Germany
BC17	1324^b	—	II	Infant formula	Inner Mongolia	China
BC18	32	—	I	Infant formula	Inner Mongolia	China
BC19	1069	ST-142	II	Infant formula	—	Germany
BC20	774	—	I	Rice flour	Guangdong	China
BC21	1315^b	ST-365	I	Rice flour	—	USA
BC22	1316^b	—	II	Rice flour	Zhejiang	China
BC23	1319^b	—	II	Rice flour	Zhejiang	China
BC24	1317^b	—	II	Rice flour	Fujian	China
BC25	90	—	I	Rice flour	Fujian	China
BC26	999	ST-142	II	Infant formula	Shanghai	China
BC27	999	ST-142	II	Infant formula	Guangdong	China
BC28	1325^b	—	II	Infant formula	Shanghai	China
BC29	1321^b	—	II	Infant formula	Guangdong	China
BC30	1321^b	—	II	Infant formula (goat)	Tianjing	China
BC31	1320^b	—	II	Infant formula	Shaanxi	China
BC32	1318^b	—	I	Rice flour	Heilongjiang	China
BC33	142	ST-142	II	Infant formula	Shanghai	China
BC34	504	ST-111	II	Infant formula	Inner Mongolia	China
BC35	1284	—	I	Infant formula	Fujian	China
BC36	26	—	I	Infant formula	Inner Mongolia	China
BC37	1282	ST-142	II	Infant formula	Zhejiang	China
BC38	1326^b	ST-205	I	Infant formula	Inner Mongolia	China
BC39	1322^b	ST-205	I	Infant formula	Heilongjiang	China
BC40	265	ST-23	II	Infant formula	Fujian	China

Province, city or region, and country meant the geographical distributions of infant food manufacturers.

New ST number designated by PubMLST (http://pubmlst.org/bcereus/).

ST, sequence type.

To date, there are a total 1330 STs in the B. cereus MLST database (http://pubmlst.org/bcereus/). Compared with the above-referenced data set, our isolates were grouped into five clonal complexes, including CC ST-205, CC ST-142, CC ST-111, CC ST-23, and CC ST-365. Among them, CC ST-205 and CC ST-142 were predominant.

All B. cereus isolates in our study were clustered into two lineages according to the phylogeny analysis (Fig. 1). CC ST-205, CC ST-365 members and fourteen singletons belonged to lineage I, CC ST-142, CC ST-111, CC ST-23 members and ten singletons belonged to lineage II. Distribution in two different lineages indicated that CC ST-205 and CC ST-142 might descend from different ancestors. In lineage I, CC ST-205 member ST1065 was the main evolutionary starting point, from which CC ST-205 members and eight singletons were evolved. In lineage II, CC ST-142 member ST999 was the main evolutionary starting point, from which three CC ST-142 members and 10 singletons evolved (Fig. 2.).

FIG. 2.

Minimum spanning tree (MST) of Bacillus cereus isolates. The MST was constructed by using BioNumerics 6.6 software based on multilocus sequence typing analysis. Each solid circle represents one ST. The number on branch was related to the allele differences between connected STs. The color of the solid circle represents the different geographical distributions of infant food manufacturers. ST, sequence type.

Interestingly, we revealed the special relationship between STs of B. cereus isolates and the geographical distributions of infant food manufacturers. Most of the strains isolated from infant foods made in Northern China, including Tianjing, Inner Mongolia Autonomous Region, Shaanxi Province, Heilongjiang Province, and Shandong Province, were grouped into Lineage I with strains mainly belonging to or evolved from CC ST-205. On the contrary, most of the strains isolated from infant foods made in Southern China, including Shanghai, Zhejiang province, Guangdong province, Jiangxi province, and Fujian province, were grouped into Lineage II with strains mainly belonging to or evolved from CC ST-142. Because all the infant foods are sealed packed after they were made from manufacturers, there was almost no possibility that infant foods could be contaminated by B. cereus in circulation. Therefore, B. cereus in infant foods might come from raw materials and/or production environments. The relationship described above indicated that B. cereus of different STs might have distinct ecological niches in China.

In addition, we analyzed the relationships among STs of B. cereus isolates, toxin gene profiles, antibiotic resistance patterns, and geographic characteristics (Fig. 1). The prominent correlation between STs of B. cereus isolates and antibiotic resistance patterns was absent. However, a significant preference in the distribution of toxin gene profiles in B. cereus strains was revealed. Most of the strains assigned the same ST or having a close genetic relationship tended to have the same or similar toxin gene profile patterns. Most of Lineage I members and Lineage II members had obviously distinctive toxin gene profiles. 44.4% of Lineage II strains belonged to toxin gene pattern II. Whereas only 4.5% of Lineage I strains were grouped into that pattern. 71.4% of positive strains for both nheABC and hblCDA were clustered into Lineage II. The discrepancy implicated that members of the two lineages might have different potential pathogenic abilities. Due to the close link between STs of B. cereus isolates and the geographical distributions of infant food manufacturers, B. cereus strains isolated from infant foods made in different geographical regions of China might have different potential pathogenic abilities. Furthermore, the situation that all multidrug-resistant strains carried at least one toxin gene posed a potential public health risk.

Conclusions

Overall, the relatively high contamination level of B. cereus in a proportion of retail prepackaged infant formula and ready-to-eat rice flour, especially in those suitable for the ≤6-month-old infant group in the Chinese market, posed food poisoning risks for the infant group. The toxin gene distribution profile revealed that B. cereus in infant foods tended to cause the diarrheal type of food poisoning. The antibiotic resistance of B. cereus isolates was demonstrated. High genetic diversity was present in B. cereus isolates by using MLST with a DI of 0.996. The relationship between STs of B. cereus isolates and the geographical distribution of infant food manufacturers made insights into the distinct ecological niches of B. cereus of different STs. The results of the studies presented herein provided comprehensive data on the quantitative prevalence and characteristics of B. cereus in retail infant foods in China. To address the microbial safety issue of infant foods, appropriate safety criteria and hygienic control measures should be drafted in China.

Footnotes

Acknowledgments

This work was supported by the Public Welfare Technology and Application Project of Zhejiang Province (2015C37058) and the Medical Scientific Research Foundation of Zhejiang Province (2016KYB065).

Disclosure Statement

No competing financial interests exist.

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