International Journal of Food Microbiology 204 (2015) 17–23

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Prevalence, molecular characterization, and antibiotic susceptibility of Cronobacter spp. in Chinese ready-to-eat foods Xiaoke Xu, Chengsi Li, Qingping Wu ⁎, Jumei Zhang, Jiahui Huang, Guangzhu Yang State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Public Laboratory for Applied and New Technology of Microbiology, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China

a r t i c l e

i n f o

Article history: Received 31 December 2014 Received in revised form 23 February 2015 Accepted 2 March 2015 Available online 6 March 2015 Keywords: Antimicrobial sensitivity Cronobacter spp. Multilocus sequence typing Quantitative analysis Ready-to-eat foods Serotype

a b s t r a c t Cronobacter spp. are foodborne pathogens that cause rare but life-threatening diseases in neonates and infants; they can also cause disease in adults. Cronobacter spp. contamination of ready-to-eat (RTE) foods has been reported previously. However, to date, the prevalence and contamination levels of these bacteria in RTE foods in China have not yet been determined. Therefore, the aim of this study was to investigate the prevalence of Cronobacter spp. in RTE foods marketed in China. Two-hundred and eighty RTE food samples were collected from different producers and retailers and analyzed using quantitative methods. The isolates obtained were identified to the species level based on fusA sequences, and were subtyped using a PCR-based serotyping technique. Selected isolates were further characterized by multilocus sequence typing (MLST) and antimicrobial sensitivity determination. Of 280 samples tested, 52 (18.6%) were positive for Cronobacter spp. The contamination levels were less than 110 MPN/g for 78.8% (41/52) of the samples. The results of the O-antigen serotyping for 111 isolates showed that Cronobacter sakazakii serogroup O2 (28 isolates) was the most prevalent serotype. MLST analyses produced 41 sequence types (STs), including 20 novel STs. ST8 was the most prevalent ST (9 isolates) followed by ST4 (5 isolates). Antimicrobial sensitivity testing showed that 84.5% and 46.5% of the isolates were resistant to penicillin G and cephalothin, respectively; in contrast, all of the tested isolates were susceptible to cefotaxime, ciprofloxacin, tetracycline, and nalidixic acid. To the best of our knowledge, this is the first report on Cronobacter spp. prevalence in RTE foods in China, and the findings of our study nonetheless suggested that Cronobacter spp. contamination of Chinese RTE foods poses a potential risk for the consumer. Thus, the study highlights the significance of developing more effective control strategies during the manufacturing process. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Cronobacter spp. is a novel genus that consists of 7 species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti (Forsythe et al., 2014; Iversen et al., 2008b; Joseph et al., 2012a; Stephan et al., 2014). Of these mainly, C. sakazakii, C. malonaticus, and C. turicensis are considered opportunistic human pathogens that have been associated with rare but serious human illnesses (FAO/WHO, 2008), and outbreaks have been reported in many countries recently (Caubilla-Barron et al., 2007; CDC, 2012; Ray et al., 2007; Tsai et al., 2013). Although infections caused by Cronobacter spp. have been epidemiologically linked to the consumption of contaminated powdered infant formula (Himelright et al., 2002; van Acker et al., 2001), Cronobacter spp. have been isolated from a variety of foods, including cereals, meat, herbs, spices, salads, fruits, and vegetables (Baumgartner et al., ⁎ Corresponding author at: Guangdong Institute of Microbiology, No. 100 Central Xianlie Road, Guangzhou 510070, China. Tel./fax: +86 20 87688132. E-mail address: [email protected] (Q. Wu).

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.03.003 0168-1605/© 2015 Elsevier B.V. All rights reserved.

2009; Chap et al., 2009; Hochel et al., 2012; Iversen and Forsythe, 2004; Wang et al., 2012). The occurrence of Cronobacter spp. in food products has been investigated in several countries (Hochel et al., 2012; Lee et al., 2012; Li et al., 2014; Mozrova et al., 2014); however, the epidemiology and reservoirs of Cronobacter spp. remain incompletely understood. Serotyping has been widely used for identifying isolates in epidemiological studies for bacteria (Jones et al., 2012). To date, 17 Cronobacter serogroups have been identified (Jarvis et al., 2013). PCR-based Oantigen serotyping methods for Cronobacter spp. have been successfully developed (Jarvis et al., 2011; Jarvis et al., 2013; Mullane et al., 2008; Sun et al., 2012). Therefore, PCR-based serotyping is considered a convenient method for the rapid and accurate typing of a wide array of Cronobacter spp. Moreover, multilocus sequence typing (MLST) has been established for the entire Cronobacter genus (Baldwin et al., 2009; Joseph and Forsythe, 2012; Joseph et al., 2012b). It revealed 336 definable sequence types. MLST has revealed the diversity of the Cronobacter genus, and its application has facilitated understanding of the virulence and environmental fitness of these bacteria. The Cronobacter PubMLST database offers a central, open access, reliable sequence-based repository for

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researchers (Forsythe et al., 2014). Cronobacter strains primarily isolated from infant formula include C. sakazakii ST1, whereas C. sakazakii ST8 is the primary strains in clinical sources (Joseph and Forsythe, 2011). Furthermore, MLST has revealed that the majority of neonatal meningitis cases are attributable to a single clonal complex, the C. sakazakii ST4 clonal complex (CDC, 2012; Hariri et al., 2013; Joseph and Forsythe, 2012). In addition, C. malonaticus ST7 is associated with adult infections, although the source has not been identified (Joseph and Forsythe, 2011, 2012). Thus, further studies involving the use of MLST for the identification of Cronobacter isolates are necessary to more comprehensively understand the ecology and distribution of significant strains of these bacteria. Naturally, most Cronobacter spp. isolates are susceptible to commonly used antimicrobial agents. However, the extensive use of antimicrobials in agriculture has led to the emergence of single- and multipledrug resistant bacterial strains. Cronobacter spp. have been reported to be resistant to cephalothin, cefotaxime, and streptomycin in some isolates from food samples (Chon et al., 2012; Lee et al., 2012; Molloy et al., 2009; Pan et al., 2014; Ye et al., 2010). Antimicrobial resistance, particularly multiple-drug resistance, is a public health concern, because it may cause failure of conventional treatment, resulting in prolonged illness and a greater risk of death. Therefore, it is necessary to establish a monitoring system for the objective evaluation of the antimicrobial resistance profile. In China, ready-to-eat (RTE) foods, such as deli meat and cold vegetable dishes in sauce, are very popular because of their pleasant taste and convenience. Our previous studies have shown that RTE foods available in Chinese markets are contaminated by foodborne pathogens such as Listeria monocytogenes (Chen et al., 2014) and Salmonella spp. (Chen et al., 2013). These findings underline the importance of evaluating the microbiological profile of RTE foods for ensuring food safety. However, the presence of Cronobacter spp. in RTE foods at the retail level has received less attention in China, and little information is available on the prevalence and contamination levels of Cronobacter spp. Therefore, the aim of this study was to investigate the prevalence of

Cronobacter spp. in RTE foods in China, to identify the isolates by fusA allele sequences and serological analysis, and further, to perform MLST analysis and evaluate the antibiotic resistance patterns of the isolated strains. 2. Materials and methods 2.1. Sampling A total of 280 RTE food samples, including 200 deli meat samples, 31 cold vegetables in sauce dishes samples, 22 cold noodles in sauce samples, and 27 fried rice or noodles samples were collected in retail markets from December 2011 to April 2013. Samples were obtained from 24 cities, which covered most of the provincial capitals of China (Fig. 1). The samples were placed in sterile sealed plastic bags and transported to the laboratory in a cold box (below 4 °C), and analyzed immediately. 2.2. Quantitative analysis of Cronobacter spp. The bacteriological media used herein, unless indicated, were purchased from Guangdong Huankai Co. Ltd. For quantitative detection, an enrichment method was as per the reference methods (Dong et al., 2013; Kandhai et al., 2010). In brief, 25 g of the samples was homogenized for 60 s in Stomacher bags (Huankai, Guangzhou, China) with 225 mL of modified lauryl sulfate tryptose (mLST) broth supplemented with vancomycin to a final concentration of 10 μg/mL. Serial 10-fold dilutions, up to 1:100, were prepared and 3 × 10-mL aliquots of the 1:10 dilution were inoculated into three individual tubes, representing the 1 g portion. Similarly, 3 × 1-mL aliquots of the 1:10 and 1:100 dilutions were inoculated into 9 mL of mLST. Nine tubes were incubated at 44 °C for 24 h. One loop of the selectively enriched broth was streaked onto chromogenic medium and incubated at 44 °C for 24 h. Three to five (if have) green or blue–green colonies were considered presumptive Cronobacter spp. from each plate and their identity was confirmed

Fig. 1. The location of sampling sites used in this study in China.

X. Xu et al. / International Journal of Food Microbiology 204 (2015) 17–23

using duplex PCR (Zhou et al., 2008). The species of these isolates was identified by comparing the fusA allele sequences as recently described (Joseph et al., 2012b; Xu et al., 2014). The most probable number (MPN) was determined on the basis of the number of positive tube(s) in each of the three sets and the MPN table (Kaysner and DePaola, 2004).

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(10 μg), streptomycin (10 μg), tetracycline (30 μg), and nalidixic acid (30 μg). The results were expressed as sensitive (S), intermediate (I), and resistant (R), following the CLSI guidelines. Escherichia coli ATCC 25922 was used as a quality control organism. 3. Results

2.3. Serotyping of Cronobacter spp. 3.1. Prevalence and contamination levels of Cronobacter spp. in RTE foods The serotypes of Cronobacter spp. isolates were identified using the PCR-based O-antigen serotyping technique. A primer mix for the 7 serotypes (O1 to O7) of C. sakazakii was used in multiplex PCR assays with primer concentrations and amplification conditions as recently described (Sun et al., 2012). Other serotype primers were used as previously reported (Jarvis et al., 2011; Jarvis et al., 2013); the primer sequences used are listed in Table 1. 2.4. MLST The MLST protocol was performed as described by Joseph et al. (2012b). Sequencing was outsourced (BGI, Guangzhou, China). Another gyrBR sequencing primer was used for some strains when sequencing with the original primer was less than optimal. In some cases, T-cloning was used when direct sequencing did not succeed. Sequence types (STs) were determined by using the Cronobacter MLST website (http:// pubmlst.org/cronobacter/). Sequence analysis of the concatenated sequences of the 7 loci (3036 nucleotides concatenated length) was performed using the ClustalX algorithm (version 1.83) (Thompson et al., 1997), which was followed by phylogenetic analysis using the maximum likelihood algorithm in MEGA 5 (version 5.05) (Tamura et al., 2011).

Fifty-two (18.6%) samples that were positive for Cronobacter spp. were detected among 280 RTE foods. As shown in Table 2, among the positive samples, 29 (14.5%) were obtained from deli meat samples, thirteen (41.9%) were from cold vegetables in sauce dishes, 8 (36.4%) were from cold noodles in sauce samples, and 2 (7.4%) were from fried rice or noodles samples. In the 52 positive samples, Cronobacter spp. contamination levels ranged between 0.3 and 110 MPN/g in 78.8% (41/52) of the samples. Contamination levels of Cronobacter spp. exceeded 110 MPN/g in 11 samples. Of 111 confirmed Cronobacter spp. isolates, 81 were identified as C. sakazakii, 21 as C. malonaticus, 8 as C. dublinensis, and one as C. muytjensii (Table 3). 3.2. Serotyping by PCR The distribution of O-antigen serotypes among the 111 Cronobacter spp. isolates was determined using the previously described PCR-based O-antigen serotyping technique. With the exception of C. sakazakii serotype O5, all other C. sakazakii serotypes were detected among the isolates. C. sakazakii serotype O2 was the most prevalent (28 isolates), followed by C. sakazakii serotype O1 (23 isolates). The results of the O-antigen serotyping for all 111 isolates are summarized in Table 3.

2.5. Antibiotic susceptibility 3.3. MLST The antibiotic susceptibility was assessed using the disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2006). Müller–Hinton agar and a panel of 9 antibiotic disks (Oxoid, Hampshire, UK) were selected for the resistance test. These disks were for penicillin G (10 U), cephalothin (30 μg), cefotaxime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin

Cronobacter spp. isolates were selected for further investigation according to the results of the species and serotyping analyses. All the Cronobacter spp. isolates from each positive sample were characterized by species and serotype, then compared with each other. Isolates from the same sample having the same species and serotype were considered

Table 1 List of serotypes primers used in this study. Target gene

Serogroup

Primer sequence

Size (bp)

wzy

C. sakazakii O1

364

wzy

C. sakazakii O2

wzy

C. sakazakii O3

wzy

C. sakazakii O4

wzy

C. sakazakii O5

wzy

C. sakazakii O6

wzx

C. sakazakii O7

wzx

C. malonaticus O1

wzx

C. malonaticus O2

wzx

C. muytjensii O1

wzx

C. muytjensii O2

wzx

C. dublinensis O1

wzm

C. dublinensis O2

F: 5-CCCGCTTGTATGGATGTT-3 R: 5-CTTTGGGAGCGTTAGGTT-3 F: 5-ATTGTTTGCGATGGTGAG-3 R: 5-AAAACAATCCAGCAGCAA-3 F: 5-CTCTGTTACTCTCCATAGTGTTC-3 R: 5-GATTAGACCACCATAGCCA-3 F: 5-ACTATGGTTTGGCTATACTCCT-3 R: 5-ATTCATATCCTGCGTGGC-3 F: 5-GATGATTTTGTAAGCGGTCT-3 R: 5-ACCTACTGGCATAGAGGATAA-3 F: 5-ATGGTGAAGGGAACGACT-3 R: 5-ATCCCCGTGCTATGAGAC-3 F: 5-CATTTCCAGATTATTACCTTTC-3 R: 5-ACACTGGCGATTCTACCC-3 F: 5-AGGGGCACGGCTTAGTTCTGG-3 R: 5-CCCGCTTGCCCTTCACCTAAC-3 F: 5-TGGCCCTTGTTAGCAAGACGTTTC-3 R: 5-ATCCACATGCCGTCCTTCATCTGT-3 F: 5-TGGCTGTCATGGTTTTCTTGC-3 R: 5-TAGTTGGCACCATCAACGCC-3 F: 5-CGCTGCGATTATGGTAGTGGGT-3 R: 5-TTCCCAGCTCAGCTCGTTTGC-3 F: 5-TCGTTTTGATGCTCTCGCTGCG-3 R: 5-ACAAATCGCGTGCTGGCTTGAA-3 F: 5-CTCGGTTCATGGATTTGCGGC-3 R: 5-CAGCGTGAAAACAGCCAGGT-3

152 704 890 235 424 615 323 394 258 475 435 227

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Table 2 Prevalence and levels of Cronobacter spp. in ready-to-eat foods. Product category

Total tests

Deli meat Cold vegetable dish in sauce Cold noodles in sauce Fried rice or noodles Total

Cronobacter spp. no. (%)

200 31 22 27 280

29 (14.5%) 13 (41.9%) 8 (36.4%) 2 (7.4%) 52 (18.6%)

clonal. Clonal isolates from individual sample were excluded. Thus, 71 isolates from 111 Cronobacter spp. isolates were selected for further study. Nucleotide sequences were deposited in the MLST database under PubMLST IDs 712 to 782 (www.pubMLST.org/cronobacter). The majority (n = 50) of isolates were identified as C. sakazakii, followed by C. malonaticus (n = 13), C. dublinensis (n = 7), and C. muytjensii (n = 1). A phylogenetic tree based on the 7 concatenated MLST sequences (Fig. 2) shows clear clustering across the Cronobacter spp., with the 71 isolates belonging to 4 of the 7 species. The tree also shows the relatedness between the sequence types. Analyses of the MLST results produced 41 STs. ST8 was the most prevalent ST (9 isolates), followed by ST4 (5 isolates). In addition, ST266, ST267, and ST270 to ST287 are STs newly discovered in our study. 3.4. Susceptibility tests The susceptible, intermediate, and resistance rates of the 71 examined Cronobacter spp. isolates with respect to 9 antibiotics are shown in Table 4. Isolates of Cronobacter spp. demonstrated different levels of antibiotic resistance. The isolates were most resistant to penicillin G, with resistance and intermediate rates of 84.5% and 15.5%, respectively. In addition, the isolates exhibited relatively high resistance and intermediate rates of 46.5% and 52.1%, respectively, for cephalothin. All of the examined isolates were susceptible to cefotaxime, ciprofloxacin, tetracycline, and nalidixic acid. Among the remaining tested antibiotics, the next-highest susceptible rates were observed for chloramphenicol (93.0%) and gentamicin (93.0%). 4. Discussion The methods widely used for detecting Cronobacter spp. in powdered infant formula and other dried foods typically involve a preenrichment step, in order to recover desiccation-stressed target cells, followed by culture in selective or differential enrichment broth (ISO/IDF, 2006; Iversen et al., 2008a). It has also been reported that the use of Cronobacter broth (CB) facilitated the recovery of Cronobacter spp. in RTE foods (Baumgartner et al., 2009). For quantitative detection in this study, mLST broth and 44 °C temperature were used in Table 3 Results of the species and PCR-based O-antigen serotyping of 111 Cronobacter spp. isolates. Serotype C. sakazakii

C. malonaticus C. muytjensii C. dublinensis

Total

O1 O2 O3 O4 O6 O7 O2 Uncertain O1 O1 O2 Uncertain

Amplicon size (bp)

No. of isolates analyzed

364 152 704 890 424 615 394 – 258 435 227 –

23 28 13 10 3 4 20 1 1 5 2 1 111

No. of samples containing the pathogen (MPN/g) 0.3 to 3

N3 to 10

N10 to 110

N110

13 7 6 2 28

4 0 0 0 4

5 3 1 0 9

7 3 1 0 11

accordance with the reference methods (Dong et al., 2013; Kandhai et al., 2010); this procedure was found to be effective for the recovery of Cronobacter spp. from RTE foods in this study, although it may restrict the growth of Cronobacter spp. and may have overlooked less temperature tolerant strains. The prevalence and contamination levels of Cronobacter spp. in retail RTE foods in China have not been determined. In this study, we analyzed 280 RTE food samples and detected Cronobacter spp. contamination in 52 samples. Thus, the overall prevalence of Cronobacter spp. in RTE food samples was determined to be 18.6%; this prevalence was 9.0% in Switzerland (Baumgartner et al., 2009). Notably, the prevalence of Cronobacter spp. was particularly high, 41.9% (13/31), in cold vegetables in sauce dishes samples. In our study, the high Cronobacter spp. prevalence in cold vegetables in sauce dishes may be due to contamination in the vegetable samples or the processing environment. In a study of the prevalence of Cronobacter spp. contamination in 128 vegetables, 19 (14.8%) were found to be positive (Lee et al., 2012). In another study, Cronobacter spp. were detected in 30% of vegetables (Chon et al., 2012), this finding was in agreement with that of our previous study (Dong et al., 2013). However, no Cronobacter spp. were isolated from vegetable samples in a study recently conducted in China (Li et al., 2014), the authors explained that the absence of Cronobacter spp. may be due to the limited number of samples analyzed or due to recent improvements in food industry hygiene levels. In the past few years, several reports have been published on the prevalence of Cronobacter spp. in various food samples. Very little information is available for Cronobacter spp. levels, particularly with regard to such levels in RTE food in China have never been determined. In this study, the MPN values of the contaminated samples (41/52) were less than 110 MPN/g. Even low levels of contamination of Cronobacter spp. in powdered infant formula were considered to be a risk factor. However, in China, no standard permissible limit of Cronobacter spp. in foods has been established. The maximum number of C. sakazakii ST4 detected was more than 110 MPN/g, which suggests that this result could be a potential risk for the neonates. As mentioned above, 17 Cronobacter serogroups have been identified in Cronobacter spp. In this study, C. sakazakii serotype O2 (28 isolates) was found to be the most prevalent serotype and C. sakazakii serotype O5 was absent from C. sakazakii isolates, which were in agreement with several previous studies (Muller et al., 2013; Xu et al., 2014). A total of 71 isolates of Cronobacter spp. were genotyped by MLST. The majority (70.4%) of Cronobacter spp. isolates were C. sakazakii. The remaining isolates were C. malonaticus (18.3%), C. dublinensis (9.9%), and C. muytjensii (1.4%). Our study supports the previous finding that C. sakazakii is the most common species isolated (Lehner, 2010; Muller et al., 2013). Comparisons of the MLST data from this study with international MLST databases revealed that ST1, ST4, ST7, ST8, and ST64 have been reported in numerous international studies. Thus, the major MLST types obtained for Cronobacter spp. isolates examined in this study were in accordance with international epidemic strains. Our study results nonetheless suggested that Cronobacter spp. contamination of Chinese RTE foods poses a pathogenic risk. Susceptibility tests revealed that the isolates were susceptible to most antibiotics. The highest resistance rate (84.5%) was observed for penicillin G, followed by cephalothin (46.5%); in addition, all of the

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100 775C.sakazakiiST31 761C.sakazakii ST31 100 751C.sakazakiiST40 18 763C.sakazakii ST40 765C.sakazakiiST16 18 739C.sakazakii ST21 778C.sakazakiiST46 33 100 726C.sakazakii ST46 734C.sakazakiiST46 100 767C.sakazakiiST13 7 768C.sakazakiiST13 781C.sakazakiiST73 52 100 782C.sakazakii ST73 749C.sakazakiiST1 100 759C.sakazakiiST1 14 755C.sakazakii ST1 760C.sakazakii ST285 13 747C.sakazakiiST281 36 758C.sakazakiiST12 58 16 733C.sakazakii ST208 777C.sakazakii ST3 14 750C.sakazakii ST136 32 764C.sakazakii ST287 719C.sakazakii ST267 100 722C.sakazakiiST4 771C.sakazakiiST4 64 723C.sakazakii ST4 38 49 735C.sakazakii ST4 717C.sakazakiiST266 743C.sakazakiiST4 100 774C.sakazakiiST22 741C.sakazakiiST22 731C.sakazakii ST198 772C.sakazakii ST64 97 740C.sakazakiiST64 44 99 732C.sakazakiiST64 736C.sakazakii ST64 96 30 748C.sakazakiiST282 766C.sakazakiiST8 776C.sakazakiiST8 773C.sakazakiiST8 87 780C.sakazakiiST8 770C.sakazakiiST8 725C.sakazakiiST226 100 779C.sakazakii ST8 100 721C.sakazakiiST8 769C.sakazakiiST8 730C.sakazakiiST279 737C.sakazakiiST8 753C.sakazakii ST283 729C. malonaticusST278 713C. malonaticusST271 714C. malonaticusST272 100 762C. malonaticusST286 756C. malonaticus ST284 44 742C. malonaticusST7 61 720C. malonaticusST7 37 745C. malonaticusST7 100 738C. malonaticus ST7 728C. malonaticus ST7 744C. malonaticusST201 64 752C. malonaticusST201 754C. malonaticus ST201 724C. muytjensiiST276 98 716C. dublinensisST274 746C. dublinensisST280 100 715C. dublinensis ST273 100 757C. dublinensisST77 727C. dublinensisST277 84 718C. dublinensisST275 86 712C. dublinensis ST270 98 89

0.01 Fig. 2. Maximum likelihood tree of the 7 multilocus sequence typing loci (3036 base pair concatenated length) of Cronobacter spp. isolates. This tree was generated using the ClustalX (version 1.83) algorithm and the MEGA (version 5.05) with 1000 bootstrap replicates.

examined isolates were susceptible to cefotaxime, ciprofloxacin, tetracycline, and nalidixic acid. Similarly, previous studies have shown that the occurrence of cephalothin resistance in Cronobacter spp. isolates from different sources is common (Chon et al., 2012; Kim et al., 2008; Molloy et al., 2009). Furthermore, 2.8% and 1.4% isolates were resistant to chloramphenicol and gentamicin, respectively, which is in agreement with previous study (Lee et al., 2012). Infections caused by Cronobacter spp. can be successfully treated with ampicillin–gentamicin or ampicillin–chloramphenicol during therapy (Lai, 2001). While more attention should be paid to these infections, the abuse and misuse of antimicrobial

agents may lead to the emergence of antimicrobial-resistant bacteria, posing a serious public health problem. In conclusion, the prevalence and levels of Cronobacter spp. contamination in RTE food in China are relatively high; furthermore, detection of the pathogenic C. sakazakii ST4 strongly implicates that this could be a potential risk for the neonates. Thus, the findings of this study underline the need for routine microbiological screening of RTE food. These findings may be useful for designing microbiological risk assessment studies for evaluating the presence of virulent Cronobacter spp. in RTE food in China.

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Table 4 Antimicrobial resistance profile of 71 Cronobacter spp. isolates from ready-to-eat foods. Antimicrobial agent

Penicillin G (P) Cephalothin (KF) Cefotaxime (CTX) Chloramphenicol (C) Ciprofloxacin (CIP) Gentamicin (CN) Streptomycin (S) Tetracycline (TE) Nalidixic acid (NA)

Cronobacter spp. (n = 71) No. (%) of R

No. (%) of I

No. (%) of S

60 (84.5) 33 (46.5) 0 (0.0) 2 (2.8) 0 (0.0) 1 (1.4) 2 (2.8) 0 (0.0) 0 (0.0)

11 (15.5) 37 (52.1) 0 (0.0) 3 (4.2) 0 (0.0) 4 (5.6) 32 (45.1) 0 (0.0) 0 (0.0)

0 (0.0) 1 (1.4) 71 (100.0) 66 (93.0) 71 (100.0) 66 (93.0) 37 (52.1) 71 (100.0) 71 (100.0)

R: resistant; I: intermediate resistance; S: susceptibility.

Acknowledgments Xiaoke Xu and Chengsi Li contributed equally to this work. This work was supported by the National Natural Science Foundation of China (31371780), the Five-year Major National Science and Technology Support Project (2013BAD16B05), and the Science and Technology Planning Project of Guangdong Province (2011A011303001, 2012020100010). We thank Prof. Steve Forsythe for his technical assistance in MLST. We would like to thank Editage for providing editorial assistance. References Baldwin, A., Loughlin, M., Caubilla-Barron, J., Kucerova, E., Manning, G., Dowson, C., Forsythe, S., 2009. Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes. BMC Microbiol. 9, 223. Baumgartner, A., Grand, M., Liniger, M., Iversen, C., 2009. Detection and frequency of Cronobacter spp. (Enterobacter sakazakii) in different categories of ready-to-eat foods other than infant formula. Int. J. Food Microbiol. 136, 189–192. Caubilla-Barron, J., Hurrell, E., Townsend, S., Cheetham, P., Loc-Carrillo, C., Fayet, O., Prere, M.F., Forsythe, S.J., 2007. Genotypic and phenotypic analysis of Enterobacter sakazakii strains from an outbreak resulting in fatalities in a neonatal intensive care unit in France. J. Clin. Microbiol. 45, 3979–3985. CDC, 2012. Centers for Disease Control and Prevention. CDC Update: Investigation of Cronobacter Infections Among Infants in the United States (January 13, 2012 [cited 2012 Aug 22]. http://www.cdc.gov/foodsafety/deseases/cronobacter/investigation. html). Chap, J., Jackson, P., Siqueira, R., Gaspar, N., Quintas, C., Park, J., Osaili, T., Shaker, R., Jaradat, Z., Hartantyo, S.H., Abdullah Sani, N., Estuningsih, S., Forsythe, S.J., 2009. International survey of Cronobacter sakazakii and other Cronobacter spp. in follow-up formulas and infant foods. Int. J. Food Microbiol. 136, 185–188. Chen, L., Zhang, J., Yang, X., Wu, Q., Xu, M., 2013. Prevalence and characterization of Salmonella spp. from foods in South China. Acta Microbiol. Sin. 53, 1326–1333. Chen, M., Wu, Q., Zhang, J., Yan, Z., Wang, J., 2014. Prevalence and characterization of Listeria monocytogenes isolated from retail-level ready-to-eat foods in South China. Food Control 38, 1–7. Chon, J.W., Song, K.Y., Kim, S.Y., Hyeon, J.Y., Seo, K.H., 2012. Isolation and characterization of Cronobacter from desiccated foods in Korea. J. Food Sci. 77, M354–M358. CLSI, 2006. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria. Approved Standard M45-A. Clinical and Laboratory Standards Institute, Wayne, PA. Dong, X., Li, C., Wu, Q., Zhang, J., Mo, S., Guo, W., Yang, X., Xu, X., 2013. Isolation and identification of Cronobacter (Enterobacter sakazakii) strains from food. Acta Microbiol. Sin. 429–436. FAO/WHO, 2008. Enterobacter sakazakii (Cronobacter spp.) in powdered follow-up formulae. Microbiological Risk Assessment Series No. 15. Food and Agriculture Organization of the United Nations/World Health Organization, Rome (90 pp.). Forsythe, S.J., Dickins, B., Jolley, K.A., 2014. Cronobacter, the emergent bacterial pathogen Enterobacter sakazakii comes of age; MLST and whole genome sequence analysis. BMC Genomics 15, 1121. Hariri, S., Joseph, S., Forsythe, S.J., 2013. Cronobacter sakazakii ST4 strains and neonatal meningitis, United States. Emerg. Infect. Dis. 19, 175. Himelright, I.E., Harris, V., Lorch, M., Anderson, T., Jones, A., Craig, M., Kuehnert, T., Forster, M., Arduino, B., Jensen Jernigan, D., 2002. Enterobacter sakazakii infections associated with the use of powdered infant formula—Tennessee, 2001. Morbidity and Mortality Weekly Report 51pp. 297–300. Hochel, I., Ruzickova, H., Krasny, L., Demnerova, K., 2012. Occurrence of Cronobacter spp. in retail foods. J. Appl. Microbiol. 112, 1257–1265. ISO/IDF, 2006. Technical Specification: Milk and Milk Products — Detection of Enterobacter sakazakii. (ISO/TS 22964; IDF/RM 201). Iversen, C., Forsythe, S., 2004. Isolation of Enterobacter sakazakii and other Enterobacteriaceae from powdered infant formula milk and related products. Food Microbiol. 21, 771–777.

Iversen, C., Druggan, P., Schumacher, S., Lehner, A., Feer, C., Gschwend, K., Joosten, H., Stephan, R., 2008a. Development of a novel screening method for the isolation of “Cronobacter” spp. (Enterobacter sakazakii). Appl. Environ. Microbiol. 74, 2550–2553. Iversen, C., Mullane, N., McCardel, B., Tal, B.D., Lehner, A., Fannin, S., Stephan, R., Joosten, H., 2008b. Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov. Int. J. Syst. Evol. Microbiol. 58, 1442–1447. Jarvis, K.G., Grim, C.J., Franco, A.A., Gopinath, G., Sathyamoorthy, V., Hu, L., Sadowski, J.A., Lee, C.S., Tall, B.D., 2011. Molecular characterization of Cronobacter lipopolysaccharide O-antigen gene clusters and development of serotype-specific PCR assays. Appl. Environ. Microbiol. 77, 4017–4026. Jarvis, K.G., Yan, Q.Q., Grim, C.J., Power, K.A., Franco, A.A., Hu, L., Gopinath, G., Sathyamoorthy, V., Kotewicz, M.L., Kothary, M.H., Lee, C., Sadowski, J., Fanning, S., Tall, B.D., 2013. Identification and characterization of five new molecular serogroups of Cronobacter spp. Foodborne Pathog. Dis. 10, 343–352. Jones, J.L., Ludeke, C.H., Bowers, J.C., Garrett, N., Fischer, M., Parsons, M.B., Bopp, C.A., DePaola, A., 2012. Biochemical, serological, and virulence characterization of clinical and oyster Vibrio parahaemolyticus isolates. J. Clin. Microbiol. 50, 2343–2352. Joseph, S., Forsythe, S.J., 2011. Predominance of Cronobacter sakazakii sequence type 4 in neonatal infections. Emerg. Infect. Dis. 17, 1713–1715. Joseph, S., Forsythe, S.J., 2012. Insights into the emergent bacterial pathogen Cronobacter spp., generated by multilocus sequence typing and analysis. Front. Microbiol. 3, 397. Joseph, S., Cetinkaya, E., Drahovska, H., Levican, A., Figueras, M.J., Forsythe, S.J., 2012a. Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg infection, water and food ingredients. Int. J. Syst. Evol. Microbiol. 62, 1277–1283. Joseph, S., Sonbol, H., Hariri, S., Desai, P., McClelland, M., Forsythe, S.J., 2012b. Diversity of the Cronobacter genus as revealed by multilocus sequence typing. J. Clin. Microbiol. 50, 3031–3039. Kandhai, M., Heuvelink, A., Reij, M., Beumer, R., Dijk, R., van Tilburg, J., van Schothorst, M., Gorris, L., 2010. A study into the occurrence of Cronobacter spp. in the Netherlands between 2001 and 2005. Food Control 21, 1127–1136. Kaysner, C.A., DePaola, A.J., 2004. U. S. Food and Drug Administration; bacteriological analytical manual; methods for specific pathogens; chapter 9 Vibrio. Available at. http:// www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm070830.htm. Kim, K., Jang, S.S., Kim, S.K., Park, J.H., Heu, S., Ryu, S., 2008. Prevalence and genetic diversity of Enterobacter sakazakii in ingredients of infant foods. Int. J. Food Microbiol. 122, 196–203. Lai, K.K., 2001. Enterobacter sakazakii infections among neonates, infants, children, and adults — case reports and a review of the literature. Medicine 80, 113–122. Lee, Y.D., Park, J.H., Chang, H., 2012. Detection, antibiotic susceptibility and biofilm formation of Cronobacter spp. from various foods in Korea. Food Control 24, 225–230. Lehner, A., 2010. Cronobacter (former Enterobacter sakazakii) spp. — its effect in dairy product safety, quality and trade. Aust. J. Dairy Technol. 65, 63–67. Li, Y.H., Chen, Q.M., Zhao, J.F., Jiang, H., Lu, F.X., Bie, X.M., Lu, Z.X., 2014. Isolation, identification and antimicrobial resistance of Cronobacter spp. isolated from various foods in China. Food Control 37, 109–114. Molloy, C., Cagney, C., O'Brien, S., Iversen, C., Fanning, S., Duffy, G., 2009. Surveillance and characterisation by pulsed-field gel electrophoresis of Cronobacter spp. in farming and domestic environments, food production animals and retail foods. Int. J. Food Microbiol. 136, 198–203. Mozrova, V., Brenova, N., Mrazek, J., Lukesova, D., Marounek, M., 2014. Surveillance and characterisation of Cronobacter spp. in Czech retail food and environmental samples. Folia Microbiol. 59, 63–68. Mullane, N.R., Ryan, M., Iversen, C., Murphy, M., O'Gaora, P., Quinn, T., Whyte, P., Wall, P.G., Fanning, S., 2008. Development of multiple-locus variable-number tandemrepeat analysis for the molecular subtyping of Enterobacter sakazakii. Appl. Environ. Microbiol. 74, 1223–1231. Muller, A., Stephan, R., Fricker-Feer, C., Lehner, A., 2013. Genetic diversity of Cronobacter sakazakii isolates collected from a Swiss infant formula production facility. J. Food Prot. 76, 883–887. Pan, Z., Cui, J., Lyu, G., Du, X., Qin, L., Guo, Y., Xu, B., Li, W., Cui, Z., Zhao, C., 2014. Isolation and molecular typing of Cronobacter spp. in commercial powdered infant formula and follow-up formula. Foodborne Pathog. Dis. 11, 456–461. Ray, P., Das, A., Gautam, V., Jain, N., Narang, A., Sharma, M., 2007. Enterobacter sakazakii in infants: novel phenomenon in India. Indian J. Med. Microbiol. 25, 408–410. Stephan, R., Grim, C.J., Gopinath, G.R., Mammel, M.K., Sathyamoorthy, V., Trach, L.H., Chase, H.R., Fanning, S., Tall, B.D., 2014. Re-examination of the taxonomic status of Enterobacter helveticus, Enterobacter pulveris and Enterobacter turicensis as members of the genus Cronobacter and their reclassification in the genera Franconibacter gen. nov. and Siccibacter gen. nov. as Franconibacter helveticus comb. nov., Franconibacter pulveris comb. nov. and Siccibacter turicensis comb. nov., respectively. Int. J. Syst. Evol. Microbiol. 64, 3402–3410. Sun, Y., Wang, M., Wang, Q., Cao, B., He, X., Li, K., Feng, L., Wang, L., 2012. Genetic analysis of the Cronobacter sakazakii O4 to O7 O-antigen gene clusters and development of a PCR assay for identification of all C. sakazakii O serotypes. Appl. Environ. Microbiol. 78, 3966–3974. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739.

X. Xu et al. / International Journal of Food Microbiology 204 (2015) 17–23 Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Tsai, H.Y., Liao, C.H., Huang, Y.T., Lee, P.L., Hsueh, P.R., 2013. Cronobacter infections not from infant formula, Taiwan. Emerg. Infect. Dis. 19, 167–169. van Acker, J., de Smet, F., Muyldermans, G., Bougatef, A., Naessens, A., Lauwers, S., 2001. Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula. J. Clin. Microbiol. 39, 293–297. Wang, X., Zhu, C., Xu, X., Zhou, G., 2012. Real-time PCR with internal amplification control for the detection of Cronobacter spp. (Enterobacter sakazakii) in food samples. Food Control 25, 144–149.

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Xu, X., Wu, Q., Zhang, J., Ye, Y., Yang, X., Dong, X., 2014. Occurrence and characterization of Cronobacter spp. in powdered formula from Chinese retail markets. Foodborne Pathog. Dis. 11, 307–312. Ye, Y., Wu, Q., Xu, X., Yang, X., Dong, X., Zhang, J., 2010. The phenotypic and genotypic characterization of Enterobacter sakazakii strains from infant formula milk. J. Dairy Sci. 93, 2315–2320. Zhou, Y., Wu, Q., Xu, X., Yang, X., Ye, Y., Zhang, J., 2008. Development of an immobilization and detection method of Enterobacter sakazakii from powdered infant formula. Food Microbiol. 25, 648–652.