883

Journal of Food Protection, Vol. 76, No. 5, 2013, Pages 883–887 doi:10.4315/0362-028X.JFP-12-521 Copyright G, International Association for Food Protection

Research Note Genetic Diversity of sakazakii Isolates Collected from a Swiss Infant Formula Production Facility

ANDREA MU¨ LLER,1 ROGER STEPHAN,1 CLAUDIA FRICKER-FEER,2 AND ANGELIKA LEHNER1*

1Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; and 2Department of Quality Assurance and Food Safety,

Hochdorf Nutritec AG, Hochdorf, Switzerland Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/5/883/1685933/0362-028x_jfp-12-521.pdf by guest on 26 September 2021

MS 12-521: Received 21 November 2012/Accepted 22 December 2012

ABSTRACT In this study, 141 Cronobacter isolates that were collected based on a hygienic monitoring program performed in a powdered infant formula production facility in Switzerland between September 2011 and October 2012 were further characterized. Isolates were identified to the species level by molecular methods, and strains of Cronobacter sakazakii were further subtyped by applying PCR-based O-antigen serotyping, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). C. sakazakii was the most prevalent species identified (93.6%). Among this collection of isolates, representatives of all but one O-antigen serotype (serotype O5) were recognized. MLST analysis of 19 selected isolates revealed that most of the typeable isolates belonged to sequence type (ST) 4. Correlations between ST4 and serotype O2 and between ST83 and serotype O7 were observed. PFGE analysis revealed clusters with multiple isolates, including strains from samples collected at different time points and sampling sources. Generally, the observed heterogeneity among strains collected over the 13 months of the monitoring program was high, suggesting a constant flux among strains rather than a selection for persisting organisms.

The genus Cronobacter comprises seven species: C. improved interventions for the control of these pathogens (9, sakazakii, C. turicensis, C. malonaticus, C. muytjensii, C. 15, 16, 20). dublinensis, C. universalis, and C. condimenti (10, 12). The risk posed by contaminated infant formula when it These are opportunistic pathogens that can cause is consumed by neonates raises the question of the possible septicemia and infections of the central nervous system, origin and routes of dissemination and transmission of these primarily in premature, low-birth-weight, and/or immuno- organisms into and within the infant formula processing compromised neonates (1, 5), but infections among environment and/or final products. Two possible routes immunocompromised elderly adults also have been reported have been described for dissemination of Cronobacter spp. (4, 19). Most outbreaks have been reported in neonatal into production lines and recontamination of pasteurized intensive care units, where the sources of infection have products: organisms may be attached to dust or to dry heat- been traced to temperature-abused reconstituted powdered labile supplement ingredients (8, 14, 17). infant formula and/or feeding equipment contaminated with This study was part of ongoing collaborative work Cronobacter spp. (7). on the identification and characterization of presumptive The presence of these pathogens in infant formula Cronobacter spp. isolates collected in a powdered infant products represents a challenge for the powdered infant formula facility in Switzerland as part of a hygienic formula industry. Thus, specific and accurate identification monitoring program. of members of the genus Cronobacter and discrimination between these bacteria and closely related but nonpatho- MATERIALS AND METHODS genic organisms that may be present in the same habitat Strains. For this study, 148 cryopreserved presumptive (products and environment) are critical. Several phenotypic Cronobacter spp. isolates collected as part of a hygienic features such as yellow pigmentation of colonies or alpha- monitoring program in a powdered infant formula facility in glucosidase activity, which have been attributed to members of Switzerland between September 2011 and October 2012 were the Cronobacter genus, have been of limited use in further characterized. The strain collection comprised 115 isolates from production environments (walls, floors, vacuum cleaners, identification schemes because of the lack of exclusivity. filters, rinsing water, drains), 25 isolates from finished products However, combinations of molecular identification methods (powdered infant formula, follow-on formula, growing-up formu- and culture detection and identification procedures significantly la), and 8 isolates originating from supplements, raw materials or ingredients (vitamins, whey protein, concentrates).

* Author for correspondence. Tel: z41-44-635-8670; Fax: z41-44-635-8908; Preparation of cell lysates to be used in PCR-based E-mail: [email protected]. assays. Cryopreserved strains were first streaked onto blood agar 884 MU¨ LLER ET AL. J. Food Prot., Vol. 76, No. 5

plates and incubated for 24 h at 37uC for further processing. A loopful of the blood agar culture material was suspended in 200 ml of lysis buffer (0.1 M Tris HCl [pH 8.5], 0.05% Tween 20, 0.24 mg ml21 Proteinase K) and incubated at 60uC for 1 h and then at 97uC for 15 min. Isolates were identified to genus and species by PCR assays according to the methods of Lehner et al. (16), Stoop et al. (20), and Lehner et al. (15). Three microliters of lysate was used in each PCR. Lysates of Cronobacter type strains were included as positive controls. Amplicons obtained from PCRs for genus and species identification were analyzed on 1% agarose gels.

Multiplex serotyping PCR. The PCR-based O-antigen serotyping scheme proposed by Sun et al. (21) was used to identify 132 C. sakazakii isolates. A primer mix for the seven Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/5/883/1685933/0362-028x_jfp-12-521.pdf by guest on 26 September 2021 serotypes (O1 to O7) was used in the multiplex PCR assays with primer concentrations and amplification conditions as recom- mended (21). Amplification products were separated on a 1.5% agarose gel for 105 min at 80 V. Serotypes of isolates were identified after determination of the amplicon sizes by comparison with a molecular size standard (GeneRuler 100 bp plus DNA ladder, Gibco, Grand Island, NY) (21).

MLST. Multilocus sequence typing (MLST) was used as a molecular technique to further characterize selected C. sakazakii strains (n ~ 19). The strains included (i) representative isolates from clusters containing multiple clonal pulsotypes and (ii) examples of all O-antigen serotypes identified in the study. Seven housekeeping genes were amplified using the primers and PCR conditions as described by Joseph et al. (13). The PCR products were purified with the MinElute PCR purification kit (Qiagen, Hilden, Germany), and the purified amplicons were combined with the respective sequencing primers (13). Sequencing was outsourced (Microsynth, Balgach, Switzer- land). Sequence types (STs) were determined by using the Cronobacter MLST Web site (http://pubmlst.org/cronobacter/) (11).

PFGE. Pulsed-field gel electrophoresis (PFGE) was carried out on all C. sakazakii isolates in this study (n ~ 132) following the method described by Iversen et al. (8). The XbaI-digested DNA was separated in a 1% pulsed-field certified agarose gel (Bio-Rad, Hercules, CA) in 0.5| Tris-borate-EDTA running buffer on a CHEF-DR III system (Bio-Rad). The running buffer was supplemented with 50 mM thiourea (Sigma, St. Louis, MO) (18). Salmonella Braenderup strain H9812 digested with XbaI served as a size marker. The following running conditions were applied for the separation of the macrorestriction fragments: pulse time of 5 to 50 s at 6 V cm21 and linear ramping for 20 h at 14uC, 120uC included angle. After electrophoresis, the gels were stained in ethidium bromide (5 mg ml2l) for 30 min and destained in distilled water for 10 min. PFGE patterns were visualized under UV light and captured with a charge-coupled device photography system (Bio-Rad) from

r FIGURE 1. Overview of the results for the 132 Cronobacter sakazakii strains investigated in this study. Data are given for PFGE pulsotypes (dendrogram), sampling codes, sampling source, sampling date, and O-antigen serotype. Env, environment; prod, product; suppl, supplement. The line marks the 95% pattern similarity cutoff. J. Food Prot., Vol. 76, No. 5 GENETIC DIVERSITY OF C. SAKAZAKII ISOLATES 885

TABLE 1. Results of MLST analysis and the PCR-based O-antigen serotyping of 19 C. sakazakii strains Sample code Sample source Sample date Serotype Multilocus sequence type (ST)

SU12_7 Environment 6 Feb. 12 O7 83 SU12_13 Environment 30 Apr. 12 O1 1 SU12_14 Product 3 Jan. 12 O2 NDa SU12_20 Environment 26 June 12 O2 4 SU12_26 Environment 14 June 12 O2 4 SU12_27 Environment 14 June 12 O7 83 SU12_36 Environment 12 Mar. 12 O1 16 SU12_37 Product 28 Nov. 11 O2 4 SU12_67 Environment 7 Nov. 11 O7 83 SU12_69 Environment 7 Nov. 11 O7 83 SU12_74 Environment 19 Sep. 11 O3 4 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/5/883/1685933/0362-028x_jfp-12-521.pdf by guest on 26 September 2021 SU12_77 Environment 19 Sep. 12 O1 ND SU12_83 Environment 7 Nov. 11 O2 4 SU12_62 Environment 13 Feb. 12 O6 ND SU12_94 Environment 1 Aug. 12 O1 1 SU12_110 Environment 9 Aug. 12 O2 ND SU12_116 Product 3 Sep. 12 O2 64 SU12_130 Product 3 Sep. 12 O2 4 SU12_142 Environment 30 May 12 O2 ND a ND, not determined; determination of the ST was not possible because the combination of the STs of the single loci resulted in two or three different possible STs for these isolates. which tagged image (TIFF) files were imported into Gel Compar antigen serotyping technique was applied to obtain data on II software version 5.1 (Applied-Maths, Sint-Martens-Latem, the distribution of O-antigen serotypes among a large Belgium). Cluster analysis of the PFGE patterns was accomplished collection of C. sakazakii isolates from this type of sample using the DICE coefficient and the unweighted pair group method material. With the exception of serotype O5, all other with arithmetic mean (8). Optimization and a band position serotypes were found among the isolates. Serotype O2 tolerance of 3% were chosen. The relatedness of patterns was was the most prevalent serotype (88 strains) followed compared at 95% similarity. by serotype O7 (24 strains). The results of the O-antigen RESULTS AND DISCUSSION serotyping for all 132 strains are included in Figure 1. PCR- based typing methods targeting O-antigen–specific genes Of 141 confirmed Cronobacter spp. isolates, 132 were are reliable and rapid for typing isolates of Escherichia coli, identified as C. sakazakii, 7asC. malonaticus, 1asC. Shigella, and Salmonella from clinical, food, and environ- turicensis, and 1 as C. dublinensis. Thus, C. sakazakii was mental samples (2, 3, 6). However, further data on the the most common species identified in this study (93.6%)in distribution of serotypes (e.g., among clinical isolates) are concordance with previous studies (8, 14). Details (sample needed to determine whether molecular serotyping is a codes, sample sources, sampling dates) of the isolates are useful alternative for epidemiological surveillance of C. given in Figure 1. sakazakii. The 132 C. sakazakii isolates were subjected to three MLST was performed on 19 selected C. sakazakii subtyping methods. A recently described PCR-based O- isolates (Table 1). ST4 was the most prevalent ST found among these strains in accordance with the results described by Joseph et al. (13), who reported that ST4 was the most frequent ST found in clinical isolates and isolates from nonclinical sources, including powdered infant formula. For five isolates (SU12_14, serotype O2; SU12_62, serotype O6; SU12_77, serotype O1; SU12_110, serotype O2; and SU12_142, serotype O2), we were unable to determine a definite ST because the combination of the sequence types of the single loci resulted in two or three different possible STs for these isolates. This difficulty may be explained by the relatively poor quality of the sequencing results (i.e., ambiguous bases within the sequences) for the glnS locus that were obtained for these nontypeable strains. FIGURE 2. Example of two clusters containing multiple clonal Comparison of the MLST STs with the O-antigen C. sakazakii strains isolated from different samples at different serotypes indicated correlations between ST4 and serotype time points. O2 and between ST83 and serotype O7. Table 1 combines 886 MU¨ LLER ET AL. J. Food Prot., Vol. 76, No. 5 the results of the O-antigen serotyping and the MLST 2. DebRoy, C., E. Roberts, J. Kundrat, M. A. Davis, C. E. Briggs, and analysis for the selected strains. P. M. Fratamico. 2004. Detection of Escherichia coli serogroups O26 and O113 by PCR amplification of the wzx and wzy genes. Appl. The 132 C. sakazakii isolates were also subjected Environ. Microbiol. 70:1830–1832. to PFGE analysis, and these isolates clustered into 61 3. Feng, L., W. Wang, J. Tao, H. Guo, G. Krause, L. Beutin, and distinguishable pulsotypes at a cutoff value of 95% pattern L. Wang. 2004. Identification of Escherichia coli O114 O-antigen similarity (Fig. 1). Each pulsotype contained 1 to 13 gene cluster and development of an O114 serogroup-specific PCR isolates; some pulsotypes contained multiple clonal isolates assay. J. Clin. Microbiol. 42:3799–3804. from samples taken at different times and places and strains 4. Gosney, M. A., M. V. Martin, A. E. Wright, and M. Gallagher. 2006. sakazakii in the mouth of stroke patients and isolated from diverse sample sources. Twenty-four clusters its association with aspiration pneumonia. Eur. J. Int. Med. 17:185– containing more than one isolate were identified, and 37 188. strains had distinct fingerprint patterns. Seven clusters 5. Gurtler, J. B., J. L. Kornacki, and L. R. Beuchat. 2005. Enterobacter containing more than three clonal isolates were identified. sakazakii: a coliform of increased concern to infant health. Int. J. The pulsotypes of the isolates and their relationship to the Food Microbiol. 104:1–34. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/5/883/1685933/0362-028x_jfp-12-521.pdf by guest on 26 September 2021 sampling sources and dates are depicted in Figure 1. 6. Han, W., B. Liu, B. Cao, L. Beutin, U. Kru¨ger, H. Liu, Y. Li, Y. Liu, L. Feng, and L. Wang. 2007. DNA microarray-based Clusters with multiple clonal isolates included samples identification of serogroups and virulence gene patterns of from different time points and sampling sources. In Escherichia coli isolates associated with porcine postweaning accordance with previous studies (8, 14, 17), some diarrhea and edema disease. Appl. Environ. Microbiol. 73:4082– pulsotypes contained clonal isolates originating from the 4088. environment, supplements, and products. Thus, strain 7. Hunter, C. J., M. Petrosyan, H. R. Ford, and N. V. Prasadarao. 2008. SU12_37 was isolated from a product in November 2011, Enterobacter sakazakii: an emerging pathogen in infants and neonates. Surg. Infect. 9:533–539. but isolates with identical pulsotypes were isolated from 8. Iversen, C., A. Lehner, C. Fricker-Feer, K. Gschwend, and R. environmental sources (e.g., SU12_89) or supplements Stephan. 2009. Genotyping of Cronobacter (Enterobacter sakazakii) (e.g., SU12_112) during sampling in 2012 (Fig. 2). This strains from an infant formula processing plant. Arch. Lebensmittel- finding indicates that isolates displaying this pulsotype are hyg. 60:66–72. highly persistent in the production facility environment and 9. Iversen, C., A. Lehner, N. Mullane, J. Marugg, S. Fanning, R. Stephan, frequent interactions among the environment, products, and and H. Joosten. 2007. Identification of ‘‘Cronobacter’’ spp. (Entero- bacter sakazakii). J. Clin. Microbiol. 45:3814–3816. supplements occurred. Such observations indicate that on- 10. Iversen, C., N. Mullane, B. McCardell, B. D. Tall, A. Lehner, S. going PFGE analysis of isolates from the production Fanning, R. Stephan, and H. Joosten. 2008. Cronobacter gen. nov., environment may serve to identify particular strains that a new genus to accommodate the biogroups of Enterobacter exhibit a persisting phenotype and may be of particular sakazakii and proposal of Cronobacter sakazakii gen. nov. comb. interest for further investigation. nov., C. malonaticus sp. nov., C. turicensis sp. nov., C. muytjensii In the present study, three molecular typing methods sp. nov., C. dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, C. dublinensis sp. nov. subsp. dublinensis were applied to further characterize a C. sakazakii strain subsp. nov., C. dublinensis sp. nov. subsp. lausannensis subsp. nov., collection from a powdered infant formula production and C. dublinensis sp. nov. subsp. lactaridi subsp. nov. Int. J. Syst. facility. Although MLST is usually used to obtain Evol. Microbiol. 58:1442–1447. information on the phylogenetic relationships among 11. Jolley, K. A., M.-S. Chan, and M. C. J. Maiden. 2004. mlstdbNet— strains, PFGE represents the method of choice when distributed multi-locus sequence typing (MLST) databases. BMC questions concerning the sources of contamination and Bioinformatics 5:86. 12. Joseph, S., E. Cetinkaya, H. Drahovska, A. Levican, M. J. Figueras, routes of transmission need to be answered. By applying the and S. J. Forsythe. 2011. Cronobacter condimenti sp. nov., isolated recently developed O-antigen serotyping PCR assay to a from spiced meat, and Cronobacter universalis sp. nov., a species large panel of strains and comparing the information with designation for Cronobacter sp. genomospecies 1, recovered from a the MLST typing results for the same strains, correlations leg infection, water and food ingredients. Int. J. Syst. Evol. Microbiol. between ST4 and serotype O2 and between ST83 and 62:1277–1283. serotype O7 were observed. 13. Joseph, S., H. Sonbol, S. Hariri, P. Desai, M. MacClelland, and S. J. Forsythe. 2012. Diversity of the Cronobacter genus as These data are an important contribution to an revealed by multi locus sequence typing. J. Clin. Microbiol. 50: understanding of the diversity and characteristics of these 3031–3039. organisms, which will help to identify contamination routes 14. Lehner, A., C. Fricker-Feer, K. Gschwend, and R. Stephan. 2010. and/or the nature of persisting strains and thus limit the risk Identification of and Cronobacter spp. in raw for contaminations in products. Comparisons of various milk concentrate and milk powder: prevalence and genotyping. Arch. Lebensmittelhyg. 61:22–26. typing methods, as performed in this study, may enable 15. Lehner, A., C. Fricker-Feer, and R. Stephan. 2012. Identification of researchers to identify certain pathotypes that may be linked the recently described Cronobacter condimenti by an rpoB-gene– to a specific O-antigen serotype and/or multilocus ST, thus based PCR system. Med. Microbiol. 61:1034–1035. opening up the possibility of using either method as a typing 16. Lehner, A., S. Nitzsche, P. Breeuwer, B. Diep, K. Thelen, and tool for molecular risk assessment. R. Stephan. 2006. Comparison of two chromogenic media and evaluation of two molecular based identification systems for REFERENCES Enterobacter sakazakii detection. BMC Microbiol. 6:15. 17. Mullane, N. R., P. Whyte, P. G. Wall, T. Quinn, and S. Fanning. 1. Bowen, A. B., and C. R. Braden. 2006. Invasive Enterobacter 2007. Application of pulsed-field gel electrophoresis to characterize sakazakii infection in the newborn. Emerg. Infect. Dis. 12:1185– and trace the prevalence of Enterobacter sakazakii in infant formula 1189. processing facility. Int. J. Food Microbiol. 116:73–81. J. Food Prot., Vol. 76, No. 5 GENETIC DIVERSITY OF C. SAKAZAKII ISOLATES 887

18. Ray, T., A. Mills, and P. Dyson. 1995. Tris-dependent oxidative differentiate the six proposed species within the genus Cronobacter. DNA scission during electrophoresis. Electrophoresis 16:888–894. Int. J. Food Microbiol. 136:165–168. 19. See, K. C., H. A. Than, and T. Tang. 2007. Enterobacter sakazakii 21. Sun, Y., M. Wang, Q. Wang, B. Cao, X. He, K. Li, L. Feng, and L. bacteraemia with multiple splenic abscesses in a 75-year-old woman: Wang. 2012. Genetic analysis of the Cronobacter sakazakii O4 to O7 a case report. Age Ageing 36:595–596. O-antigen gene clusters and development of a PCR assay for 20. Stoop, B., A. Lehner, C. Iversen, S. Fanning, and R. Stephan. 2009. identification of all C. sakazakii O serotypes. Appl. Environ. Microbiol. Development and evaluation of rpoB based PCR systems to 78:3966–3974. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/76/5/883/1685933/0362-028x_jfp-12-521.pdf by guest on 26 September 2021