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

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1 883 Journal of Food Protection, Vol. 76, No. 5, 2013, Pages doi: / x.jfp Copyright G, International Association for Food Protection Research Note Genetic Diversity of Cronobacter sakazakii Isolates Collected from a Swiss Infant Formula Production Facility ANDREA MÜLLER, 1 ROGER STEPHAN, 1 CLAUDIA FRICKER-FEER, 2 AND ANGELIKA LEHNER 1 * 1 Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland; and 2 Department of Quality Assurance and Food Safety, Hochdorf Nutritec AG, Hochdorf, Switzerland MS : 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. sakazakii, C. turicensis, C. malonaticus, C. muytjensii, C. dublinensis, C. universalis, and C. condimenti (10, 12). These bacteria are opportunistic pathogens that can cause septicemia and infections of the central nervous system, primarily in premature, low-birth-weight, and/or immunocompromised neonates (1, 5), but infections among immunocompromised elderly adults also have been reported (4, 19). Most outbreaks have been reported in neonatal intensive care units, where the sources of infection have been traced to temperature-abused reconstituted powdered infant formula and/or feeding equipment contaminated with Cronobacter spp. (7). The presence of these pathogens in infant formula products represents a challenge for the powdered infant formula industry. Thus, specific and accurate identification of members of the genus Cronobacter and discrimination between these bacteria and closely related but nonpathogenic organisms that may be present in the same habitat (products and environment) are critical. Several phenotypic features such as yellow pigmentation of colonies or alphaglucosidase activity, which have been attributed to members of the Cronobacter genus, have been of limited use in identification schemes because of the lack of exclusivity. However, combinations of molecular identification methods and culture detection and identification procedures significantly * Author for correspondence. Tel: z ; Fax: z ; lehnera@fsafety.uzh.ch. improved interventions for the control of these pathogens (9, 15, 16, 20). The risk posed by contaminated infant formula when it is consumed by neonates raises the question of the possible origin and routes of dissemination and transmission of these organisms into and within the infant formula processing environment and/or final products. Two possible routes have been described for dissemination of Cronobacter spp. into production lines and recontamination of pasteurized products: organisms may be attached to dust or to dry heatlabile supplement ingredients (8, 14, 17). This study was part of ongoing collaborative work on the identification and characterization of presumptive Cronobacter spp. isolates collected in a powdered infant formula facility in Switzerland as part of a hygienic monitoring program. MATERIALS AND METHODS Strains. For this study, 148 cryopreserved presumptive Cronobacter spp. isolates collected as part of a hygienic monitoring program in a powdered infant formula facility in Switzerland between September 2011 and October 2012 were further characterized. The strain collection comprised 115 isolates from production environments (walls, floors, vacuum cleaners, filters, rinsing water, drains), 25 isolates from finished products (powdered infant formula, follow-on formula, growing-up formula), and 8 isolates originating from supplements, raw materials or ingredients (vitamins, whey protein, concentrates). Preparation of cell lysates to be used in PCR-based assays. Cryopreserved strains were first streaked onto blood agar

2 884 MÜLLER 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 ml 21 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 serotypes (O1 to O7) was used in the multiplex PCR assays with primer concentrations and amplification conditions as recommended (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, Switzerland). Sequence types (STs) were determined by using the Cronobacter MLST Web site ( (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 cm 21 and linear ramping for 20 h at 14uC, 120uC included angle. After electrophoresis, the gels were stained in ethidium bromide (5 mg ml 2l ) 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.

3 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 ND a 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 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 II software version 5.1 (Applied-Maths, Sint-Martens-Latem, Belgium). Cluster analysis of the PFGE patterns was accomplished using the DICE coefficient and the unweighted pair group method with arithmetic mean (8). Optimization and a band position tolerance of 3% were chosen. The relatedness of patterns was compared at 95% similarity. RESULTS AND DISCUSSION Of 141 confirmed Cronobacter spp. isolates, 132 were identified as C. sakazakii, 7asC. malonaticus, 1asC. turicensis, and 1 as C. dublinensis. Thus, C. sakazakii was the most common species identified in this study (93.6%) in concordance with previous studies (8, 14). Details (sample codes, sample sources, sampling dates) of the isolates are given in Figure 1. The 132 C. sakazakii isolates were subjected to three subtyping methods. A recently described PCR-based O- FIGURE 2. Example of two clusters containing multiple clonal C. sakazakii strains isolated from different samples at different time points. antigen serotyping technique was applied to obtain data on the distribution of O-antigen serotypes among a large collection of C. sakazakii isolates from this type of sample material. With the exception of serotype O5, all other serotypes were found among the isolates. Serotype O2 was the most prevalent serotype (88 strains) followed by serotype O7 (24 strains). The results of the O-antigen serotyping for all 132 strains are included in Figure 1. PCRbased typing methods targeting O-antigen specific genes are reliable and rapid for typing isolates of Escherichia coli, Shigella, and Salmonella from clinical, food, and environmental samples (2, 3, 6). However, further data on the distribution of serotypes (e.g., among clinical isolates) are needed to determine whether molecular serotyping is a useful alternative for epidemiological surveillance of C. sakazakii. MLST was performed on 19 selected C. sakazakii 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. Comparison of the MLST STs with the O-antigen serotypes indicated correlations between ST4 and serotype O2 and between ST83 and serotype O7. Table 1 combines

4 886 MÜLLER ET AL. J. Food Prot., Vol. 76, No. 5 the results of the O-antigen serotyping and the MLST analysis for the selected strains. The 132 C. sakazakii isolates were also subjected to PFGE analysis, and these isolates clustered into 61 distinguishable pulsotypes at a cutoff value of 95% pattern similarity (Fig. 1). Each pulsotype contained 1 to 13 isolates; some pulsotypes contained multiple clonal isolates from samples taken at different times and places and strains isolated from diverse sample sources. Twenty-four clusters containing more than one isolate were identified, and 37 strains had distinct fingerprint patterns. Seven clusters containing more than three clonal isolates were identified. The pulsotypes of the isolates and their relationship to the sampling sources and dates are depicted in Figure 1. Clusters with multiple clonal isolates included samples from different time points and sampling sources. In accordance with previous studies (8, 14, 17), some pulsotypes contained clonal isolates originating from the environment, supplements, and products. Thus, strain SU12_37 was isolated from a product in November 2011, but isolates with identical pulsotypes were isolated from environmental sources (e.g., SU12_89) or supplements (e.g., SU12_112) during sampling in 2012 (Fig. 2). This finding indicates that isolates displaying this pulsotype are highly persistent in the production facility environment and frequent interactions among the environment, products, and supplements occurred. Such observations indicate that ongoing PFGE analysis of isolates from the production environment may serve to identify particular strains that exhibit a persisting phenotype and may be of particular interest for further investigation. In the present study, three molecular typing methods were applied to further characterize a C. sakazakii strain collection from a powdered infant formula production facility. Although MLST is usually used to obtain information on the phylogenetic relationships among strains, PFGE represents the method of choice when questions concerning the sources of contamination and routes of transmission need to be answered. By applying the recently developed O-antigen serotyping PCR assay to a large panel of strains and comparing the information with the MLST typing results for the same strains, correlations between ST4 and serotype O2 and between ST83 and serotype O7 were observed. These data are an important contribution to an understanding of the diversity and characteristics of these organisms, which will help to identify contamination routes and/or the nature of persisting strains and thus limit the risk for contaminations in products. 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