Prevalence, phenotypic and genotypic characterization, virulence potential and antimicrobial resistance of Cronobacter species from ready-to-eat foods

Cronobacter , an emerging foodborne pathogen, contaminates various foods such as ready-to-eat (RTE) food due to its ubiquitous nature. Consumption of food contaminated with Cronobacter can cause severe infections in children, elderly or immunocompromised people. Therefore, we aimed to assess the presence of Cronobacter spp. in RTE foods, popularly consumed products, and pose potential health threat for consumers, especially for risk groups.Results Out of 340 RTE 59 (17.4%) were contaminated with Cronobacter spp. in this study. The highest contamination rate was found in free cig koftes followed by cereals and A total of 64 Cronobacter isolates were identified phenotypically and genotypically from the 59 ready-to-eat foods samples. Molecular characterization was accomplished by PCR targeting 16S rRNA, gluA , rpoB and cgcA genes. The 64 Cronobacter isolates were completely identified by the biochemical and rpoB , while the PCR targeting the cgcA gene failed to identify to eight isolates. In this study, we investigated major virulence characteristics contributing to the pathogenicity of Cronobacter spp. including the outer membrane protein A, zinc-metalloprotease, siderophore production and biofilm formation. Many Cronobacter isolates (>87%) had these virulence characteristics. All isolates and a type strain were characterized using ERIC-PCR and genetic profiles of cluster analysis showed that the isolates were highly heterogeneous and genetically diverse. Antimicrobial susceptibility of RTE isolates to 18 different antimicrobial agents was determined by the disc diffusion method. Most Cronobacter isolates with a rate of 81.3% were resistant to cephalothin, 32.8% to cefoxitin and 20.3% to ampicillin. All Cronobacter isolates were susceptible to gentamicin and trimethoprim/sulfamethoxazole. detection and isolation method developed for Cronobacter species. The oxidase and catalase test; motility assessment; indole production; utilization reaction;

at least three or more antimicrobial agents was detected in 18.8% of Cronobacter

isolates.Conclusions
Results indicate that RTE food harbors potential pathogenic Cronobacter species and is a possible transmission vehicle for Cronobacter infection in vulnerable person. So, there is a need to adopt hygienic practices and rigorous sanitization treatments to ensure microbiological safety of RTE food.
Cronobacter is considered to be an opportunistic pathogen implicated in several diseases including necrotizing enterocolitis, bacteremia, and meningitis [1,6]. In 2002, the

International Commission for Microbiological Specifications for Foods (ICMSF) classified
Cronobacter as a serious threat for restricted populations, causing life-threatening or considerable illness of extended period, with the high-risk populations being newborns and immunocompromised infants [7]. The symptoms of Cronobacter infections are high fever, headache with nausea, a swelling on the head, body and neck stiffness, skin rash, seizures, urosepsis, pneumonia [8]. All Cronobacter species have been associated with human infections except for C. condimenti. Cronobacter infections in neonates and infant seem to be particularly concerned with species of C. sakazakii [8,9,10]. In addition, C. malonaticus has been commonly linked to adult infections. Moreover, a single case of Drug Administration (FDA) and the International Organization for Standardization (ISO) improved protocols for the isolation and detection of Cronobacter spp. [21,22].
Cronobacter species have been identified and classified genotypically using amplification of 16S rRNA gene, the gluA gene, the enterobacterial repetitive intergenic consensus (ERIC) sequences, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), ribotyping and plasmid typing [5,[23][24][25]. Moreover, the rpoB gene-based PCR method was improved to differentiate between the seven Cronobacter species. It has been known that the bacterial RNA polymerase β-subunit is encoded by this universal gene and its suitability in species definition has been evaluated [26,27]. Furthermore, gene cgcA encoding a diguanylate cyclase which is involved in virulence, biofilm production and survival of the organism has also been used as the genetic marker to distinguish Cronobacter species [28].
However, antimicrobial resistant strains of Cronobacter spp., some of which have multiple drug resistance, were reported in various studies [31,32]. The emergence of resistant Cronobacter spp. and transmission of these resistant species from food to human may be a significant problem for treatment of Cronobacter infections [5,11] Today, a growing number of people are buying ready-to-eat foods since they are easy and practical to prepare, and delicious foods to consume [33]. Ready-to-eat foods that are usually high in protein and ready to eat without further heating or cooking are known as high-risk foods and provide excellent conditions for bacterial growth [34]. Cronobacter , a ubiquitous foodborne pathogen, can cause severe infections in immunocompromised people over the world. In Turkey, several studies focused on isolation and identification of Cronobacter spp. from samples of milk powder, powdered infant formula and cereal-based products [35,36], but there is little data on the prevalence of Cronobacter spp. in readyto-eat foods. Therefore, this study aims to determine the prevalence of Cronobacter species in ready-to-eat foods, to identify the Cronobacter species by phenotypic and genotypic methods, and to evaluate virulence factors, antimicrobial susceptibility and genetic relatedness of Cronobacter isolates.

Sample collection
A total of 340 ready-to-eat food samples were purchased from markets, local bazaar, patisserie and herbalist from January 2016 to December 2016 in Bolu (Western Turkey).
Isolation and phenotypically identification of Cronobacter spp.
Genus-specific identification of Cronobacter spp. using PCR The genomic DNA extraction to use in all PCR experiments was performed using a previously described method [59]. All Cronobacter isolates were grown on TSA for 24 h at 37°C. One colony of each culture was added into 5 mL Brain Heart Infusion (Merck) broth and was incubated at 37°C for 18 h. The extracted DNA was kept at -20°C for further use.
For the genus-specific identification, the 16S rRNA gene [25] and the gluA gene [24] were used in all isolates. The primer sequences, expected size of amplification products and references are presented in Table 3. PCR amplification of 16S rRNA was performed in a total volume of 30 µL, with the following reagent concentration: 3 µL of 10× PCR buffer Species-specific identification of Cronobacter spp. using PCR Two PCR assays targeting the rpoB gene and the cgcA gene were used to identify Cronobacter species. Identification of the seven species of Cronobacter including C.
universalis was carried out with the appropriate primer sets for the rpoB gene [26,27].
Species-specific detection and differentiation of Cronobacter species by Multiplex-PCR (M-PCR) assay employing amplification of the cgcA gene were performed as described earlier [28]. PCR was performed in a reaction volume of 50 μL containing 5 μL of 10× PCR buffer (Vivantis), 4 mM MgCl 2 (Vivantis), 0.2 mM dNTP mix (Vivantis), 0.4 μM of each primers (Biomers), 5 μL of DNA template (50 ng/μL) and 2 U of Taq DNA polymerase (Vivantis). The final volume to 50 μL was adjusted by adding molecular grade water (AppliChem). The PCR condition of the cgcA M-PCR assays was carried out as described by Carter et al. [28]. The primer sequences, expected size of amplification products and references for both methods are given in Table 3.
Siderophore production was studied based on the Chrome azurol S (CAS) assay of Schwyn and Neilands [60] with some modifications made by Fiss and Brooks [61]. Briefly, overnight culture of Cronobacter isolates were spot inoculated onto a chrome azurol S (CAS) agar plate and incubated for 5 days at 37°C. After incubation, siderophore positive isolates were seen as an orange halo around a colony [60]. were performed triple times. The isolates were classified as strong, moderate and weak for biofilm formation with respect to Stepanovic et al. [62].

Antimicrobial susceptibility test
All Cronobacter spp. were tested for antimicrobial susceptibility to 18 antimicrobials using the disk diffusion method according to the Clinical and Laboratory Standards Institute  Table   1.
In this study, all phenotypically-identified isolates were completely confirmed by the genus-specific 16S rRNA gene while the presence of gluA gene was 93.8% among the Cronobacter isolates. All Cronobacter species differentiated using species-specific the rpoB gene and the cgcA gene along with the results of the biochemical identification, the 16S rRNA and gluA are shown in Table 2.
Virulence characteristics of Cronobacter spp.
The pathogenic potential of the obtained Cronobacter isolates was confirmed by the presence of virulence related genes: ompA is associated with the invasion ability; zpx encodes the zinc-containing metalloprotease that contributes pathogenicity of Cronobacter. All 64 Cronobacter isolates (100%) were found to be positive for the ompA gene while 63 (98.4%) isolates harbored the zpx gene. The biofilm formation was detected in the 56 (87.5%) of the 64 isolates by the microtiter plate assay. Overall, 3.1%, 31.3% and 53.1% of the Cronobacter isolates were strong, moderate and weak biofilm producer, respectively. On the other hand, the production of siderophore as a significant virulence property was detected in all Cronobacter isolates (100%) ( Table 2).
Genetic variation among the Cronobacter isolates from ready-to-eat foods The 64 Cronobacter isolates and type strain C. sakazakii ATCC 29544 were subjected to ERIC-PCR. All isolates amplified with the ERIC primers. The ERIC-PCR generated the 3 to 12 amplified bands with sizes ranging from 100 to 3000 bp. We identified 64 different ERIC patterns with a similarity index ranging from 10% to 100% using the BioNumerics software (version 7.6) (Fig. 1). The ERIC patterns were grouped into two major clusters (A and B).
The predominant cluster was cluster B, which contained 56 (86.2%) isolates, 2 of which had the same pattern. Cluster A was composed of 9 (13.8%) of the isolates that presented 9 distinct patterns. The Cronobacter isolates from the same food categories were represented in the distinct patterns; therefore, there was no correlation between the ERIC-PCR profiles and origin of the isolates (Fig. 1).

Antimicrobial resistance
The antimicrobial susceptibility results of all 64 Cronobacter isolates from RTE food to 18 antimicrobial agents interpreted as susceptible, intermediate or resistant are shown in  Table 2.

Discussion
Cronobacter species as pathogens cause severe infections in vulnerable adults and infants. These bacteria which are ubiquitous have been isolated from a wide range of sources such as contaminated ready-to-eat foods [5,6,34]. In this study, the prevalence of Cronobacter spp. in RTE foods was detected to be 17.4%. In previous studies, the level of contamination with Cronobacter spp. in RTE foods ranged from 9.0% to 45% [4,[37][38][39].
The different rates of the presence of the Cronobacter in various foods have been documented in many different countries such as in Ireland, China, India, Brazil and Egypt [9,[40][41][42]. The variation in the occurrence of bacteria can be explained by different factors such as source of the isolation, nature of samples and geographic location [5].
In recent studies, among food products, cereal, flour, pasta, herbs and spices have been highlighted as being most frequently contaminated by Cronobacter spp. [9,38,41]. In the current study, Cronobacter spp. were commonly isolated from 51.9%, 46.7%, 30.8% and 30.2% samples of meat-free cigkoftes, spices, cereals and desserts, respectively. In Turkey, meat-free cig kofte is a popular food consisting of bulgur, various spices and vegetables and it is consumed uncooked. In particular, a high occurrence of Cronobacter spp. (51.9%) in meat free cig koftes may be due to contaminated ingredients including cereal (46.7%) and spices (30.8%). In the study of Aksu et al. [10] in Turkey, the 50% of the Cronobacter isolates obtained from cig kofte (cereal-based ball) was similar to our research. Four samples of cereals (bulgur, red lentil, peanut, and goji berry) and seven samples of spices (cumin, chili pepper, thyme, mint, flower flour, coconut) were contaminated with Cronobacter spp. (Table 1). These findings were similar to some studies focused on isolation from cereals and spices [38,40,41]. The reported isolation rates of samples varied from 3.6% to 34% [37,41,43].
In several studies, Cronobacter were isolated from flours and dairy products such as milkbased desserts, cheeses, raw milk, curd, ice-cream [38,[41][42][43]. In this study, Cronobacter spp. were detected from desserts including milky desserts (2/16) and dough sweets (11/27) (Table 1). Desserts can be made by using ingredients such as milk, flour, cream, flavors, sweeteners, cereals, nuts and fruits. Our study indicates that contamination of desserts may be resulted from contaminated ingredients. In the study of Saad and Ewida [42], the prevalence of Cronobacter spp. was detected in 5.5% of milk-based desserts in accordance with the result of our study. The occurrence of Cronobacter in cheeses (7.5%) was found to be higher than results of studies of Iversen and Forsythe [44] and Singh et al. [41]. In another study, Cronobacter was not isolated (0/20) from cheeses [40]. Saad and Ewida [42] isolated Cronobacter from 1/30 samples of ice-cream, but Kandhai et al. [43] did not found Cronobacter in ice-cream (0/89). These findings were lower than our results (14.8%) in ice-cream samples.
The occurrence of Cronobacter spp. was 12.2% in doner, 9.8% in pastramis and 8.6% in kavurmas as retail meat products in this research. Several studies demonstrated varying prevalence of Cronobacter spp.in meat and meat products: 0.0% in Turkey [10], 3.2% in the Netherlands [43], and 14.5% in China [4]. In this study, Cronobacter spp. were isolated from 2.6% samples of salads, while were not detected in herbs and vegetables. The result in salads (1/38) was lower than the result (13/30) obtained by Vasconcellos et al. [39], while was similar to the result (1/15) obtained by Lee et al. [45]. Cronobacter were not detected from dried herbs and vegetables in some studies, similar to our results [10,40].
In another research, the Cronobacter spp. was detected in 25% of vegetables [41]. In the light of our results and results of many previous studies, it has been considered that the contamination of RTE foods may be the result from the contaminated ingredients, improper food handling practices, storage conditions and environmental factors.
In the present study, all Cronobacter spp. to the genus level were identified using the 16S rRNA and the gluA gene. Many researchers have been used the gluA gene as an additional tool for identification of Cronobacter spp. [24,46]. Iversen et al. [46] reported that gluA gene were 100% sensitive and specific for determination and confirmation of Cronobacter spp. However, in this research, out of the 64 isolates, the 60 (93.8%) isolates were positive for the gluA gene ( Table 2). Some other researchers did not also detect the gluA gene in some of Cronobacter strains, similar to our findings [47,48].
In this study, in addition to phenotypic identification, the genotypic identification of Cronobacter isolates were performed using the species-specific rpoB and cgcA genes [26][27][28]. Although the Cronobacter isolates were completely identified by the biochemical test and the rpoB PCR method, the eight isolates were not identified by the cgcA M-PCR method. Compared to the results of identification using the rpoB gene, failure results in the cgcA method were associated with the species C. malonaticus, C. turicensis, C. muytjensii and C. dublinensis (Table 2). Of 64 Cronobacter isolates, 42 (65.6%) were completely consistent according to the identification results of the biochemical tests, rpoB PCR method and cgcA M-PCR method. Among the remaining Cronobacter isolates, there was inconsistency to phenotypic and genotypic identification methods. In this study, the identification of 41 C. sakazakii isolates was same in rpoB PCR and cgcA M-PCR method.
Brandao et al. [38] identified successfully Cronobacter isolates to the species level using cgcA primers. However, some reports documented the inefficiency of cgcA gene to identify some Cronobacter isolates for the species-specific identification [30,39].
C. sakazakii is most common among the species of the Cronobacter genus and plays a significant role in human diseases [5,20]. In this study, C. sakazakii was also the most common Cronobacter species isolated, followed by C. malonaticus and other species of Cronobacter. Many researchers found C. sakazakii as the most common species among the Cronobacter genus, similar to our result [10,30,38]. Moreover, in many previous studies, the C. malonaticus andother species of Cronobacter were isolated from various foods [4,30,39].
In the present study, various virulence characteristics such as outer membrane protein A (OmpA), zinc-containing metalloprotease, siderophore production, and biofilm formation were found among Cronobacter isolates from RTE foods. The ompA gene encoding OmpA, has a significant role in Cronobacter invasion, was detected in 100% of the Cronobacter isolates. Similarly, the occurrence of ompA gene was found 100% among the Cronobacter isolates by many researchers [43,49]. On the contrary, in some studies, the incidence of Cronobacter spp. containing the ompA gene has been reported as 64.7% in the USA [50] and 33.3% in the Bangladesh [17]. In addition, in this study, many isolates of Cronobacter (98.4%) harbored the zpx gene encoding zinc-containing metalloprotease as an indicator of pathogenicity. The prevalence of zpx gene in Cronobacter spp. ranging from 1.3% to 65.5% was reported in previous studies [17,47,51]. Siderophore required for the iron uptake is a virulence factor for Cronobacter [19]. In several researches, all Cronobacter isolates exhibited siderophore production, similar to the result in this research [17,52].
Biofilms are considered to be a potential way of pathogen transmission [17,18]. In this study, the biofilm formation of Cronobacter (87.5%) was higher than the result obtained by Fakruddin et al. [17] and Lee et al. [45], while was similar to the result obtained by Ye et al. [53].
The genetic diversity of the 64 Cronobacter isolates in RTE food products and type strain C. sakazakii ATCC 29544 was assessed by ERIC-PCR in the present research (Fig. 1). The similarity threshold of 10%. Most isolates (>86%) harbored virulence factors and antibiotic resistance were distributed on the cluster B. In this study, the results of ERIC-PCR indicated that there was no apparent clustering tendency among the genotypes related to the origin of isolates, virulence profile and antimicrobial resistance. In many studies, genetic variation of Cronobacter isolates from various food samples have been also detected using ERIC-PCR method [17,54,55].
In this study, the resistance rates of the Cronobacter isolates ranging from 1.5% to 20.3% to piperacillin, ampicillin, amoxicillin-clavulanic acid, nalidixic acid, aztreonam were obtained. Previous reports revealed the occurrence of resistance to the antimicrobial agents in Cronobacter spp. isolated from different sources [31,32,56]. Increasing resistance of Cronobacter against a range of cephalosporins and penicillins due to the production of β-lactamases has been reported [11,29,30]. In our study, the cephalosporins that are used in the treatment of Cronobacter infections had resistance ranging from 4.7% to 81.3% through the first-second-third-fourth generation. In addition, in the present study, it is noteworthy that Cronobacter isolates displayed a high intermediate resistance to cefoxitin (32.8%), cefepime (21.9%), cefotaxime (20.3%), ampicillin (17.2%), piperacillin (17.2%) and cephalothin (10.9%) (Fig. 2). In several studies, the resistance to ampicillin, cephalothin, cefoxitin, and cefotaxime in Cronobacter was reported to range from 5.6% to 50.0% [31,32]. Cronobacter infections have been treated with carbapenems or the newer cephalosporins in concert with an aminoglycoside or trimethoprim-sulfamethoxazole [11,12]. As prescribed for the treatment, in our study, more than 90% of the Cronobacter isolates were also susceptibility to piperacillintazobactam, gentamicin, amikacin, imipenem, meropenem, tetracycline, trimethoprim/sulfamethoxazole, ciprofloxacin, and chloramphenicol. Susceptibility to these antimicrobials has been reported in many countries: Brazil [30], China [9], and Iran [56]. In the current study, the multidrug resistance to at least three or more antimicrobial agents was detected in the 12 (18.8%) of Cronobacter isolates. The findings of Kim et al. [57] and Li et al. [58] were contrary to our results that any isolates were resistant against three or more antimicrobial agents.

Conclusions
Cronobacter spp. were isolated from samples of ready-to-eat (RTE) foods, with a higher presence of C. sakazakii, an emerging foodborne pathogen. The present study provides current knowledge on the incidence, identification, genotyping, potential virulence and antimicrobial resistance of Cronobacter spp. in RTE foods. The consumption of RTE foods Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable. Tables   Table 1 Prevalence   b Siderophore Production. c Not identified due to non-PCR amplification or non-specific PCR amplification.