High-affinity truncated aptamers for detection of Cronobacter spp with magnetic separation-assisted DNAzyme-driven 3D DNA walker.

After optimizing the original aptamer sequence by truncation strategy, a magnetic separation-assisted DNAzyme-driven 3D DNA walker fluorescent aptasensor was developed for detecting the food-borne pathogen Cronobacter species. Iron oxide magnetic nanoparticles (MNPs) modified with a hybrid of truncated aptamer probe and DNAzyme strand (AP-E1) denoted as MNPs@AP-E1, were employed as capture probes. Simultaneously, a DNAzyme-driven 3D-DNA walker was utilized as the signal amplification element. The substrate strand (Sub) was conjugated with the gold nanoparticles (AuNPs), resulting in the formation of AuNPs@Sub, which served as a 3D walking track. In the presence of the target bacteria and Mg2+, E1-DNAzyme was activated and moved along AuNPs@Sub, continuously releasing the signal probe. Under optimized conditions, a strong linear correlation was observed for Cronobacter sakazakii (C. sakazakii) in the concentration range 101 to 106 CFU mL-1, with a low detection limit of 2 CFU mL-1. The fluorescence signal responses for different Cronobacter species exhibited insignificant differences, with a relative standard deviation of 3.6%. Moreover, the aptasensor was successfully applied to determine  C. sakazakii in real samples with recoveries of 92.86%-108.33%. Therefore, the novel method could be a good candidate for ultra-sensitive and selective detection of Cronobacter species without complex manipulation.

Assessment of the Sensitivity of Foodborne Cronobacter spp. Strains and Other Enterobacteria to Temperature Stresses and Chlorine-Containing Biocides.

We examined the effects of elevated temperatures and biocides on survivability of food isolates of Cronobacter spp. (C. sakazakii) and concomitant enterobacteriaceae obtained in microbiological control of infant nutrition products. Increased resistance of certain strains of Cronobacter, Enterobacter cloacae, and Pantoea spp. to thermal processing was revealed. Salmonella, Pantoea, and Cronobacter bacteria were least sensitive to antimicrobial action of chlorine-containing agents. The above properties varied in the strains of the same species. Specifically, only two of three examined isolates of Cronobacter spp. demonstrated lower sensitivity to heat in comparison with the enterobacterial test-cultures of other species.

Invited review: Stress resistance of Cronobacter spp. affecting control of its growth during food production.

Members of the Cronobacter genus include food-borne pathogens that can cause infections in infants, with a mortality rate as high as 40 to 80%. The high fatality rate of Cronobacter and its isolation from numerous types of food, especially from powdered infant formula, demonstrate the serious nature of this organism. The source tracking of Cronobacter spp. and the analysis of high-frequency species from different sources are helpful for a more targeted control. Furthermore, the persistence during food processing and storage may be attributed to strong resistance of Cronobacter spp. to environment stresses such as heat, pH, and desiccation. There are many factors that support the survival of Cronobacter spp. in harsh environments, such as some genes, regulatory systems, and biofilms. Advanced detection technology is helpful for the strict monitoring of Cronobacter spp. In addition to the traditional heat treatment, many new control techniques have been developed, and the ability to control Cronobacter spp. has been demonstrated. The control of this bacteria is required not only during manufacture, but also through the selection of packaging methods to reduce postprocessing contamination. At the same time, the effect of inactivation methods on product quality and safety must be considered. This review considers the advances in our understanding of environmental stress response in Cronobacter spp. with special emphasis on its implications in food processing.

Dual-signal amplification strategy: Universal asymmetric tailing-PCR triggered rolling circle amplification assay for fluorescent detection of Cronobacter spp. in milk.

Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula that cause many serious diseases in neonates and infants. In this study, a novel assay based on dual signal amplification strategy was developed by coupling asymmetric tailing PCR (AT-PCR) with rolling circle amplification (RCA) for the detection of Cronobacter spp. in milk. The tailing single-stranded DNA was generated through AT-PCR and used to initiate RCA, generating tandem repetitive G-quadruplex sequences. In the presence of the fluorescence dye thioflavin T that could intercalate into the G-quadruplex structures, the fluorescence signal was detected with a microplate reader. The AT-PCR coupled with RCA assay was specific for Cronobacter spp. detection because of the highly specific primers chosen for the AT-PCR. The limits of detection were 4.3 × 101 cfu/mL in pure culture and 4.5 × 102 cfu/mL in spiked milk, respectively. The fixed sequences designed in the hairpin DNA allowed this AT-PCR coupled with RCA assay to serve as a universal platform for the detection of other pathogens by modifying the specificity of the PCR primers.

Quantifying the uncertainty of transfer of Cronobacter spp. between fomites and floors and touch points in dairy processing plants.

Cronobacter spp. are opportunistic pathogens that must be controlled in infant powder manufacturing plants. This study evaluated the spread of Cronobacter cells via contact surfaces within a dairy manufacturing environment. Transfer rates of Cronobacter spp. were determined from vectors for transmission including moveable fomites (e.g. trolley wheels and boots) and gloved hands to various types of recipient surfaces (stainless steel, linoleum and resin-coated concrete) typical for dairy manufacturing environments. Overall, with a starting inoculum of 106 CFU/mL, approximately 104 CFU/mL Cronobacter cells were transferred from each fomite onto each recipient surface during the initial transfer event. Gloved hands transferred the highest number of Cronobacter cells, followed by polyvinylchloride boots and then polyurethane trolley wheels. We demonstrate, using a combination of experimental data and uncertainty analysis, that if a movable fomite (boots or trolley wheels), or gloves became contaminated, Cronobacter could be spread over a wide area within a manufacturing plant. To the authors' knowledge, this is the first quantitative estimation of the spread of Cronobacter within a dairy manufacturing plant, that can also be practically applied as a tool for providing information in making risk management decisions. In particular, the estimation of spread suggests areas for cleaning and sanitation within a dairy manufacturing environment during a contamination event.

Efficient inhibition of Cronobacter biofilms by chitooligosaccharides of specific molecular weight.

Biofilms enable Cronobacter spp. to contaminate food, infect infants and resist different environmental stresses, especially desiccation, which is the main reason why Cronobacter can survive in powdered infant formula (PIF) for a long time. Considering the high lethality of Cronobacter infection in infants, it is important to find efficient and safe inhibitors of Cronobacter biofilms. In this study, we found that chitooligosaccharides (COS) with a molecular weight of 2000 Da efficiently inhibited Cronobacter biofilms, especially in skim milk broth. The minimum biofilm inhibitory concentration (MBIC77) of COS was as low as 20 µg/mL, which is lower than that reported in most previous studies. Besides, the elimination rate of COS for Cronobacter mature biofilms was 50% when the concentration was 10 mg/mL. COS could significantly inhibit soluble polysaccharide secretion and biofilm cell growth, as well as change the cell membrane permeability of Cronobacter. These might be the possible reasons for COS's efficient inhibition of Cronobacter biofilms. However, during the inhibition, five important genes-related to biofilm formation-flhD, flgJ, luxR, ompA, and wcaJ-were all up-regulated after COS treatment, except the gene bcsA. In summary, our findings showed that COS could be used as an efficient and safe inhibitor against Cronobacter biofilms for better control of Cronobacter contamination and infection.

Characterisation of Cronobacter strains isolated from hospitalised adult patients.

Bacteria belonging to the genus Cronobacter are opportunistic pathogens known for causing rare but serious infections in neonates, including meningitis, necrotising enterocolitis and sepsis. Cronobacter infections occur also in adult populations, however, they generally have milder manifestations and their prevalence is uncertain. In this study, the presence of Cronobacter strains from adult patients in the University Hospital in Bratislava was investigated and overall 18 confirmed isolates from 321 patients (5.3%) were recovered. No Cronobacter positive sample was detected in 215 sputum samples from outpatients. The highest occurrence of Cronobacter strains was observed from stroke patients and this may be associated with an abnormal swallowing ability. The isolated strains belonged to the species Cronobacter sakazakii and Cronobacter malonaticus. In silico genotyping (MLST, CRISPR-cas array profiling) of whole genome sequences assigned the strains to three different MLST clones. The majority (12/18) of the isolated strains were sequence type ST513 or single locus variants ST514 and ST515, thereby being members of C. sakazakii pathovar clonal complex CC4. However, according to core genome MLST analysis the ST513-ST515 strains created a unique cluster substantially different from other CC4 strains. The isolated strains were susceptible to 18 tested antibiotics. All strains possess a genomic island encoding for increased thermal tolerance. As Cronobacter strains are frequently present in dried foods of plant origin, spread of a specific clone within a hospital may be caused by food transmission and may be facilitated by its tolerance to environmental stresses such as desiccation and temperature.

Development of an isothermal amplification-based assay for the rapid detection of Cronobacter spp.

Cronobacter spp. is an opportunistic pathogen that is associated with rare but life-threatening neonatal infections resulting from the consumption of contaminated powdered infant formula milk (PIF). In the present study, we developed recombinase polymerase amplification (RPA) and real-time RPA for the detection of Cronobacter spp. in PIF for the first time by targeting the ompA gene. The specificity and sensitivity of the RPA and real-time RPA were validated and the practical applicability of these methods for the detection of Cronobacter spp. in artificially contaminated PIF samples was proved by comparing their reaction time, sensitivity, and efficacy with those of real-time PCR and the Chinese traditional method. The RPA and real-time RPA assays reduced the analysis time to less than 15 min and the results were as reliable as those of real-time PCR. Taken together, the RPA and real-time RPA assays served as fast, reliable, and sensitive techniques for the detection of Cronobacter spp.

Isolation, molecular and phenotypic characterization, and antibiotic susceptibility of Cronobacter spp. from Brazilian retail foods.

Several Cronobacter species are opportunistic pathogens that cause infections in humans. The aim of this study was to detect Cronobacter spp. from 90 samples of retail foods in Brazil, and characterize the strains by phenotypic tests, molecular assays and antibiotic susceptibility. Three isolation methodologies were evaluated using different selective enrichments and the isolates were identified using Vitek 2.0, PCRs protocols, fusA allele sequencing and multilocus sequence typing (MLST). Thirty-eight samples (42.2%) contained Cronobacter spp., and the highest percentage was found in flours (66.7%, 20/30), followed by spices and herbs (36.7%, 11/30), and cereal mixes for children (23.3%, 7/30). The 45 isolates included four species: C. sakazakii (n = 37), C. malonaticus (n = 3), C. dublinensis (n = 3), and C. muytjensii (n = 2); that presented 20 different fusA alleles. MLST analysis revealed 32 sequence types (STs), 13 of which were newly identified. All strains were sensitive to all antibiotics (n = 10) tested. The combination of CSB/v enrichment with DFI plating was considered the most efficient for Cronobacter spp. isolation. This study revealed the presence of Cronobacter spp. in foods commercialized in Brazil and the isolates showed a high diversity after MLST analysis and included two strains of the C. sakazakii ST4 neonatal meningitic pathovar.

Novel PCR-RFLP system based on rpoB gene for differentiation of Cronobacter species.

Bacteria from the genus Cronobacter are opportunistic foodborne pathogens that can cause severe infections. More rapid, cost-effective and reliable methods are still required for the species identification of Cronobacter spp. In this study, we present a novel PCR-RFLP-based method that uses a newly designed pair of primers for the PCR-amplification of a partial rpoB gene sequence (1635 bp). The amplified products of DNA from 80 Cronobacter strains were separately digested with three restriction endonucleases (Csp6I, HinP1I, MboI). Using the obtained restriction patterns, a PCR-RFLP identification system was created to enable differentiation between all seven currently-known Cronobacter species. The functionality of our method was successfully verified on real food samples. Moreover, the relationships between the Cronobacter species were determined via a phylogenetic tree created from the RFLP patterns.

Microbiological quality of selected ready-to-eat leaf vegetables, sprouts and non-pasteurized fresh fruit-vegetable juices including the presence of Cronobacter spp.

Bacteria of the genus Cronobacter are emerging food-borne pathogens. Foods contaminated with Cronobacter spp. may pose a risk to infants or adults with suppressed immunity. This study was aimed at determining the microbiological quality of ready-to-eat (RTE) plant-origin food products available on the Polish market with special emphasis on the prevalence of Cronobacter genus bacteria. Analyses were carried out on 60 samples of commercial RTE type plant-origin food products, including: leaf vegetables (20 samples), sprouts (20 samples) and non-pasteurized vegetable, fruit and fruit-vegetable juices (20 samples). All samples were determined for the total count of aerobic mesophilic bacteria (TAMB) and for the presence of Cronobacter spp. The isolates of Cronobacter spp. were subjected to genetic identification and differentiation by 16S rDNA sequencing, PCR-RFLP analysis and RAPD-PCR and evaluation of antibiotic susceptibility by the disk diffusion assay. The TAMB count in samples of lettuces, sprouts and non-pasteurized fruit, vegetable and fruit-vegetable juices was in the range of 5.6-7.6, 6.7-8.4 and 2.9-7.7 log CFU g-1, respectively. The presence of Cronobacter spp. was detected in 21 (35%) samples of the products, including in 6 (30%) samples of leaf vegetables (rucola, lamb's lettuce, endive escarola and leaf vegetables mix) and in 15 (75%) samples of sprouts (alfalfa, broccoli, small radish, lentil, sunflower, leek and sprout mix). No presence of Cronobacter spp. was detected in the analyzed samples of non-pasteurized fruit, vegetable and fruit-vegetable juices. The 21 strains of Cronobacter spp. isolated from leaf vegetable and sprouts included: 13 strains of C. sakazakii, 4 strains of C. muytjensii, 2 strains of C. turicensis, one strain of C. malonaticus and one strain of C. condimenti. All isolated C. sakazakii, C. muytjensii, C. turicensis and C. malonaticus strains were sensitive to ampicillin, cefepime, chloramphenicol, gentamycin, streptomycin, tetracycline, ciprofloxacin and cotrimoxazol, whereas the C. condimenti isolate showed intermediate resistance to streptomycin and cotrimoxazole.

Reassessment of Cronobacter spp. originally isolated as Enterobacter sakazakii from infant food.

Cronobacter spp. cause infant disease, several cases have been associated with powdered infant formulae (PIF). In the early 2000s, contamination of German PIF with these opportunistic pathogens was quite common. Before 2008, all isolates Cronobacter spp. had been classified as Enterobacter sakazakii, therefore little is known about species diversity within such isolates. Genetic, serologic, and biochemical traits of 80 Cronobacter isolates, originally obtained 2003-2006 within infant food surveys in Germany, were reassessed in this study. By sequencing of the fusA gene, all isolates were unambiguously assigned to two species, C. sakazakii (n = 73) and C. malonaticus (n = 7). PCR serotyping identified five C. sakazakii serotypes and two C. malonaticus serotypes, biochemical profiling yielded five biogroups. PFGE analysis also showed high heterogeneity in both species. Multilocus sequence typing of 26 selected isolates yielded 16 different sequence types (ST), including C. sakazakii ST 1 (n = 6) and the highly virulent ST 4 (n = 2). The results suggest that just two, but highly heterogeneous species were responsible for the Cronobacter contamination problem which challenged the German PIF industry in the beginning of this century. This fact may have influenced the success of efforts to identify and eliminate sources of contamination.

Development and application of a sensitive, rapid, and reliable immunomagnetic separation-PCR detection method for Cronobacter spp.

Cronobacter spp. have been linked to clinical cases of infection in both adults and infants. Enrichment of Cronobacter spp. before detection has been necessary but is quite time consuming. Hence, we sought to develop an immunomagnetic separation (IMS) PCR method that could shorten the time of enrichment before the detection of Cronobacter spp. The polyclonal antibody used in this immunomagnetic separation was prepared based on the outer membrane protein A of Cronobacter sakazakii China Center of Industrial Culture Collection 21560 and had high specificity to the target. The primers used in the IMS-PCR method also showed high specificity. The detection limit of IMS-PCR for pure C. sakazakii culture was 5.2 × 102 cfu/mL. Cronobacter sakazakii in artificially contaminated powdered infant formula (PIF) was also detected at a detection limit of 5.2 × 102 cfu/mL. After 8 h of enrichment, the detection limit in PIF was lower than 5.2 × 101 cfu/mL. An interference test using Escherichia coli in artificially contaminated PIF showed that the IMS-PCR method developed in this study had a good ability to resist interference. Finally, the IMS-PCR method was applied to the detection of Cronobacter in food samples and was shown to be reliable. Thus, this newly developed IMS-PCR detection method was quite sensitive, rapid, and reliable and could be applied to the detection of Cronobacter in foods.

Two Cases of Multi-antibiotic Resistant Cronobacter spp. Infections of Infants in China.

Infections by Cronobacter spp. are hazardous to infants since they can lead to neonatal meningitis, bacteremia, and necrotizing enterocolitis. Cronobacter spp. are frequently resistant to ß-lactam derivatives, macrolides, and aminoglycosides. In addition, multi-resistant strains have also been detected. In China, the isolation rate of Cronobacter spp. from commercial powdered infant formula (PIF) or follow-up formula (FUF) is relatively high. Nevertheless, clinical cases of Cronobacter infection have been ignored to date. Here we describe two cases of Cronobacter infection detected at the Wuhan Women and Children Medical Care Center Hospital (Wuhan City, China). We provide the genomic analysis of the isolates and the antibiotic-resistance profiles of the two strains. The Cronobacter strains identified in this study were not susceptible to third-generation cephalosporins, aminoglycoside, and/or trimethoprim-sulfamethoxazole. Whole genome sequencing revealed various genes known to encode antibiotic resistance. Future studies are needed to determine whether the genes predicted in this study are functional. As with Enterobacter spp., the antibiotic resistance of Cronobacter is a serious issue that requires more attention.

Surveillance and molecular typing of Cronobacter spp. in commercial powdered infant formula and follow-up formula from 2011 to 2013 in Shandong Province, China.

BACKGROUND: Infection with Cronobacter spp. leads to neonatal meningitis, necrotizing enterocolitis and bacteremia. Cronobacter spp. are reported to comprise an important pathogen contaminating powdered infant formula (PIF) and follow-up formula (FUF), although little is known about the contamination level of Cronobacter spp. in PIFs and FUFs in China. RESULTS: In total, 1032 samples were collected between 2011 and 2013. Forty-two samples were positive, including 1.6% in PIFs and 6.5% in FUFs. The strains were susceptible to most antibiotics except for cefoxitin. Pulsed-field gel electrophoresis after XbaI digestion produced a total of 36 banding patterns. The 38 strains were found in 27 sequence types (STs), of which nine types (ST454 to ST462) had not been reported in other countries. The clinically relevant strains obtained from the 38 isolates in the present study comprised three ST3, two ST4, two ST8 and one ST1. CONCLUSION: The contamination rate in the PIF and FUF has stayed at a relatively high level. The contamination rate of PIF was significantly lower than FUF. The isolates had high susceptibility to the antibiotics tested, except cefoxitin. There were polymorphisms between the Cronobacter spp. as indicated by pulsed-field gel electrophoresis and multilocus sequence typing. Therefore, contamination with Cronobacter spp. remains a current issue for commercial infant formulas in China. © 2016 Society of Chemical Industry.

Contribution of the thermotolerance genomic island to increased thermal tolerance in Cronobacter strains.

Cronobacter spp. are opportunistic pathogens associated with serious infections in neonates. Increased stress tolerance, including the thermotolerance of some Cronobacter strains, can promote their survival in production facilities and thus raise the possibility of contamination of dried infant formula which has been identified as a potential source of infection. Some Cronobacter strains contain a genomic island, which might be responsible for increased thermotolerance. By analysis of Cronobacter sequenced genomes this determinant was found to be present in only 49/73 Cronobacter sakazakii strains and in 9/14 Cronobacter malonaticus strains. The island was also found in 16/17 clinical isolates originating from two hospitals. Two configurations of the locus were detected; the first one with the size of 18 kbp containing the thrB-Q genes and a shorter version (6 kbp) harbouring only the thrBCD and thrOP genes. Strains containing the thermotolerance island survived significantly better at 58 °C comparing to a C. sakazakii isogenic mutant lacking the island and strains with the longer version of the island were 2-10 times more tolerant than those with the shortened sequence. The function of the genomic island was further confirmed by its cloning into a low-copy vector and transforming it into the isogenic mutant. Different levels of rpoS, encoding for stress-response sigma factor, expression were also associated with variability in strain thermotolerance.

The Survey of Cronobacter spp. (formerly Enterbacter sakazakii) in Infant and Follow-up Powdered Formula in China in 2012.

OBJECTIVE: To determine Cronobacter spp. contamination in infant and follow-up powdered formula in China. METHODS: All of 2282 samples were collected from the retail markets in China from January 2012 to December 2012, and analyzed for Cronobacter spp. by the Chinese National Food Safety Standard. Characterization of the isolates was analyzed by pulsed-field gel electrophoresis (PFGE) with XbaI and SpeI restriction enzymes. RESULTS: Cronobacter spp. strains were isolated from 25 samples, and the positive rates in infant powdered formulas and follow-up powdered formulas were 0.90% (10/1011) and 1.18% (15/1271), respectively. Analysis of variable data regarding different purchasing store formats, seasonality, and production locations as well as comparison of infant versus follow-up formulas did not reveal statistically significant factors. During the sampling period, one of six surveillance zones did exhibit a statistically significant trend towards higher positive rate. PFGE characterization of Cronobacter spp. to elucidate genetic diversity revealed only three pairs of Cronobacter spp. out of 25 having the same PFGE patterns. CONCLUSION: The current investigation indicated a lower positive rate of Cronobacter spp. in the powdered formula in China. This evidence suggested contamination originating from multiple different sources during the manufacturing process.

Diversity of Cronobacter spp. isolates from the vegetables in the middle-east coastline of China.

Cronobacter spp. has caused life-threatening neonatal infections mainly resulted from consumption of contaminated powdered infant formula. A total of 102 vegetable samples from retail markets were evaluated for the presence of Cronobacter spp. Thirty-five presumptive Cronobacter isolates were isolated and identified using API 20E and 16S rDNA sequencing analyses. All isolates and type strains were characterized using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and genetic profiles of cluster analysis from this molecular typing test clearly showed that there were differences among isolates from different vegetables. A polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) based on the amplification of the gyrB gene (1258 bp) was developed to differentiate among Cronobacter species. A new PCR-RFLP assay based on the amplification of the gyrB gene using Alu I and Hinf I endonuclease combination is established and it has been confirmed an accurate and rapid subtyping method to differentiate Cronobacter species. Sequence analysis of the gyrB gene was proven to be suitable for the phylogenetic analysis of the Cronobacter strains, which has much better resolution based on SNPs in the identification of Cronobacter species specificity than PCR-RFLP and ERIC-PCR. Our study further confirmed that vegetables are one of the most common habitats or sources of Cronobacter spp. contamination in the middle-east coastline of China.

Effect of heat shock and recovery temperature on variability of single cell lag time of Cronobacter turicensis.

The effect of heat stress and subsequent recovery temperature on the individual cellular lag of Cronobacter turicensis was analysed using optical density measurements. Low numbers of cells were obtained through serial dilution and the time to reach an optical density of 0.035 was determined. Assuming the lag of a single cell follows a shifted Gamma distribution with a fixed shape parameter, the effect of recovery temperature on the individual lag of untreated and sublethally heat treated cells of Cr. turicensis were modelled. It was found that the shift parameter (Tshift) increased asymptotically as the temperature decreased while the logarithm of the scale parameter (θ) decreased linearly with recovery temperature. To test the validity of the model in food, growth of low numbers of untreated and heat treated Cr. turicensis in artificially contaminated infant first milk was measured experimentally and compared with predictions obtained by Monte Carlo simulations. Although the model for untreated cells slightly underestimated the actual growth in first milk at low temperatures, the model for heat treated cells was in agreement with the data derived from the challenge tests and provides a basis for reliable quantitative microbiological risk assessments for Cronobacter spp. in infant milk.

Prevalence and subtyping of Cronobacter species in goat milk powder factories in Shaanxi province, China.

Cronobacter spp. are opportunistic pathogens that can cause serious diseases in neonates and infants via consumption of contaminated milk powder. To determine Cronobacter spp. contamination status, 632 samples, including 15 evaporated milk, 45 intermediate powder, 150 finished products, and 422 manufacturing environment samples, were collected from 3 goat milk powder factories in Shaanxi province, China, from July 2013 to April 2014. The recovered Cronobacter isolates were subtyped using pulsed-field gel electrophoresis to trace the potential dissemination routes during the whole production processing. Sixty-seven Cronobacter spp. isolates were recovered. The prevalence rates in manufacturing environment, intermediate powder, and finished products were 92.5, 6.0, and 1.5%, respectively. The predominant species were Cronobacter sakazakii (88.1%); no Cronobacter turicensis, Cronobacter condimenti, or Cronobacter dublinensis were detected. Sixty-seven Cronobacter isolates were grouped in 26 clusters by pulsed-field gel electrophoresis, and substantial genetic similarity was observed among isolates from different sampling sites in the same factory. Isolates in the main clusters were commonly recovered from intermediate powder, floor powder, and shoes. These data indicated that air, powder, and personnel movement were potential routes for Cronobacter dissemination, and manufacturing environment is the key control point for Cronobacter contamination.