Introduction

Cronobacter spp. (formerly Enterobacter sakazakii) are Gram-negative opportunistic pathogens but fatal in neonates and immunocompromised infants (Ling et al., 2018). The genus Cronobacter currently comprises seven species: C. sakazakii, C. dublinensis, C. turicensis, C. malonaticus, C. muytjensii, C. condiment, and C. universalis. It is well known that Cronobacter had been classified as pathogenic organisms to a restricted population, especially causing life-threatening infections in neonates and immunocompromised infants (Forsythe, 2018). These organisms are widely distributed in foods including powdered infant formulas (PIF), infant cereal foods, dairy products, fruits, and elements in the environment like soil, water, and dust (Tutar et al., 2018; Ueda, 2017). Cronobacter infections in infants occur mostly through the consumption of contaminated PIF. As a non-sterile product, PIF is a major source of nutrition for infants under 6 months of age who could not be breastfed; however, the production of PIF involves stages where the product is susceptible to microbial, especially Cronobacter contamination, the most vulnerable phase occurring after pasteurization and prior to packaging (Drapala et al., 2018; Kalyantanda et al., 2015; Pires et al., 2020). In several outbreaks caused by Cronobacter, the source of infection was traced to contaminated PIF or to spoons and blenders used in preparation of PIF (Chenu and Cox, 2009; Friedemann, 2008). Additionally, Cronobacter spp. display remarkable resistance to desiccation compared with other Enterobacteriaceae, which may contribute to their long-term survival in PIF and on surfaces of the production environment (Gurtler and Beuchat, 2007). Cronobacter contamination can occur in any step during PIF production, especially in raw ingredients, during processing—originating from the surrounding environment, as a result of improper flushing, and in the prepared PIF for infant consumption. Besides, multiple studies illustrated that some strains of Cronobacter are able to form biofilms on glass, stainless steel, polyvinyl chloride, polycarbonate, silicone, and enteral feeding tubes through different media (Henry and Monica, 2019; Iversen et al., 2004). Biofilm formation by Cronobacter species is a major concern to the PIF industry, as biofilms on raw ingredients or PIF contact surfaces represent possible sources of product contamination with Cronobacter.

Our previous study showed that four (3.4%, 4/119) out of 119 retailed powdered formula samples collected from 16 provinces between 2014 and 2015 were positive for Cronobacter (Gan et al., 2018). Based on this result, we carried out a 5-month successive surveillance for Cronobacter along the entire PIF production chain in two PIF manufactures of which their PIF at retail level were contaminated by Cronobacter. The aim of this study was to determine the Cronobacter contamination pathways, to characterize the antimicrobial susceptibility as well as biofilm formation of the major species of Cronobacter recovered in these two factories. Meanwhile, pulsed-field gel electrophoresis (PFGE) of Cronobacter isolating from different origins was also conducted in order to track the source of contamination across the entire PIF production chain.

Materials and Methods

Sample collection

Two factories (referred to as A and B) located in Shan Xi province, China were chosen to carry out a 5-month surveillance from July to November 2016. Sampling plan was set up based on production flow of dry process. A total of 620 samples including 174 powder samples, 33 liquid samples, 135 air sedimentation samples, and 278 swab samples were obtained from 54 sampling areas and items including raw ingredients, surface of equipment, environment (soil and dust), swabs of the staff’s hand, intermediate products, the end products etc. Among them, 195 samples including 28 powder samples, six liquid samples, 48 air sedimentation samples, and 113 swab samples were collected from Factory A. While, 425 samples including 146 powder samples, 27 liquid samples, 87 air sedimentation samples and 165 swab samples were obtained from Factory B. Brain Heart Agar (BHA) plates were used to collect air samples. Briefly, plates were exposed in the air in different areas of PIF production for 15–20 min. Additionally, 50 mL sterile centrifuge tubes were used to collect liquid samples including raw goat milk, concentrated milk, and water; they were collected from production pipeline. Powder samples including base powder, final PIF powder, soil around factory, floor powder, and raw ingredients were collected at amounts shown in Table 1 and stored in sampling bags. Swab samples were collected over a 25 cm² surface with sterile cotton swabs moistened in buffered peptone water (BPW). Details were given in Table 1. All samples were placed in insulation box with ice bag and tested on the same day.

Isolation and species identification of Cronobacter

Samples were detected for Cronobacter using a modified method based on China National Food Safety Standard (GB4789.40, 2016). For powder and liquid samples, an aliquot of 100 g (or) mL test portion was mixed with 900 mL BPW (unless otherwise indicated, all media from Beijing Land Bridge Technology Ltd., Beijing, China) and incubated at 37°C for 18 h ± 2 h. Swabs were inoculated into 10 mL BPW and incubated at 37°C for 18 h ± 2 h. The pre-enrichment samples were mixed well followed by transferring 1 mL into a tube containing 10 mL mLST/Vancomycin medium and incubated at 41.5°C for 24 h ± 2 h. A loop full of mLST/vancomycin culture was streaked on Brilliance Enterobacter sakazakii Agar (DFI) (Oxoid, England), and incubated at 41.5°C for 24 h ± 2 h. Five suspected colonies (all suspected colonies would be picked up when the number of colonies less than five) were selected and inoculated onto tryptone soya agar (TSA) plate and incubated at 37°C for 24 h. Colonies on TSA plate were further identified by both VITEK® 2 Compact (bioMérieux, France) and ITS (ITS-F 5’-GGGTTGTCTGCGAAAGCGAA-3’, ITS-R 5’-GTCTTCGTGCTGCGAGTTTG-3’) by polymerase chain reaction (PCR) based on the method published by Liu (Liu et al., 2006). During determination of Cronobacter in the air of PIF production areas by disk sedimentation on TSA plate, all plates were incubated at 25°C for 48 h ± 4 h. Yellow pigment colonies were selected and streaked on TSA plate again and identified by both VITEK® 2 Compact and PCR, respectively.

For Cronobacter identification at species level, seven pairs of primers designed according to RNA polymerase β subunit (rpoB) gene were used to differentiate the seven species of Cronobacter according to the methods published previously (Lehner et al., 2012; Stoop et al., 2009). A total of 25 μl PCR reaction mixtures were prepared containing 12.5 μl of 2 × GoTaq Green Master Mix, each primer (10 μmol/L) for 1 μl, 9.5 μl of distilled deionized water and 1 μl of template. The primer sequences and PCR conditions are shown in Table 2.

Antimicrobial susceptibility testing

Minimal inhibitory concentrations (MIC) of 12 antimicrobials including Tetracycline (TET), Nalidixic acid (NAL), Ciprofloxacin (CIP), Chloramphenicol (CHL), Sulfamethoxazole-Trimethoprim (SXT), Amoxicillin (AMX), Ampicillin (AMP), Ampicillin-Sulbactam (SAM), Gentamicin (GEN), Cefotaxime (CTX), Ceftazidime (CAZ), and Imipenem (IMP) were determined via broth microdilution method for all confirmed Cronobacter isolates. Escherichia coli ATCC 25922 was used as a quality control in antimicrobial susceptibility testing (AST) experiment. All susceptibility results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) interpretive standards (CLSI M100S, 2016).

PFGE subtyping of Cronobacter

PFGE analysis was performed on all Cronobacter isolates. Cell suspensions were adjusted to 4–4.5 malt concentration. After solidification, plugs [0.5% (w/v)] were lysed in 5 mL cell lysis buffer (50 mmol/L Tris-HCl, 50 nmol/L EDTA, 1% sodium lauroyl-sarcosine) and 25 μl proteinase K (20 mg/mL) at 55°C with 170 r/min shaking for 2 h. Plugs were washed twice with 15 mL deionized water for 10 min followed by washing with 15 mL TE buffer at 50°C shaking at a speed of 170 r/min for 3 times, 15 min for each. After cutting into 3 mm slices, the plug was digest with XbaI at 37°C for 2 h. Treated plugs were cast into a 1% (w/v) SKG gel and separated by electrophoresis in 0.5 × TBE buffer at 14°C for 18 h. The PFGE gel was then dyed with 0.01% (w/v) GelRed for 30 min and then soaked in deionized water for 30 min. DNA fingerprints were analyzed by BioNumerics V7.6 (Applied Math, Belgium) using DICE coefficient and unweighted pair group method with arithmetic mean (UPGMA). Patterns indistinguishable by computer and visual inspection were assigned the same pattern designation.

Biofilm formation assay

Quantification of biofilms grown in microtiter dishes was performed by following the procedure as described by Yan (Yan et al., 2015). M9 minimal medium (6 g/l Na₂HPO₄, 3 g/l KH₂PO₄, 0.5 g/l NaCl, 1 g/l NH₄Cl, 2 mM MgSO₄, 0.1% glucose and 0.1 mM CaCl₂) adjusted to 0.1 Malt concentration was used as the growth medium. Cronobacter isolates were inoculated into tryptic soy broth (TSB) medium and incubated at 37°C overnight. Briefly, a triplicate 200 μl cultures was transferred into 96-well microplates and incubated at 28°C and 37°C for 72 h, respectively. Each well was washed with 200 μl of phosphate-buffered saline (PBS) for 3 times, fixated with 200 μl of methanol for 20 min and stained with 200 μl 0.4% (w/v) crystal violet (CV) for 15 min after air-drying for 15 min. The plate was washed with 200 μl PBS for 3 times, dissolved biofilm with 200 μl of 33% (v/v) acetic acid for 30 min, and measured the optical density (OD) at 570 nm. Salmonella Typhimurium ATCC 14028 with a strong biofilm-forming capacity was selected as the positive control (Martins et al., 2013). All tests were carried out in triplicate.

Results

Prevalence of Cronobacter contamination in samples

Table 3 shows that among 620 samples, 14 (2.26%, 14/620) were positive for Cronobacter and 33 isolates were obtained. Among the positive samples, seven (50%, 7/14) samples were soil origin, four were swabs of drain, air inlet and soles of staff, two were PIF raw ingredients of lactose, and one was air sedimentation, respectively. In terms of 33 Cronobacter isolates, 21 (63.64%, 21/33), seven (21.21%, 7/33), four (12.12%, 4/33), and one (0.30%, 1/33) were cultured from soil, lactose, environmental swabs, and air sedimentation samples, respectively. Regarding PIF factories, 13 isolates cultured from eight samples (57.14%, 8/14) including four soil samples, four swabs of air inlet of purification room, drain, and soles from Factory A and 20 isolates from six samples (42.86%, 6/14) including three soil samples, two lactose samples and one rear packaging room air sedimentation samples from Factory B were contaminated by Cronobacter. No significant difference on the incidence of Cronobacter contamination between these two PIF factories were found (Chi-square test: X² = 3.251, P = 0.071); however, contamination of raw ingredients was found in Factory B. In 14 Cronobacter positive samples, 12 samples including soil, environmental swabs, and lactose were contaminated by C. sakazakii and two (including soil and air sedimentation) by C. malonaticus, respectively. Among 33 Cronobacter isolates, C. sakazakii was the predominant species detected and accounted for 87.88% (29/33) followed by C. malonaticus (12.12%, 4/33). All 13 isolates from Factory A were C. sakazakii. Whereas, 20 Cronobacter isolates from Factory B were diverse at the species level: 16 (80%, 16/20) for C. sakazakii from soil and lactose, and four for C. malonaticus from soil and air sedimentation, respectively. Precisely, two different species were detected from soil samples collected in Factory B (Table 3).

AST of Cronobacter

Results of AST show that all 33 Cronobacter isolates cultured from different matrix samples were susceptible to all the 12 antimicrobials tested except one isolate with an intermediate resistance to Chloramphenicol.

PFGE subtyping analysis

The 33 Cronobacter isolates comprising 29 C. sakazakii and four C. malonaticus were analyzed using PFGE with XbaI and differentiated into nine PFGE pulsotypes with similarities above 95% (Figure 1). The predominant pulsotype of C. sakazakii was C8, which included seven isolates (7/33, 21.2%) cultured from lactose collected from Factory B, followed by C4 (6/33, 18.2%) and C5 (6/33, 18.2%) cultured from soil collected from Factory A and Factory B, respectively. Pulsotypes of C3 and C6 consisted of four C. malonaticus isolates from both soil and air sedimentation and three C. sakazakii isolates from Factory B, respectively. Three pulsotypes (C1, C2, and C7) of C. sakazakii cultured from soil, air inlet, drain, and workers’ sole collected from Factory A, respectively were found to contain two isolates for each. And the remaining C9 pulsotype contained one isolate obtained from soil in Factory A. The PFGE results revealed that the drain and air inlet in the workshop of Factory A had cross contamination of Cronobacter with soil around the factory, respectively. Factory B also existed cross contamination between soil and rear packaging room environment. And the isolates collected form staff’s sole of Factory A were different from other strains isolated from this factory. The contamination of Cronobacter during the production of PIF may come from both raw ingredients like lactose and the environment including soil, air, workers movement, and so forth.

Figure 1. PFGE typing of 33 Cronobacter isolates.

Biofilm formation

All 33 Cronobacter isolates were studied for biofilm formation using the microplate method with M9 minimal medium as the growth medium at two different incubation temperatures: 28°C and 37°C. A temperature of 28°C was chosen to simulate that of PIF processing environment, while 37°C was the one similar to human body temperature. Biofilm formation by Cronobacter isolates is shown in Table 4 and Figure 2. In all the 33 Cronobacter isolates tested, 17 isolates (51.52%, 17/33) of C. sakazakii produced biofilm which were deemed “weak” on their rating scale with an optical density (OD) at 570 nm (OD₅₇₀) <0.2 at both 28°C and 37°C on the basis of criteria described by Lee (Lee et al., 2012). Eight (8/33, 24.24%) isolates including three C. malonaticus and five C. sakazakii showed intermediate capacity of biofilm formation with an OD₅₇₀ ranged from 0.5 to 1.0 at 28°C and two of C. sakazakii isolates exhibited strong biofilm formation with an OD₅₇₀ >1 at 28°C. Whereas, two (2/33, 6.06%) C. sakazakii and three C. malonaticus (3/33, 9.09%) isolates showed intermediate and strong biofilm formation ability at 37°C, respectively. No significant difference of isolates in OD₅₇₀ >0.5 biofilm-forming ability between 28°C and 37°C was found (Fisher test: P = 0.240). Additionally, intermediate and strong biofilm formation ability of four C. malonaticus isolates at 28°C and 37°C, respectively, was observed. No significant difference in the biofilm-forming capacity at 28°C was found between C. malonaticus and C. sakazakii isolates (Fisher test: P = 0.073). Whereas, the capacity of 3 C. malonaticus isolates in forming biofilm at 37°C was significantly stronger than those of C. sakazakii (Fisher test: P = 0.007). Regarding the effect of temperature on biofilm formation, Figure 2 revealed that there is no significant difference in biofilm formation by Cronobacter between 28°C and 37°C (T tested: P = 0.176). Compared with S. Typhimurium ATCC 14028, 11 Cronobacter isolates were of stronger capacity in biofilm formation at 37°C.

Figure 2. Comparison of biofilm formation by 33 Cronobacter isolates at 28°C and 37°C

Discussion

The results of this study indicated that Cronobacter can enter processing plants through raw ingredients, soil, and PIF production environment and be disseminated throughout, especially in the situation of areas of high foot traffic were contaminated by staff’s soles with Cronobacter, and consequently can cross-contaminate processing equipment and persist for long periods in these facilities. This persistence is also attributable to biofilms—a well-known factor for protecting bacteria from harsh environmental stresses such as desiccation, antimicrobials, and disinfectants—which is of concern to public health and food safety. The result of PFGE in this study further illustrated that carry-over of Cronobacter from raw ingredient as well as PIF processing environment contamination by this bacterium are the major sources of Cronobacter contamination in PIF. Therefore, PIF manufactures should strengthen the management of raw ingredients and production environments, especially they should pay more attention to external and internal environment contamination of PIF processing workshop.

Among the 33 Cronobacter isolates, 29 (87.88%, 29/33) were C. sakazakii and four (12.12%, 4/33) were C. malonaticus. C. sakazakii is the dominant species isolated, which was the same as previous studies (Fei et al., 2015, 2017). Since all seven species of Cronobacter have pathogenicity and the clinical reports of neonatal Cronobacter infection are primarily because of C. sakazakii, C. malonaticus, and C. turicensis. According to Stephen J. Forsythe, grouped Cronobacter species were divided into two groups: Group 1 comprises C. sakazakii and C. malonaticus as the majority of clinical isolates in all age groups. C. sakazakii is mainly related to neonatal infections while C. malonaticus is related to adult infections (Forsythe, 2018; Jaradat et al., 2014). Hence, we can speculate that infants who consumed PIF produced by these two factories that is contaminated with one or more Cronobacters like C. sakazakii and C. malonaticus would be at a high risk of Cronobacter infection on the basis of our study.

Antibiotic resistant bacteria has been the focus of global attention; the spread of antibiotic resistant bacteria directly leads to the reduction or ineffectiveness of clinical antibiotic treatments, extend patient’s course, and could cause complications. All the 33 Cronobacter isolates obtained in the present study were susceptible to all 12 antimicrobials tested except one isolate that was of intermediate resistance to Chloramphenicol. Our previous studies revealed that eight out of 417 (1.92%) Cronobacter isolates cultured from four kinds of infant foods from 27 provinces in China during 2012–2014 were resistant to antimicrobials, including AMX (3), AMP (2), CHL (2), CTX (1), NAL (1), SXT (1), and TET (1) (Gan et al., 2015). These data imply that Cronobacter isolates were generally sensitive to antimicrobials, similar to the results reported by Gu, Chen and Pei (Chen et al., 2011; Gu et al., 2008; Pei et al., 2007).

Lee defined OD_600nm > 1 as a strong biofilm-forming strain, 0.5 < OD_600nm < 1 as a moderate biofilm-forming strain, and OD_600nm < 0.5 as a weak biofilm strain (Lee et al., 2012). According to his definition, the biofilm formation ability of all 33 isolates were weaker than S. Typhymurium ATCC 14028 at 28°C. There were two C. sakazakii isolates, codex YL32 and YL33, which had strong biofilm formation ability. PFGE showed that both isolates cultured from the same source (swabs of staff’s sole in Factory A); once these isolates are found to persist in the workshop, they will cause a potential long term threat such as PIF contamination. There were eight moderate biofilm-forming strains, including YL15, YL21, and YL23 which belonged to pulsotype C3 with 100% similarity and isolated from disk sedimentation of rear packaging room and soil sample. And YL16, YL19, YL20, YL25, and YL26 with moderate biofilm formation ability belonged to C8 and came from lactose powders. These isolates could cause persistent contamination on the surface of productive facilities and affect the safety of end products. Two C. malonaticus isolates were strong biofilm-forming strains at 37°C and moderate at 28°C. The National Institutes of Health (NIH) of America announced that over 80% of microbial infections were found involved in biofilm formation (Römling and Balsalobre, 2012). Cronobacter could form biofilm on different materials such as silicon, latex, polycarbonate, stainless steel, glass, and polyvinyl chloride (Choi et al., 2014). Cells of Cronobacter in biofilm could survive for several weeks under dry conditions and resistant to osmotic pressure (Dancer et al., 2009). Biofilm has a great impact on PIF industry, persistent strains with biofilm formation ability could store on the surface of packaging materials and processing equipment for a long time, and resistant to common cleaning agents, disinfection and sterilization methods, and cause a long term and persistent contamination in production environment. A strict regulatory strategy for PIF manufactures including successive monitoring for environment, raw ingredients, semi and end products, particular disinfection procedures are needed. Hazard analysis and critical point (HACCP) systems could be the effective control measures and decrease the risk of Cronobacter contamination along the entire production chain.

Conclusion

Totally, 33 isolates of Cronobacter were isolated form raw ingredients, PIF processing environment, and workers’ sole from two PIF processing factories in Shanxi province, China between July and November 2016. C. sakazakii was found to be the predominant species in different sources of our surveillance. The results suggest that two factories exited multi-source contamination of Cronobacter, especially in the PIF processing environment. C. sakazakii and C. malonaticus could form biofilm at either 28°C or 37°C, with strong biofilm formation ability at 37°C for C. malonaticus. The data obtained in the present study would provide the scientific basis for prevention and control measures of PIF contamination by Cronobacter during manufacturing. It is necessary to minimize the contamination of this bacterium from different sources and reduce the risk of infants being exposed to Cronobacter via PIF.