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Characterizing the Rogues and Smooth Pairs of Cronobacter sakazakii Involved in Biofilm Formation and Long-Term Survival

Cronobacter species are peritrichous flagella, Gram-negative bacilli, and emerging opportunistic foodborne pathogens (Farmer et al., 1980; Iversen et al., 2008; Jarada et al., 2014). Although the epidemiology and reservoir of Cronobacter are still not understood very well, powdered infant formula has been considered as the principal route of transmission among infants (Beuchat et al., 2009; Himelright et al., 2002; Hunter and Bean, 2013; Tall et al., 2015). Cronobacter also cause urinary tract infections, septicemia, and catheter-associated wound infections in people of all ages, especially in the elderly and those with other diseases or immune-compromised individuals (Joseph et al., 2012). Cronobacter can survive in dried food, fresh produces, and fruit juices (Lin and Beuchat, 2007; Gurtler and Beuchat, 2007; Tall et al., 2015).


Introduction
Cronobacter species are peritrichous flagella, Gram-negative bacilli, and emerging opportunistic foodborne pathogens (Farmer et al., 1980;Iversen et al., 2008;Jarada et al., 2014). Although the epidemiology and reservoir of Cronobacter are still not understood very well, powdered infant formula has been considered as the principal route of transmission among infants (Beuchat et al., 2009;Himelright et al., 2002;Hunter and Bean, 2013;Tall et al., 2015). Cronobacter also cause urinary tract infections, septicemia, and catheter-associated wound infections in people of all ages, especially in the elderly and those with other diseases or immune-compromised individuals (Joseph et al., 2012). Cronobacter can survive in dried food, fresh produces, and fruit juices (Lin and Beuchat, 2007;Gurtler and Beuchat, 2007;Tall et al., 2015).
In response to environmental stresses, many pathogens undergo phase variation between rugose (also known as wrinkled or rdar) and smooth colony morphology. Although both variants of Vibrio and Salmonella are pathogenic and are found both in vivo and in vitro, the rugose phase variants form corrugated colonies, exhibit increased resistances to acids, low temperatures, osmotic pressure and oxidative stresses, and form more biofilms (Anriany et al., 2001;Beyhan et al., 2007;de Rezende et al., 2005;Hollenback et al., 2014;Morris et al., 1996;Rice et al., 1992;Wai et al., 1998;Yildiz and Schoolnik 1999;Yildiz et al., 2004). In Vibrio species, more surface polysaccharide (VPS) are produced in the rugose variants. The increase is mediated by genes located in the VpsI (VpsA-K) and VpsII (VpsL-Q) loci (Yildiz and Schoolnik 1999). A microarray study of V. cholerae shows that smooth and rugose phase variation are directed by complex regulatory genes to raise the resistance of the organisms (Yildiz et al., 2004). A rugose (R) variant of V. vulnificus produces a large number of extracellular polysaccharides (EPS) matrix and form biofilms (Grau et al., 2005), while a smooth variant forms less biofilms. The rugose colony morphology and biofilm formation of S. typhimurium are associated with extracellular substances including curli, cellulose, LPS, EPS, and capsules, which are important in the bacterial resistances (Anriany et al., 2001;de Rezende et al., 2005).
Biofilm consists of surface attached microbial communities (single species or several species) and extracellular substances. Formation of biofilms on surfaces can be regarded as universal bacterial strategy for bacterial survival and growth (Kaplan, 2010;Hollenback et al., 2014). C. sakazakii formed biofilms on stainless steel and enteral feeding tubes have increased their resistances to disinfectants (Beuchat et al., 2009), and the biofilms also facilitated survival and persistence of the bacteria in food and food manufacturing environments (Breeuwer et al., 2003).

Cronobacter
and their pathogenic mechanisms. Rugosity may play an important role in Cronobacter survival and persistence in food and food processing environments. In this study, we tried to determine the possible advantages of rugose state in Cronobacter. The rugose and smooth pairs of C. sakazakii were compared in biofilm formation, cell autoaggregation, and long-term survival.

Congo red and calcofluor binding assays
These assays were applied to check the expression of rugose morphotypes and cellulose production (Chapman et al., 2002;Hu et al., 2015). In brief, bacteria were cultured on LB agar overnight at 37 C, and inoculated onto LBNS agar plates supplemented with 40 µg/ml Congo red (Sigma, St. Louis, MO) and 20 µg/ml Coomassie brilliant blue (Sigma), or 200 µg/ml fluorescence brightener 28 (Sigma). The inoculated agar plates were incubated at 28 C for 24 h, and then moved to room temperature, and evaluated at 24 h, 48 h, and 72 h. Cellulose production was judged by viewing the florescence of the colonies under a 366 nm UV light. All isolates were screened in duplicates in each assay and repeated the assay three times. The images of colonies were taken by a Bio-Rad digital camera.

Biofilm formation assay
Detection and quantification of biofilm were performed with glass tubes (11x75 mm) using the procedure of Hu et al., (2015) with slight modification. These strains were incubated in TSB at 37 C or 28 C, shaking at 150 rpm for 48 h. Biofilm formation in the tubes (pellicles at the air-liquid interfaces) was visually tested. The tubes were washed twice with PBS, air-dried for 30 min, stained with 0.1% crystal violet at room temperature for 30 min, and washed twice with distilled water. The crystal violet bound to the biofilm was dissolved with 5 ml of 95% ethanol for 30 min by shaking, 100 µl per well was added to a 96-well plate (three wells for each sample), and absorbance was determined at 570 nm by a UV max microplate reader (Molecular Devices, Corp., Sunnyvale, CA). The biofilm assay was carried out in duplicates, and three independent experiments were performed for each strain. Results are presented as the mean ± SD.

Cell autoaggregation
To determine cell aggregation, the strains grown overnight were inoculated into 5 ml LB broth in a small glass tube with shaking at 37 C or 28 C for 48 h, then left standing at room temperature for 1 h. Cell aggregation was determined by visual estimation of the cell sediment at the bottom of the glass tubes (Gualdi et al., 2008). The assay was carried out at least three independent experiments.

Long-term survival assay
The assay was done following the method of Vestby et al., (2009) with slight modification. Ten microliter overnight cultures of C.
Sakazakii strain 713 and 632 were inoculated in 50 ml sterile centrifuge tubes. Each tube contained 20 ml LB broth and an autoclaved microscope slide, and all tubes was incubated with shaking at 37°C or 28°C. Two tubes without added bacteria served as negative controls. During incubation, biofilms were allowed to form on both sides of the slides. After 48 h, the slides were washed twice in sterile saline, moved to empty sterile centrifuge tubes, and air-dried in a sterile Class II Biosafety cabinet hood overnight. The lids of the tubes with slides were loosely tightened, and placed at room temperature for 5 months for detecting bacterial long-term survival. After 5 months, the slides were moved to new sterile tubes with 20 ml PBS, the biofilms on the slides were thoroughly scraped using a sterile cell scraper and were disrupted with sterile glass beads (20-30 nm diameter) by vortexing. The number of viable bacteria remained in each sample was determined by serially diluting the cell mixtures and plated in duplicates onto 5% sheep blood TBS agar plates (Remel, Lenexa, KS) and incubated at 37 C. Data were presented as the mean number of recovered colonies ± SD of triplicate wells from three independent experiments.

Molecular typing
Pulsed-field gel electrophoresis (PFGE) analysis of XbaI-digested genomic DNA was used to examine the genetic relatedness of Cronobacter rugose and smooth pairs. High molecular mass DNA in agarose plugs was prepared as reported by Herschleb et al., (2007). The DNA samples were digested with the restriction enzyme Xbal, and the plugs were loaded onto a 1.5% agarose gel in 0.5% TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.0). The DNA samples were subjected to separation in a CHEF-Mapper (Bio-Rad) using the flowing parameter: pulsetimes, 1-14 s for 5 h, 12-25 s for 10 h, 8-50 s for 4 h, 6 V cm-1; 120 reorientation angle. The gels were stained with 0.1% ethidium bromide solution, and imaged with a Bio-Rad Gel/Chemo Doc System (Bio-Rad, Hercules, CA).
Metabolic typing by VITEK 2 VITEK® 2 compact system (BioMerieux, Hazelwood, MO) was employed to identify metabolic differences between the C. sakazakii rugose and smooth phase variants following the manufacturer's instruction. The bacteria were grown overnight in TSA broth at 37 C with shaking, the colonies of a pure culture were suspended to a turbidity reading of ~0.5, and a Gram negative (GN) card was used.

Motility assay
Bacteria were grown on TSA overnight, and a single colony was inoculated into a motility test medium (Remel), and incubated in 37 C for 24 h. The bacterial motility was confirmed and the images of the bacterial motility were taken by a Bio-Rad digital camera.

Transmission electron microscope (TEM)
Cronobacter were grown overnight on TSA at 37 C. A bacteria suspension in 0.25-0.5% phosphotungstic acid (pH 6.8) containing approximately 10 6 CFU/ml was applied to a formavar-carbon-coated 300 mesh copper grids for 2 min. The liquid was removed by blotting. The samples were air-dried and visualized under a JEOL JEM-1011 electron microscope using an accelerating voltage of 80 KV.

PCR assay
Cronobacter strains were cultured on TSA overnight at 37 C, and single colony was transferred to TSB, and incubated with shaking overnight. The DNA of the Cronobacter strains was prepared by boiling cell culture in 100 µl distilled water for 10 min served as DNA templates. Primers for detecting cellulose and flagella genes of Cronobacter were designed from the conservative sequences of bcsABC, fliC, flgE, and flgK of C. sakazakii strains (Table 1). GoTaq Green Master Mix (Promega Corp., Madison, WI) was used as manufacture's instruction. One µM of each primer and 1 µl bacterial cell lysate (approximately 50 ng DNA) were missed with PCR buffer. Distilled water served as a negative control. All PCR reactions were run in an Applied Biosystem's models 2720 thermal cycler (Applied Biosystems, Foster City, CL). The PCR assay started by 5 min incubation at 94 C, followed by 30 cycles of 30 sec at 94 C, and 30 sec at 55-60 C, and 1-2 min at 72 C, and terminated with a final extension period of 7 min at 72 C. PCR products were separated with 1.5% agarose gels and stained with 0.1% ethidium bromide solution, and imaged with a Bio-Rad Gel/Chemo Doc System.

Statistical analyses
Each experiment was performed in triplicate. The means of the biofilm formation were compared using Student's t-test.

Results and Discussion
Isolation of rugose and smooth phase variants C. sakazakii 713 and 632 were grown on TSA plates at 37 C. To develop stable, phase variants, serial passages (>20) of colonies from rugose or smooth phase variants were performed by sequentially streaking a single rugose or smooth colony every 3 days onto a fresh TSA plate at 37°C. The stable rugose (rough and dry) colonies were designated as -rugose‖ (R) while the smooth and wet colonies were named -smooth‖ (S). The Cronobacter colonies were grown on LBNS supplemented with Congo red and Coommassie blue at 28 C. Figure 1 shows the colony morphotypes of C. Sakazakii strain 713 and 632 on LBNS agar supplemented with Congo red and Coomassie blue. Strain 713R and 632R were presented as brown, dry, and rough (Bdar) colonies while the 713S was presented as smooth and light yellow colony (YAS) (Fig. 1). 632S showed smooth and light brown colonies.

Comparing rugose and smooth pairs on biofilm formation and cell aggregation
After incubating in TSB in glass tubes for 48 h at 37 C or 28 C with shaking, the formed biofilms were characterized by the presence of a pronounced ring at the air-liquid interface, and the production of the biofilm was assayed by crystal violet staining method. The quantity of biofilm formation of the strains was slightly different when the strains were grown at varying temperatures (Fig. 2). The biofilm formation of 713R at OD570 was ~1.80 at 37 C ( Fig. 2A) and ~1.90 at 28 C (Fig. 2B). The rugose strains produced significantly more biofilms than their smooth counterparts at 37 C and 28 C (all ρ<0.01). These results indicated that Cronobacter rugose strains expressed greater biofilm formation at both temperatures than the smooth counterparts.
Cell aggregation is the auto-clumping of bacterial cells. C. sakazakii 713R and 632R produced large clumps in TSB, leading to rapid bacterial aggregation. Lesser cell aggregates were observed in the 713S and 632S cultures compared to their rugose counterparts (Table 2).

Long-term survival of rugose and smooth pairs in biofilms
After bacterial biofilms formed on the glass slides, the slides were washed, air-dried, and kept in sterile tubes for 5 months at room temperature. Then the biofilm on the slides were thoroughly scraped and the number of viable bacteria remained in each slide was determined by plate counts. Survival of 732R and 632R were significant higher than 713S and 632S (ρ<0.001) ( Table 3). The results clearly indicated that Cronobacter could survive in desiccated biofilm communities for at least 5 months, and the ability of producing large quantities of biofilms by rugose variants provided greater survival and persistence advantages than that of their smooth phase variant counterparts.

PFGE and VITEK typing of rugose and smooth pairs
The genetic typing of strain 713 and 632 was assessed by XbaI-digested genomic DNA using PFGE. The PFGE analysis showed the DNA patterns of the rugose and smooth pairs. There were two distinctly different bands between 713R and 713S in Figure 3. No obvious different patterns were identified between 632R and 632S. The patterns suggested that no remarkable genetic differences were found between the rugose and smooth variants. The difference in strain 713R and 713S may not result from the phase change from rugose to smooth due to no similar common pattern changes between 632R and 632S. The VITEK 2 compact system is a fully automated system that performs bacterial identification by biochemical analysis using colorimetry. All strains were identified as C. sakazakii in this typing method. There are three different reactions between strain 713 (GGT+, BXYL-, and GlyA+) and strain 632 (GGT-, BXYL+, and GlyA-), and one reaction differed between 713R (ILATK+) and 713S (ILATK). No common differences were found between two rugose and smooth pairs. To understand if flagella, curli and cellulose cause the phase changes between strain 713R and 713S, further experiments were carried out.

Detection of motility of C. sakazakii 713 and 632
The motility test agar and transmission EM were used to check the bacterial motility and presence of flagella. The tubes with 0.6% TSA agar were incubated at 37 C, and were observed at 24 h, 48 h, and 72 h. Figure 4 I shows the images of motility tubes incubated for 24 h at 37 C. All strains except 713S showed positive motility in the assay. EM observation of negative stained Cronobacter strains grown on TSA plates at 37 C revealed flagella in 713R, 632R and 632S, but not in 713S (Fig. 4 II). The result confirmed that the lacking of motility in strain 713S was resulted from no expression of its flagella.

Detection of cellulose bcsABC genes and expression in the rugose and smooth pairs
The rugosity of E. coli and Salmonella species were reported to relate to cellulose and curli of the organisms. PCR assay was employed to detect cellulose bcsABC genes in the rugose and smooth pairs, and all strains presented bcsABC genes ( Table 4). The PCR results indicated that cellulose bcsABC genes were present in both rugose and smooth strains.   Note: -+‖ and --‖ represent: undetectable (-), weak (+), strong reaction (++), Stronger reaction (+++) in the assay. 2.00 x 10 ± 2.89 x 10 6.57 x 10 ± 4.39 x 10 _ *Bacteria were cultured at 37 C or 28 C for 2 days.  (Fig. 5). Because of the PCR results, the deficient expression of cellulose in strain 713S was clearly not caused by lacking bcsABC genes.

Detection of flagella genes in the rugose and smooth pairs
Bacterial motility is mostly driven by flagella (Ridgway et al., 1977). A set of primers were designed to check flagella genes in the two pairs (Table 1). fliC encodes flagellin (FliC), which is a flagellin monomer while flgE encodes a flagellar hook protein and flgK encodes a hook-associated protein. The PCR results are shown in Table 4. flgE and flgK were positive in all of the tested strains. fliC presented in all of the strains except strain 713S. The negative PCR result of flagellin gene fliC in 713S may be the reason why motility or flagella were absent in 713S in the motility test or the EM analysis.
Once excreted from the host, enteric pathogens face to harsh environments characterized by limited nutrients, high or low temperatures, acid, osmotic stress, and dryness (Winfield and Froisman, 2003). The rugose morphology is considered to help Vibrio and Salmonella survival in the stressful environments (White and Surette, 2006). In this study, we identified and defined rugose and smooth phenotypes in C. sakazakii. Based on these results, we showed the advantages of rugose phenotype in survival and persistence of C. sakazakii. Rugose variants produced more biofilms and cell aggregation than their smooth phase variant counterparts. The capacity of Cronobacter to switch between smooth to rugose phase phenotypes increase bacterial resistances to temperature and desiccative stresses; thus contributes to their survival in harsh environments. In contrast with Vibrio, the rugose morphology of Salmonella and other bacteria do not produce Vps. The rugosity of Salmonella species and pathogenic E. coli are reported to related to the expression of cellulose, curli, lipopolysaccharide (LPS), capsules or other polysaccharides (Kim and Wei, 2009). We try to find if the rugosity of Cronobacter related to curli and cellulose.
C. sakazakii do not possess curli csgBAC and csgDEFG genes (Hu, unpublished data). Both rugose and smooth pairs tested here possessed cellulose bcsABC genes using the PCR assay (Table 4). Furthermore, the rugose variants could form biofilms as much at 37 C as at 28 C, and the biofilms could form when the bacteria were cultured in rich culture media with 1% sodium chloride. In contrast to that, the biofilms formation associated with curli and cellulose usually occurred at lower temperature and low osmotic media (no salt or low salt); however, the biofilm formation and cell aggregation of the rugose strains could occur at 37 C and high osmotic condition. Thus, the difference between the rugose and smooth pairs in biofilm formation, cell aggregation, and long-term survival indicated that some unknown factors might mediate the smooth to rugose phase change except curli and cellulose Hu, unpublished data).
To understand what differences exist between rugose and smooth strains, a molecular typing (PFGE) and a colorimetric typing (VITEK) were used to detect potential differences. After genomic DNA of the organisms was separated by PFGE, no remarkable differences between the rugose and smooth pairs were found in PFGE typing except for two different bands between 713R and 713S. Furthermore, no differences in VITEK typing were shown between the rugose and smooth pairs. These results suggest that no obvious genomic and biochemical differences are found between the rugose and smooth variants using the assays.
Cronobacter could form biofilm, however, the quantity of biofilm formation varied among strains. Rugose variant 713R produced more biofilms and auto aggregation than rugose variant 632R. It was not clear why some rugose variant produced more biofilms than the others. The VITEK typing showed three biochemical reactions differed between strain 713 and 632. Whether the different reactions affect the quantity of biofilm formation still needs further investigation. A bacterial flagellum is comprised of a basal body, a hook and a filament (Apel and Surette, 2008). PCR primers targeted flagella hook and filament structure genes showed only filament gene fliC was absent in strain 713S. The failed expression of flagella in 713S may be due to the absent of fliC gene. It is still unknown if the failed expression of the filament or other factors in 713S caused no significant fluorescence in strain 713S in Calcofluor binding assay. However, it is clear that the absent of fliC in strain 713S did not relate to the rugose and smooth phase changes. From our results, the rugosity of C. sakazakii strain 713R and 632R were not involved in curli or cellulose. Other possible contributing factors may be LPS, extracellular polysaccharides, and capsules, or regulatory factors (Anriany et al., 2001;de Rezende et al., 2005).
Taking together, our data revealed that the rugose phenotype of Cronobacter formed more biofilms and cell autoaggregation, and enhanced the resistance to desiccated stress. The rugose variants of Cronobacter could survive longer than their smooth counterparts in the desiccated environment. Furthermore, Cronobacter rugosity did not associate with expression of curli and cellulose. PFGE and VITEK typing analyses of the pairs showed no significant differences between the rugose and smooth pairs in the assays. These results suggest that the smooth to rugose phenotype change may play an important role in Cronobacter survival and persistence in environments.