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1 These authors contributed equally to this research.
Affiliations
School of Food Science and Engineering, Hefei University of Technology, Hefei 230009, ChinaState Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China
1 These authors contributed equally to this research.
Na Ling
Footnotes
1 These authors contributed equally to this research.
Affiliations
State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China
State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China
State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China
State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China
Cronobacter sakazakii is an important foodborne pathogen associated with rare but severe infections through consumption of powdered infant formula. Tolerance to osmotic stress in Cronobacter has been described. However, the detailed factors involved in tolerance to osmotic stress in C. sakazakii are poorly understood. In this study, roles of outer membrane protein W (OmpW) on survival rates, morphologic changes of cells, and biofilm formation in C. sakazakii under different NaCl concentrations between wild type (WT) and OmpW mutant (ΔOmpW) were determined. The survival rates of ΔOmpW in Luria-Bertani medium with 3.5% or 5.5% NaCl were reduced significantly, and morphological injury of ΔOmpW was significantly increased compared with survival and morphology of WT. Compared with biofilm formation of the WT strain, biofilms in ΔOmpW were significantly increased in Luria-Bertani with 3.5% or 5.5% NaCl using crystal violet staining assay after 48 and 72 h of incubation. Detection of biofilms using confocal laser scanning microscopy and scanning electron microscopy further confirmed the changes of biofilm formation under different NaCl stresses. This study demonstrates that OmpW contributes to survival of cells in planktonic mode under NaCl stresses, and biofilm formation is increased in ΔOmpW in response to NaCl stress.
Cronobacter (formerly known as Enterobacter sakazakii) is an opportunistic foodborne pathogen causing rare but severe infections in newborns (Healy et al., 2012). Cronobacter spp. are widely distributed in food and environments including food-processing environments, water, soil, and clinical samples (
). To date, the genus of Cronobacter consists of 7 species: C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. condiment, C. universalis, and C. dublinensis (
Cronobacter condimenti sp. nov., isolated from spiced meat and Cronobacter universalis sp. nov., a novel species designation for Cronobacter sp. genomospecies1, recovered from a leg infection, water, and food ingredients.
Int. J. Syst. Evol. Microbiol.2012; 62 (22661070): 1277-1283
demonstrated that Cronobacter cells were more resistant to osmotic stress than Escherichia coli, Salmonella spp., and other strains of Enterobacteriaceae. Increasing the compatible solute concentrations in medium was effective in increasing osmotic resistance of E. coli and Salmonella (
). Growth of 18 of Cronobacter strains from dried edible macrofungi samples in 1.0, 2.5, 4.0, and 5.5% NaCl indicated that Cronobacter has unusual ability to survive under NaCl stress (
). To understand the mechanisms involved in osmotic stress response, 53 proteins were identified using 2-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS (
Outer membrane protein W (OmpW) is an important outer membrane protein involved in response to osmotic stress or salt regulation in Photobacterium damselae (
). However, little attention has been focused on the role of OmpW in tolerance to osmotic stress in Cronobacter species.
In this study, we determined the survival rates and morphologic changes between C. sakazakii wild type (WT) and OmpW mutant (ΔOmpW) strains under different NaCl stresses. In addition, we assessed the biofilm formation of strains under NaCl stress using crystal violet staining (CVS) assay, scanning electron microscopy, and confocal laser scanning microscopy (CLSM).
MATERIALS AND METHODS
Growth and Survival of C. sakazakii Under NaCl Stress
Cronobacter sakazakii GDMCC1409C (WT) and its OmpW mutant (ΔOmpW) were from Guangdong Microbiology Culture Center (GDMCC; Guangzhou, China). Two C. sakazakii strains were incubated into normal Luria-Bertani (LB) medium (control), LB with 1.5% NaCl, LB with 3.5% NaCl, and LB with 5.5% NaCl for 8 h. The number of C. sakazakii under different media was counted using a colony counting method. Survival rates were calculated as the number of C. sakazakii cells in the control sample divided by the number of cells in different NaCl samples. Each experiment was done in triplicate.
Morphologic Changes of C. sakazakii Cells Under Different Osmotic Stresses
Cronobacter sakazakii GDMCC1409 was incubated in LB, LB + 1.5%NaCl, LB + 3.5%NaCl, and LB + 5.5%NaCl, and the cells were harvested to detect morphologic changes using scanning electron microscopy (Hitachi, Tokyo, Japan). The treatment procedure was performed according to description by
For detection of biofilms by CVS assay, 2 C. sakazakii strains were inoculated into LB, LB + 1.5% NaCl, LB + 3.5% NaCl, and LB + 5.5% NaCl and incubated at 37°C for 10 to 12 h with constant shaking. Twenty microliters of overnight culture [optical density (OD) at 600 nm = 0.5] was inoculated into 48-well polystyrene plates containing 1.98 mL of sterile LB broth and incubated at 37°C for 24, 48, and 72 h. The plates were rinsed 3 times with deionized water, and the adherent bacteria cells were stained with 1% crystal violet for 30 min. After being rinsed 3 times with deionized water, the crystal violet was liberated by 30% acetic acid following a 10-min incubation. The OD values of each well were measured at 590 nm. Each experiment was done in triplicate.
For scanning electron microscopy, glass coverslips (Jingan, Shanghai, China) were placed into the 48-well plates containing 1.98 mL of LB, LB + 1.5% NaCl, LB + 3.5%NaCl, and LB + 5.5%NaCl, respectively. Then, C. sakazakii overnight culture (20 μL) was mixed with the LB with different NaCl concentrations in 48-well plates (Baiyan, Shanghai, China) for incubation at 37°C for 24, 48, and 72 h. During the biofilm formation period, old medium was replaced with fresh LB at 24-h intervals. The glass coverslips from different incubation times were prepared for scanning electron microscopy as described previously by
For CLSM, glass coverslips were prepared in the same manner as for scanning electron microscopy detection, and bacterial biofilms on glass slips were stained by using the LIVE/DEAD BacLight bacterial viability kit according to instructions (Invitrogen/Thermo Fisher Scientific, Shanghai, China), and were then observed by CLSM (Zeiss, Berlin, Germany).
Statistics Analysis
Survival rates and biofilm-forming ability using CVS between WT and ΔompW under osmotic stresses were assessed in triplicate and analyzed using OriginPro 8.5.1 (OriginLab Corp., Northampton, MA).
RESULTS AND DISCUSSION
As important foodborne pathogens, Cronobacter spp. have unusual ability to survive under different environmental stresses (
). In the current study, we determined the role of OmpW in response to osmotic stress. As shown in Figure 1, survival rates of WT and ΔOmpW under different NaCl concentrations (1.5, 3.5, and 5.5% NaCl) were 8.06 ± 0.59% and 7.29 ± 0.315, 1.64 ± 0.30% and 0.19 ± 0.02%, and 0.57 ± 0.19% and 0.14 ± 0.02%, respectively, compared with those in the control (LB medium). In addition, under 3.5% and 5.5% NaCl stress, survival rates of WT were significantly increased (P < 0.05) compared with those of ΔompW.
noted that the growth of different Cronobacter strains from dry edible macrofungi samples under the same NaCl concentrations was distinct. In Aeromonas hydrophila, expression of OmpW protein was found to be salt-dependent (
). Expression of OmpW in Vibrio cholerae was found to depend environmental factors and might be linked to the adaptive response of the organism under stress conditions (
). With increasing NaCl concentrations, morphological injuries, including cell disruption and shrinkage, were observed among the WT and ΔompW strains in the current study, as shown in Figure 2, and injuries of cells were more predominant in ΔompW than in WT, suggesting that OmpW contributes to survival of C. sakazakii cells under NaCl stress.
Figure 1Survival rates of Cronobacter sakazakii wild type (WT) and outer membrane protein W mutant (ΔOmpW) under different NaCl stresses. Error bars indicate standard deviations. Asterisks indicate difference between WT and mutant at *P < 0.05 and **P < 0.01.
Figure 2Morphological changes of Cronobacter sakazakii wild type (WT) and outer membrane protein W mutant (ΔOmpW) under different NaCl stresses using scanning electron microscopy.
Bacterial biofilms formed on surfaces of food processing environments and equipment might be critical factors in persistent contamination by foodborne pathogens (
). In the current study, biofilm formation (assessed using CVS assay) by ΔOmpW was significantly increased compared with that by WT under NaCl stress (Table 1). After 24 h, no significant quantitative changes in biofilm were observed, whereas biofilms were significantly reduced after 48 and 72 h compared with control groups. As shown in Figure 3, after 24 h of incubation in LB medium, the 2 strains could not form biofilms and cells were loosely adhered to the glass coverslips. After 48 h of incubation, biofilms with mature and spatial structure were observed in the 2 strains, and disassembly of biofilms occurred after 72 h of incubation. In particular, biofilms in ΔOmpW were more compact and complete compared with those of the WT strain under the same salt concentrations, suggesting the ΔOmpW mutant strain could adapt to NaCl stress by enhancing biofilm formation. As shown in Figure 4, mature and compact biofilms of WT and ΔOmpW were formed after 48 h of incubation at 37°C; after 72 h, dead cells were obvious and were more predominant under 5.5% NaCl compared with other groups. In addition, the biofilms of ΔOmpW were more compact than those of the WT strain. Outer membrane proteins A and X are required for invasion of C. sakazakii into biotic cells (
demonstrated that incubation conditions, including temperature, time, and NaCl concentration, play important roles in biofilm formation in Cronobacter strains. In Pseudoalteromonas sp. D41, OmpW mutant formed reduced and altered biofilms (
). Proteomic analysis in Acinetobacter baumannii showed that expression of putative OmpW in biofilm mode was significantly reduced compared with that in planktonic mode (
). These results suggest that roles of OmpW under NaCl stress depend on the species or environmental conditions. The detailed mechanism of OmpW on biofilm formation in C. sakazakii remains to be revealed.
Table 1Biofilm formation (measured as optical density at 590 nm; means ± SD) of wild type (WT) and outer membrane protein W mutant (ΔOmpW) under NaCl stress and incubated for 24, 48, and 72 h
Figure 3Biofilm formation of Cronobacter sakazakii wild type (WT) and outer membrane protein W mutant (ΔOmpW) under different NaCl stresses and incubation for 24, 48, and 72 h using scanning electron microscopy.
Figure 4Biofilm formation of Cronobacter sakazakii wild type (WT) and outer membrane protein W mutant (ΔOmpW) under different NaCl stresses and incubation for 24, 48, and 72 h using confocal laser scanning microscopy. Color version available online.
We gratefully acknowledge the financial support of the National Natural Science Foundation of China (Beijing; 31671951), the Anhui provincial Grand Project special of Science and Technology (15czz03109), the Science and Technology Planning Project of Guangdong Province (2016A050502033), Project of Science and Technology in Guangzhou (201604020036), and State Key Laboratory of Applied Microbiology Southern China Open Foundations, Guangzhou, China (SKLAM004-2015).
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Cronobacter condimenti sp. nov., isolated from spiced meat and Cronobacter universalis sp. nov., a novel species designation for Cronobacter sp. genomospecies1, recovered from a leg infection, water, and food ingredients.
Int. J. Syst. Evol. Microbiol.2012; 62 (22661070): 1277-1283
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