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1 These authors contributed equally to the manuscript.
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 the manuscript.
Na Ling
Footnotes
1 These authors contributed equally to the manuscript.
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
Cronobacter sakazakii is associated with severe infections including sepsis, neonatal meningitis, and necrotizing enterocolitis. Antibiotic resistance in Cronobacter species has been documented in recent years, but the genes involved in resistance in Cronobacter strains are poorly understood. In this study, we determined the role of outer membrane protein W (OmpW) on survival rates, morphologic changes, and biofilm formation between wild type (WT) and an OmpW mutant strain (ΔOmpW) under neomycin sulfate stress. Results indicated that the survival rates of ΔOmpW were significantly reduced after half minimum inhibitory concentration (½ MIC) treatment compared with the WT strain. Filamentation of C. sakazakii cells was observed after ½ MIC treatment in WT and ΔOmpW, and morphologic injury, including cell disruption and leakage of cells, was more predominant in ΔOmpW. Under ½ MIC stress, the biofilms of WT and ΔOmpW were significantly decreased, but decreasing rates of biofilm formation in mutant strain were more predominant compared with WT strain. This is the first report to determine the role of OmpW on survival, morphological changes, and biofilm formation in C. sakazakii under neomycin sulfate stress. The findings indicated that OmpW contributed to survival and reduction of morphological injury under neomycin sulfate stress. In addition, enhancing biofilm formation in ΔOmpW may be an alternative advantage for adaptation to neomycin sulfate stress.
). Unfortunately, the true source of Cronobacter in powdered infant formula was not clearly understood. Consequently, Cronobacter posed a high risk to public health, especially for newborns.
In recent years, tolerance or resistance of foodborne pathogens to antibiotics is of great concern for human health. Although Cronobacter has been reported as being more sensitive than other Enterobacter species to some antibiotics, including aminoglycosides, ureidopenicillins, ampicillin, and carboxypenicillins (
Correlation between ceftriaxone resistance of Salmonella enterica serovar Typhimurium and expression of outer membrane proteins OmpW and Ail/OmpX-like protein, which are regulated by BaeR of a two-component system.
). OmpW is distributed in gram-negative bacteria, and its 3-dimensional structure consists of an 8-stranded β-barrel with a long and narrow hydrophobic channel (
). Neomycin sulfate, as an aminoglycoside antibiotic, can transfer through hydrophilic ion channels on the surface of bacteria to interfere with synthesis of proteins and further acquire antibacterial activity. However, the role of OmpW under neomycin sulfate stress in Cronobacter species is poorly understood.
In this study, we determined survival rates and morphologic changes of C. sakazakii cells under neomycin sulfate stress between the wild type (WT) and OmpW mutant (ΔOmpW) strains. We also assessed biofilm formation under neomycin sulfate stress using crystal violet staining (CVS), scanning electron microscopy, and confocal laser scanning microscopy (CLSM).
Cronobacter sakazakii WT and ΔOmpW from Guangdong Microbiology Culture Center (GDMCC; Guangzhou, China) were incubated in unsupplemented neomycin sulfate Luria Bertani (LB) broth at 37°C for overnight. Then, 1% of the overnight culture was transferred into fresh LB containing different concentrations of neomycin sulfate for 16 h at 37°C. The MIC of neomycin sulfate were 8 µg/mL and 7 µg/mL for WT and ΔOmpW. The 2 strains (106 cfu/mL) were transferred into normal LB and LB with 50% MIC for each for 6 h at 37°C. Survival rates were counted according to the following formula: Survival rate = number of cells in LB with ½ MIC/No. of cells in normal LB.
Each experiment was performed in triplicate, and significance was determined using Origin pro 8.5.1 (OriginLab, Northampton, MA). The survival rate of the WT strain was higher (P < 0.01) than that of ΔOmpW, as shown in Figure 1A. In Escherichia coli, the relatively high expression of OmpW was consistent with strong tolerance to tetracycline and ampicillin (
), indicating that OmpW functions as a porin to export antibiotics from the cell. In another study, survival capability significantly decreased in an OmpW mutant compared with a wild strain under tetracycline stress (
Correlation between ceftriaxone resistance of Salmonella enterica serovar Typhimurium and expression of outer membrane proteins OmpW and Ail/OmpX-like protein, which are regulated by BaeR of a two-component system.
). These studies indicate that the role of OmpW in antibiotic resistance depends on pathogen species and type of antibiotic.
Figure 1Survival rates (A) and morphological changes, including filamentation and disruption of cells under neomycin sulfate (B), of Cronobacter sakazakii wild type (WT) and an outer membrane protein mutant (ΔOmpW) under neomycin sulfate (NM) stress (NM at ½ MIC; the MIC were 8 and 7 µg/mL for WT and ΔOmpW, respectively). Survival rates = means ± SD. **P < 0.01 between WT and ΔOmpW.
, using scanning electron microscopy (Hitachi, Tokyo, Japan). In addition, we detected filamentation of C. sakazakii under neomycin sulfate treatment, and injury of mutant cells was more prevalent than that of the WT, as shown in Figure 1B.
Biofilm formation of 2 strains in LB broth and LB with ½ MIC neomycin sulfate was determined using CVS described by
. For CVS detection, C. sakazakii WT and ΔOmpW strains were inoculated into 5 mL of LB and grown for 12 to 14 h at 37°C with constant shaking. Fifty microliters of culture (optical density at 600 nm = 0.5) was inoculated into 96-well polystyrene plates containing 250 µL of sterile LB broth with neomycin sulfate or without neomycin sulfate, and then 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 optical density values of each well were measured at 590 nm.
Furthermore, biofilm formation by C. sakazakii WT and ΔOmpW strains were determined using scanning electron microscopy. Cronobacter sakazakii were inoculated in LB broth at 37°C overnight and 0.05 mL of the culture transferred to 24-well plates (Baiyan, Shanghai, China) containing 5 mL of fresh LB with ½ MIC neomycin sulfate. To test biofilm formation on glass, glass coverslips (Jingan, Shanghai, China) were immersed in LB broth and then inoculated with C. sakazakii. The coverslips were incubated in 24-well plates at 37°C for 24, 48, and 72 h, after which time bacterial biofilm formation was observed. The coverslips at different incubation times were rinsed in PBS and fixed with 2.5% glutaraldehyde overnight at 4°C. Post-fixation was carried out using 1% osmium tetroxide for 2 h before dehydration in an ethanol series (50, 70, 80, 90, and 100%; 30 min for each concentration). The dehydrated biofilms were coated with a thin layer of gold and examined under a Hitachi SU1510 scanning electron microscope using an accelerating voltage of 5 kV (Hitachi).
For CLSM detection, biofilms of WT and ΔOmpW on glass coverslips were prepared according to the same procedure described above, stained with LIVE/DEAD BacLight bacterial viability kit (Invitrogen, Beijing, China), and detected by CLSM (Zeiss, Berlin, Germany).
In Cronobacter strains, Omp A and OmpX contribute to adhesion of Cronobacter to Caco-2 and INT-407 cells (
). In the current study, under neomycin sulfate stress, biofilm formations in ΔOmpW and WT were significantly different compared with those in LB broth. In addition, significant differences (P < 0.01) of biofilms in WT and ΔOmpW under 50% MIC neomycin sulfate were also observed, showing the mutant might protect itself against neomycin sulfate stress through enhanced biofilm production. As can be seen in Figure 2B and 2C, structural changes in biofilms were detected between WT and ΔOmpW at different times using scanning electron microscopy and CLSM. Biofilm formation by ΔOmpW in LB broth was significantly increased compared with that by the WT strain, and mature biofilm was formed in both strains after 48 h of incubation. In addition, the biofilms of WT and ΔOmpW strains were decreased under 50% MIC neomycin sulfate stress. After incubation for 72 h, the dead cells (yellow color in Figure 2B) were predominant compared with incubation of 24 and 48 h biofilms. From Figure 2C, the biofilms were not integrated and compact under 50% MIC neomycin sulfate stress. Our results also indicated that disassembly of biofilms appeared after 72 h in C. sakazakii.
Figure 2Biofilm formation of Cronobacter sakazakii wild type (WT) and an OmpW mutant (ΔOmpW) in Luria Bertani (LB) broth and LB with neomycin sulfate stress (NM at ½ MIC; the MIC were 8 and 7 µg/mL for WT and ΔOmpW, respectively). (A) Crystal violet staining, (B) confocal laser scanning microscopy, and (C) scanning electron microscopy. OD590nm = optical density at 590 nm; survival rates = means ± SD. *P < 0.01, **P < 0.05 indicate difference between WT and ΔOmpW strains. Color version available online.
In summary, we determined, for the first time, the role of OmpW in survival and biofilm formation in C. sakazakii. The findings indicated that OmpW contributed to survival and reduced morphological injury under neomycin sulfate stress. In addition, enhanced biofilm formation in ΔOmpW may be an alternative advantage for adaptation to neomycin sulfate stress.
Acknowledgments
We gratefully acknowledge the financial support of the National Natural Science Foundation of China (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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Correlation between ceftriaxone resistance of Salmonella enterica serovar Typhimurium and expression of outer membrane proteins OmpW and Ail/OmpX-like protein, which are regulated by BaeR of a two-component system.
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